Draft Guidelines on Similar Biologics - Regulatory Requirements for Marketing Authorization in India, 2025

1. No. r-DNA-15011(11)/17/2024-eoffice Government of India Directorate General of Health Services Central Drugs Standard Control Organization (Biological Division) FDA Bhawan, Kotla Road. New Delhi 110002 06 MAY 2025 Date:

NOTICE

Subject: Inviting comments on Revised Guidelines on Similar Biologics- Regulatory

requirements for Marketing Authorization in India, 2025 drafted by CDSCO In view of advances in scientific knowledge and experience, it was decided to revise the existing guidelines in line with recent international quidelines. To facilitate this process, a Committee comprising of technical subject experts, representatives from NIB, DBT and

representatives from Industries involved in manufacturing of similar biologics was

Constituted. The committee meetings were convened to discuss the revisions in the guidelines.

The Draft Guidelines is now being placed in the public domain for inviting comments/suggestions from concerned stakeholders. This window of opportunity will close within 30 days of publishing the draft guidelines on CDSCO website, and, once finalized all interested this Therefore, document. in change for scope be minimal will there stakeholders are requested to provide comments/suggestions within the window of 30 days per the format. annexed document as in word at biological@cdsco.nic.in shall days the 30 within address the on email above received The suggestions/comments be taken into consideration for finalisation of the draft Guidance document.

Stakeholder's Comments format

Name and Designation:

Firm Name:

Line S.No. Page Section/Sub- Current Proposed Explanation/Reference section/Heading text text No. No.

Dr-Rajsev Singh Raghuvanshi Drugs Controller General (India) To: All Stakeholders through CDSCO website Encl: Copy of Draft CDSCO Guidelines on Similar Biologics-Regulatory Requirements for

Marketing Authorization in India, 2025

Page 1 of 1

DRAFT GUIDELINES ON SIMILAR BIOLOGICS

Regulatory Requirements for Marketing Authorization in India, 2025

Central Drugs Standard Control Department of Biotechnology Organization Ministry of Science & Technology, 1 Ministry of Health & Family Welfare Government of India Government of India Document Name: GUIDELINES ON SIMILAR BIOLOGICS Effective From Year: 2025 Validity: Till Further Revision

Contents

Message ................................................................................................ 4

Foreword ............................................................................................... 5

Preface .................................................................................................. 6

List of Acronyms .................................................................................... 8

1. Introduction ........................................................................................................... 8
2. Background .......................................................................................................... 9
3. Purpose & Scope ................................................................................................ 10
4. Applicable Regulations and Guidelines .............................................................. 10
5. Competent Authorities ........................................................................................ 11
6. Scientific Considerations and Concept for Licensing Similar Biologics ............... 12
7. Key Principles for the Licensing of Similar Biologics .......................................... 12
8. Reference Biological Product (RBP) ................................................................... 13
9. Quality ................................................................................................................ 14 9.1 Reference standards ........................................................................................... 14

9.2 Manufacturing process ........................................................................................ 15

9.3 Analytical considerations ..................................................................................... 17

9.4 Comparative analytical assessment .................................................................... 22

9.5 Specifications ...................................................................................................... 25

9.6 Stability ................................................................................................................ 26

1. Data Requirements for Preclinical Studies ....................................................... 26 10.1 In vitro studies ................................................................................................... 27

10.2 Determination of the need for in vivo animal studies ......................................... 28

10.3 In vivo studies .................................................................................................... 30

1. Data Requirements for Clinical Trial Application .............................................. 32 11.1 Pharmacokinetic (PK) Studies ........................................................................... 33

11.2 Pharmacodynamic Studies ................................................................................ 35

11.3 Confirmatory PK and/or PD studies ................................................................... 35

11.4 Efficacy studies .................................................................................................. 37

11.5 Safety ................................................................................................................ 38

11.6 Immunogenicity ................................................................................................. 39

11.7 Waiver of safety and efficacy study .................................................................... 42

11.8 Extrapolation of Efficacy and Safety Data to Other Indications ......................... 42

1. Data Requirements for Market Authorization Application ................................. 43
2. Risk management plan (RMP) ......................................................................... 43
3. Post-Market Data for Similar Biologics ............................................................. 43 14.1 Pharmacovigilance Plan .................................................................................... 44

14.2 Adverse Drug Reaction (ADR) Reporting .......................................................... 44

14.3 Post Marketing Studies (Phase IV Study) .......................................................... 44

1. Labelling and Prescribing Information .............................................................. 45
2. Application Forms ............................................................................................ 45
3. Archiving of Data/Retention of Samples: .......................................................... 46
4. Glossary ........................................................................................................... 46
5. References ....................................................................................................... 50 Annexure I: Pathway for approval to manufacture and market indigenously developed

Similar Biologics ......................................................................................................... 52

Annexure IA: Pathway for approval to import and market Similar Biologics ............... 53

Annexure II: Critical Quality Attributes (CQA) ............................................................ 54

Annexure III: Statistical tools for Biosimilarity assessment ........................................ 55

Annexure IV: Requirements of Toxicological Studies ................................................ 62

Annexure V: Statistical consideration in sample size determination for Clinical Study

................................................................................................................................... 64

Message

Foreword

Preface

The Guidelines on Similar Biologic-Regulatory Requirements for Marketing 2 Authorization in India was published in the year 2012 by CDSCO in collaboration with 3 Department of Biotechnology (DBT) to address the regulatory pathway for Similar 4 Biologics in India. The Guidelines was then revised in the year 2016 with more focus on 5 scientific principles and stepwise approach to be applied during the demonstration of 6 similarity between a similar biological product and its reference biological product. 7 Keeping in view the advances in scientific knowledge and experience, it was decided to 8 update the existing guidelines in line with recent international guidelines. A Committee 9 was constituted for the same including technical subject experts, representatives from 10 NIB, DBT and representatives from Industries involved in manufacturing of similar 11 biologics. The committee meetings were convened to discuss the revisions in the 12 guidelines. 13 In view of committee recommendations, the present Guideline document, 2025 was 14 framed which represents the outcome of the revision process and replaces 15 GUIDELINES ON SIMILAR BIOLOGICS: Regulatory Requirements for Marketing 16 Authorization in India, 2016. This guideline considers the current scientific evidence 17 and scientific updates from the International Guidelines majorly WHO TRS 1043: 18 Guidelines for evaluation of biosimilars. Since, major countries are moving for waiver of 19 non-clinical studies for similar biologics, the current revision principally focus on 20 strengthened orthogonal analytical tools and in vitro studies to establish similarity 21 between the similar biologic product and Reference Biological Product. 22 The salient features of the revision include- 23

1. Introduction of scientific considerations and key principles for licensing of similar 24 biologics. 25 b. Sections of quality, and nonclinical and clinical evaluation are updated to make 26 them more consistent with current international practices and to provide more 27 clarity and flexibility. 28 c. Revised pathway for approval of similar biologics in India 29 d. Specific topics addressed in the revision include but are not limited to:  30 • Next generation analytical methodologies introduced for establishing analytical 31 similarity 32 • Use of reference standards and development of in-house reference standards 33 elaborated 34 • Elaborative list of in vitro studies included 35 • New guidance on determining the need for in vivo animal studies and on the 36 37           the use of animals in testing 38 6

• Statistical intervals for establishment of similarity ranges to provide clarity and focus on statistical consideration in calculation of sample size for clinical studies.

List of Acronyms

Guidelines on Similar Biologics 44

Regulatory Requirements for Marketing Authorization in India 45

1. Introduction 46 8 ADA Anti-Drug Response FC Fragment Crystallizable ADCC Antibody-Dependent Cellular Cytotoxicity ADCP Antibody-Dependent Cellular Cytotoxicity BP British Pharmacopoeia CDSCO Central Drugs Standard Control Organization CDC Complement Dependent Cytotoxicity CRS Chemical Reference Standards CQA Critical Quality Attributes DBT Department of Biotechnology DCGI Drug Controller General of India EMA European Medicines Agency EP European Pharmacopoeia GEAC Genetic Engineering Appraisal Committee GMP Good Manufacturing Practice IBSC Institutional Biosafety Committees ICH International Council of Harmonisation IRS In-house reference Standards IU International Units JP Japanese Pharmacopoeia LMO Living Modified Organism MA Market Authorization mAbs Monoclonal Antibodies MoHFW Ministry of Health & Family Welfare NDCT New Drugs and Clinical Trial Rules 2019 NIBSC National Institute for Biological Standards and Control NIST National Institute of Standards and Technology PD Pharmacodynamic PK Pharmacokinetic PSUR Periodic Safety Update Reports QA Quality Attribute RBP Reference Biological Product RCGM Review Committee on Genetic Manipulation SBP Similar Biological Product TNF Tumour Necrosis Factor USFDA United States Food and Drug Administration USP United States Pharmacopeia WHO World Health Organization Biotherapeutic products have a proven track record in treating numerous life-threatening and chronic diseases. As patents and data protection periods for many of these products expire, a new wave of products has emerged that are designed to be highly "similar" to the licensed "originator" products. These similar products can partly rely the safety and efficacy data of the originator products, based on a thorough head-to-head comparison demonstrating high 51 similarity. 52 CDSCO is the national regulatory authority in India that evaluates safety, efficacy, and quality of 53 drugs in the country. Th54 Control Organization (CDSCO) and the Department of Biotechnology (DBT) lay down the 55 regulatory pathway for a Similar Biologic claiming to be Similar to an already authorized 56 Reference Biologic. 57

Similar B58 of quality, safety and efficacy to Reference Biological Product (RBP) licensed or approved in 59 India, or any innovator product approved in International Council of Harmonisation (ICH) 60 member countries. The      widely used by many Drug regulatory 61 agencies such as United States Food and Drug Administration (USFDA), European Medicines 62 Agency (EMA), WHO etc. Both the terms 63 to the same terminology and can be used interchangeably. 64 Presently, several organizations are actively engaged in manufacturing and marketing similar 65 biologics in India. In the past, these Similar Biologics were approved by RCGM and Central 66 Drugs Standard Control Organization (CDSCO) using an abbreviated version of the pathway 67 applicable to new drugs on a case-by-case basis. 68 These guidelines are for the guidance of all stakeholders and are not meant to substitute or 69 rephrase the Rules made under Drugs and Cosmetics Act, 1940 or any other relevant Acts and 70 are subject to being in conformity with the Drugs and Cosmetics Act and Rules as may be 71 amended from time to time. 72

1. Background 73 CDSCO in collaboration with Department of Biotechnology (DBT) published the first guidelines 74 titled as  Similar Biologic- Regulatory Requirements for Marketing Authorization 75 in India  2012 to address the regulatory pathway regarding manufacturing process and 76 quality aspects for Similar Biologics. The said guidelines also address the pre-market regulatory 77 requirements including comparability exercise for quality, preclinical and clinical studies and 78 post market regulatory requirements for similar biologics. 79 Keeping it at par with latest regulatory requirements and to provide more clarity, the guidelines 80 were revised in the year 2016 with more focus on scientific principles and stepwise approach to 81 be applied during the demonstration of similarity between a similar biological product and its 82 reference biological product. I83 revised in line with advances in scientific knowledge and experience. 84 9

It was decided that a review of existing guidelines should be undertaken of current scientific evidence and international guidelines including Guidelines on Evaluation of Similar Biologics WHO Technical Report Series, No. 1043, 2022 (Replacement of Annex 2 of WHO Technical Report Series, No. 977). This revised guideline would provide an opportunity to evaluate new developments and identify areas where the current guidance could be more flexible without 89 compromising its basic principles and allow for the provision of additional explanation of the 90 possibility of tailoring the amount of data needed for regulatory approval. 91

1. Purpose & Scope 92
   The objective of this document is to provide guidance to applicants to enable them to 93 understand and comply with the regulatory requirements for market authorization of Similar 94 Biologics in India. 95 These guidelines apply to Similar Biologics that contain well characterized proteins as their 96 active substance, derived through modern biotechnological methods such as use of 97 recombinant DNA technology. The demonstration of similarity depends upon detailed and 98 comprehensive product characterization, preclinical and clinical studies carried out in 99 comparison with a Reference Biological Product. 100 Similar Biologics can only be developed against the Reference Biological Product that has been 101 approved using a complete data package in India. In case the RBP is not authorized in India, it 102 should have been approved / licensed and marketed in an ICH (The International Council for 103 Harmonisation of Technical Requirements for Pharmaceuticals for Human Use) country namely 104 USA, UK, Japan, Australia, Canada and EU. 105 Any product can be considered as a similar biologic, only if it is proven to be similar using 106 totality of the evidence concept requiring that sufficient structural, functional, nonclinical, and 107 clinical data is acquired in stepwise manner to demonstrate that there are no clinically 108 meaningful differences between the similar biological product (SBP) and the reference 109 biological product (RBP) in terms of the safety, purity, and potency of the product. 110 The reference biological product (RBP) is central to the licensing of a similar biological product, 111 and the choice of a suitable RBP is fundamental for a similar biologic development. The RBP 112 should have been marketed for a suitable duration, have a significant volume of marketed use 113 in the relevant country or area, and have a long established history of good safety and efficacy. 114 These guidelines are applicable for similar biologics to be developed in India or imported into 115 the country for marketing authorization. Detailed regulatory pathway for approval of Similar 116 Biologics is given in Annexure I and Annexure IA. 117

2. Applicable Regulations and Guidelines 118
   The similar biologics are regulated as per the Drugs and Cosmetics Act, 1940, the Drugs Rules, 119 1945 (as amended from time to time), New Drugs and Clinical Trial Rules 2019 (NDCT) and 120 Rules for the manufacture, use, import, export and storage of hazardous microorganisms/ 121 10

genetically engineered organisms or cells, 1989 (Rules, 1989) notified under the Environment (Protection) Act, 1986. Various applicable guidelines are as follows:

• Guidelines for generating preclinical and clinical data for rDNA vaccines, diagnostics and other Biologicals, 1999.

• CDSCO guidance for industry, 2024 126
  Submission of Clinical Trial Application for Evaluating Safety and Efficacy 127 Requirement for permission of New Drug Approval. 128 Preparation of Quality Information for Drug Submission for New Drug Approval: 129 Biotechnological/Biological Products 130

• Post approval changes in biological products: Quality, Safety and Efficacy Documents, 131 2024 132

• Regulation and Guidelines for Recombinant DNA Research and Biocontainment, 2017 133

• Guidelines and Handbook for Institutional Biosafety Committees (IBSCs), 2020. 134

1. Competent Authorities 135 The competent authorities involved in the approval process are as follows: 136

Institutional Biosafety Committee (IBSC) 137 IBSC is required to be constituted by any person including research institutions handling 138 hazardous microorganisms and/ or genetically engineered organisms. IBSC is responsible for 139 ensuring biosafety on-site and is also assigned with the responsibility to review and authorize 140 firm for exchange of aforesaid organisms for the purpose of research. 141

Review Committee on Genetic Manipulation (RCGM) 142 RCGM is functioning under the Department of Biotechnology (DBT), Ministry of Science and 143 Technology, Government of India. In the context of Similar Biologics, RCGM is responsible for 144 authorizing the conduct of research and development involving Risk Group 3 and 4 organisms 145 and exchange of genetically engineered cell banks for the purpose of research and 146 development . 147

Central Drugs Standard Control Organization (CDSCO) 148 CDSCO, headed by the Drug Controller General of India (DCGI) is the apex regulatory body 149 under Ministry of Health & Family Welfare (MoHFW), Government of India, which is responsible 150 for the approval of New Drugs, Clinical Trials in the country, laying down the standards for 151 Drugs, control over the quality of Imported Drugs, coordination of the activities of State Drug 152 Control Organizations and providing expert advice with a view of bring about the uniformity in 153 the enforcement of the Drugs and Cosmetics Act. 154

In the context of Similar Biologics, CDSCO is responsible for clinical trial approval (also grants permission for import of drugs for clinical trial) and permission for import and manufacturing for sale or for distribution.

1. Scientific Considerations and Concept for Licensing Similar Biologics
   The regulatory framework for licensing generic medicines is well-established in many countries. 159 Typically, demonstrating structural similarity and bioequivalence between a generic medicine 160 and its RBP is sufficient to infer therapeutic equivalence. However, this approach is not 161 applicable to the licensing of similar biologics, as biological products are generally large and 162 complex proteins that are more difficult to characterize and manufacture than small molecules. 163 The first step in developing a similar biologic should be the characterization and evaluation of 164 the quality attributes of the RBP. This is followed by a comparability exercise using sensitive, 165 orthogonal analytical methods and assays to demonstrate structural, functional, and clinical 166 similarity. Comprehensive characterization and comparison at the quality and nonclinical (in 167 vitro) levels serve as the basis for establishing comparability, with a tailored confirmatory clinical 168 data package required for licensing. If relevant differences between the similar biologic and the 169 RBP are identified, the underlying causes should be explored. Unless these differences can be 170 explained and justified in terms of their lack of clinical impact, additional data, such as on safety, 171 may be needed. 172 In addition to quality and nonclinical (in vitro) data, clinical data are typically required for any 173 similar biologic. The type and extent of such data needed will depend on factors such as the 174 specific product or product class, the level of characterization achievable through advanced 175 analytical methods, observed or potential differences between the similar biologic and the 176 reference biological product (RBP), and clinical experience with the RBP. 177 Manufacturers must demonstrate a thorough understanding of their product, ensure consistent 178 and reliable manufacturing processes, and provide a comprehensive quality dossier that 179 includes detailed product characterization. The dose and route of administration for the similar 180 biologic must be same as that of RBP. Clinical studies must be conducted using the final 181 formulation of the similar biologic derived from the final process; otherwise, additional evidence 182 is needed to confirm that the marketed product matches the one used in clinical trials. 183 In case more than one indication is approved for the RBP, the similar biologic also qualifies for 184 all the indications only if it is justified and if meets the conditions set forth in the section 185 tion of 186 indication shall be based on comparability in quality, preclinical and clinical studies, available 187 literature data and whether or not the same mechanism of action is involved in specific 188 indications. 189

2. Key Principles for the Licensing of Similar Biologics 190

• Characterization of the quality attributes of the RBP should be the first step in guiding the development of the similar biologic. The subsequent comparability exercise should demonstrate structural, functional and clinical similarity. • Demonstration of similarity of a similar biologic to an RBP in terms of structural and functional aspects is a prerequisite for establishing comparability, with a tailored clinical data 195 package required as needed. 196 • Comparative clinical trial, assessment of pharmacokinetic (PK) and pharmacodynamic (PD) 197 parameters (if available), and immunogenicity in human subjects, will typically be a core part 198 of the clinical comparability assessment, unless scientifically justified. 199 • The decision to license a similar biologic should be based on evaluation of the whole data 200 package generated during the overall comparability exercise. 201 • If relevant differences between the proposed similar biologic and the RBP are found at the 202 structural, functional or clinical level, the product is unlikely to qualify as a similar biologic. 203 • If comparability exercises are not performed as outlined in this document, then the final 204 product should not be referred to as a similar biologic. 205 • The authorization process of generic medicines does not apply for similar biologics. 206 • As with other biological products, similar biologics require effective regulatory oversight pre- 207 and post-approval in order to manage the potential risks they pose and to maximize their 208 benefits. 209

1. Reference Biological Product (RBP) 210 Comprehensive information on the reference biological product (RBP) provides the basis for 211 establishing the quality, safety and efficacy profile against which the similar biologic will be 212 compared. The RBP has to be used in all the comparability exercises with respect to quality, 213 preclinical and clinical considerations. 214 The choice of RBP is therefore critically important in the evaluation of a similar biologic. The 215 following factors should be considered for selection of the RBP. 216

• The RBP should be licensed / approved in India or ICH countries and should be the 217 innovator's product. The RBP should be licensed based on a full safety, efficacy and 218 quality data. Therefore, another similar biologic cannot be considered as a choice for 219 RBP. 220 • In case the RBP is not marketed in India, the RBP should have been licensed in any ICH 221 countries. The RBP can be imported for developing the similar biologic for quality, pre-222 clinical and clinical comparability. 223 • The same RBP should be used throughout the studies supporting the safety, efficacy 224 and quality of the product (i.e. in the development Programme for the similar biologic). 225 • The dose and route of administration of the similar biologic should be the same as that of 226 the RBP. However, the strength e.g. fills volume, pharmaceutical form, formulation, 227 excipients and presentation (for example, use of a different medical device or number of 228 syringes in a pack) of the similar biologic might differ from the RBP, if justified. 229 • Packaging configuration can be decided by the manufacturer if justified. 230

• The acceptance of an innovator product as a RBP for evaluation of similar biologic does not imply approval for its use in India. Note: ICH countries in this context include USA, UK, Japan, Australia, Canada and EU.

1. Quality 235 The comparison showing molecular similarity between the similar biologic and the RBP 236 provides the essential rationale for predicting that the clinical safety and efficacy profiles of the 237 RBP apply to the similar biologic. Therefore, a high degree of analytical and functional similarity 238 between the similar biologic and the RBP is the basis for developing a similar biologic. 239 Development of a similar biologic involves the thorough characterization of multiple RBP 240 batches in order to obtain an understanding of the overall quality profile as well as range of 241 variability of the RBP batches on the market. Based on the knowledge gained from the RBP 242 characterization studies, as well as available in-house and public information, the manufacturing 243 process of the similar biologic is developed to produce a product that is highly similar to the 244 RBP in all clinically relevant quality attributes (that is, attributes that may impact clinical 245 performance). 246 The manufacturer of the similar biologic should additionally carry out a comprehensive and 247 comparative state-of-the-art physicochemical and biological characterization of the similar 248 biologic and the RBP and document the results in the submitted marketing authorization 249 application. 250

9.1 Reference standards 251 252 Biological reference materials which serve as reference sources of defined biological activity 253 expressed in internationally agreed units. International units (IU) are assigned to such 254 255   manner. The Reference Standard is usually assigned an estimated potency value after a multi-256 centre collaborative study. These s        257 which regional, national and international laboratories and manufacturers calibrate their own 258 working standards. Typically, it is established by a public agency (e.g. WHO), Government (e.g. 259 Indian Pharmacopeia Commission, National Institute of Standards and Technology (NIST), 260 National Institute for Biological Standards and Control (NIBSC), or compendia (e.g., Indian 261 Pharmacopoeia, United States Pharmacopeia (USP), Ph. Eur.), and is officially recognized as 262 standard by individual regulatory authorities. There are also other types of external reference 263 standards such as the Chemical Reference Standards (CRS), which are higher in concentration 264 as compared to biological reference. 265 In the absence of established Reference Standards, the development of in-house reference 266  should be established. 267 Extensive characterization of in-house standards is performed through comprehensive 268 analytical testing to confirm identity, potency, purity, impurity profiles etc. While RBP may be 269 14

used for establishing bio similarity/comparability, relying on reference standards or in-house standards guarantees authenticity, consistency, and alignment with the  production process, which is vital for maintaining the production integrity and consistent quality of the product. 274

9.2 Manufacturing process 275 276 The manufacturing process of the similar biologic should be developed based on a 277 comprehensive understanding of the RBP gained through detailed characterization studies of a 278 sufficient number of RBP batches. 279 The similar biologics manufacturer should develop the manufacturing process to yield a 280 comparable quality product in terms of identity, purity and potency to the RBP. The 281 manufacturing process for similar biologics should be validated and demonstrated to be highly 282 consistent and robust. 283 The manufacturer must demonstrate the consistency and robustness of the manufacturing 284 process by implementing state-of-the-art quality control and assurance procedures, in-process 285 controls and process validation. The similar biologic manufacturing process should meet the 286 same standards required for originator products, including manufacture under current good 287 manufacturing practices. 288 As for any biological product, if process changes are introduced during the development of a 289 similar biologic, then the impact of the changes should be assessed through a comparability 290 exercise. Although many of the same principles are followed, the assessment of manufacturing 291 process changes should be addressed separately from the comparability exercise performed to 292 demonstrate similar biological activity with the RBP. It is, however, strongly recommended that 293 the pivotal data used to demonstrate similarity are generated using similar biologic batches 294 manufactured using the commercial manufacturing process and therefore representing the 295 quality profile of the batches to be commercialized. 296 Although the similar biologic does not need to be expressed in the same type of host cell as that 297 used for the RBP, it is recommended that a similar host cell type is used (for example, 298 Escherichia coli, Chinese hamster ovary cells, etc.). This will reduce the potential for critical 299 changes in the quality attributes of the protein, or in post-translational modifications, product-300 related impurities or the process-related impurity profile, that could potentially affect clinical 301 outcomes and immunogenicity. If a different host cell is used (for example to avoid unwanted 302 and potentially immunogenic glycan structures present in the RBP) then changes introduced in 303 terms of product-related substances, as well as product- and process-related impurities, need to 304 be carefully considered. 305 The manufacturing process used can significantly affect the structure of the drug substance and 306 thereby impact upon the potency of the product. For example, in the case of mAbs, when 307 deciding upon the expression system to employ, manufacturers should be guided by the 308 15

potential for both enzymatic and non-enzymatic modifications, such as incomplete disulfide bond formation, formation of aggregates, glycosylation, N-terminal pyroglutamine cyclization, C- terminal lysine processing, deamidation, isomerization and oxidation, modification of the N- terminal amino acids by maleuric acid, and amidation of the C-terminal amino acid. The data requirements for review of manufacturing process at developmental stage includes a 313 complete description of the manufacturing process from development and characterization of 314 cell banks, stability of clone, cell culture/fermentation, harvest, excipients, formulation, 315 purification, primary packaging interactions (if different from RBP), etc. and the consequences 316 on product characteristics as indicated below: 317 9.2.1 Molecular Biology Considerations 318 319 The details regarding host cell cultures (including viral clearance), vectors, gene sequences, 320 promoters etc. used in the production of similar biologics should be provided with appropriate 321 drawings/figures. The detail of post-translational modifications (glycosylation, oxidation, 322 deamidation, phosphorylation etc.), if any should be explained. 323 324 9.2.2 Upstream Process Development 325

• Upstream process should be described in detail including media components used for cell 326 growth. 327 • At least three batches of reproducible fermentation data at pilot scale (batch size 328 adequate to give enough purified product to generate preclinical/developmental data). 329 • Upstream process should be well controlled and monitored. 330 • Details of upstream process kinetics data from consistency batches indicating cell growth, 331 product formation, pH, temperature, dissolved oxygen, major nutrient consumption pattern 332 and agitation rate. 333 • Concentration to be defined in terms of product/ liter, yield and volumetric productivity. 334 • Data to verify that the specific protein yield (amount of protein per unit cell mass) remains 335 constant for all upstream batches. 336 • Demonstrate that the overall productivity is reproducible and scalable. 337 338 9.2.3 Downstream Process Development 339 340 • Detail description of the methods followed for the cell harvesting and extraction of the 341 protein. 342
• Steps involved in purification of protein. 343
• Batch size for protein purification. 344

• Description of each unit operation step during purification and recovery of protein along 345 with quantitative recovery of product at each stage. 346

• Consistency of recovery in three consecutive batches of purification from three independent batches of cell culture/fermentation. Describe post translational variation, if any.

• Details of removal of impurities like product related variants & impurities, and host cell & process related impurities considered to pose a risk of Immunogenicity (EMA 2017) 351

• Virus clearance validation studies should be part of Marketing Authorization application. 352
  For clinical trial application, additional requirements are applicable as per CDSCO guidelines. A 353 well-defined manufacturing process with its associated process controls assures that an 354 acceptable product is produced on consistent basis in accordance with Good Manufacturing 355 Practice (GMP). Data for submission should include: 356 357 • Detailed description of the drug substance and drug product processes 358

• Critical Quality Attributes (CQA) of the product 359

• Manufacturing process controls 360

• Critical process parameters 361

• Stability data 362

• Comparability of product manufactured at intended commercial scale against RBP 363

• Data from consistency batches and/ or process validation batches at commercial scale as 364 applicable. 365
  9.3 Analytical considerations 366 Thorough characterization of both the RBP and the similar biologic should be carried out using 367 state-of-the-art chemical, biochemical, biophysical and biological analytical techniques. The 368 goal of the comparability investigation is to be as comprehensive as possible in order to 369 minimize the possibility of undetected differences between the RBP and the similar biologic that 370 may affect safety and clinical activity. 371 Details should be provided on primary and higher-order structure, post translational 372 modifications (including, but not limited to, glycoforms), biological activity, purity, impurities, 373 product-related (active) substances (variants) and immunochemical properties, where relevant. 374 The methods should be scientifically sound and demonstrated to be of appropriate sensitivity 375 and specificity for their intended use. The analytical methods should be chosen for establishing 376 product comparability as per the critical quality attributes of the product. For certain attributes 377 (e.g. product aggregation) it is customary to use multiple, orthogonal methods for 378 characterization. Extensive state of the art analytical methods should be applied to detect even 379            like 380 USP / European Pharmacopoeia (EP)/ British Pharmacopoeia (BP) / Japanese 381 Pharmacopoeia (JP) / etc. monograph should be followed, if available. However, if advanced 382 17

analytical methods superior to Pharmacopoeia are used, those methods can be employed based on method validation with suitable justification. The analytical limitations of each technique (for example, limit of detection or resolving power) should be considered when determining the similarity of a similar biologic to its RBP. Representative raw data should be provided for analytical methods (for example, high-quality 387 reproductions of gels and chromatograms) in addition to tabular data summarizing the 388 complete dataset and showing the results of all release and characterization analyses carried 389 out on the similar biologic and the RBP. Graphical presentation of datasets comparing similar 390 biologic and RBP analytical data should also be produced where possible. The results should 391 be accompanied by sufficient interpretation and discussion of the findings. 392 The measurement of quality attributes in characterization should entail the use of appropriately 393 qualified assays, which are reproducible and reliable. The methods used to measure quality 394 attributes for batch release, stability studies and in- process controls should be validated in 395 accordance with ICH guidelines (ICHQ2, Q5C, Q6B), as appropriate. The characterization 396 studies should include samples of the applicant 's r-DNA derived product, RBP as control, 397 known positive standard and negative control, wherever relevant. A complete description of the 398 analytical techniques employed for release and characterization of the product, along with 399 method validation or qualification data (as appropriate), should be provided in the dossier. 400 Due to the unavailability of drug substance for the RBP, the similar biologic manufacturer will 401 usually be using a commercial drug product for the similarity exercise. The commercial drug 402 product will, by definition, be in the final dosage form containing the drug substance(s) 403 formulated with excipients. It should be verified that these excipients do not interfere with the 404 analytical methods used and thus have no impact on test results. If the drug substance in the 405 RBP needs to be purified from a formulated reference drug product in order to be suitable for 406 characterization then studies must be carried out to demonstrate that product heterogeneity 407 and relevant attributes of the active moiety are not affected by the isolation process. The 408 approach used for isolating the drug substance of the RBP and comparing it with the similar 409 biologic should be justified and demonstrated (with accompanying data) to be appropriate for 410 the intended purpose. 411 Physicochemical and Biological characterization methods (Quality Attributes) to be used for r-412 DNA derived products are given in Annexure II. It may be noted that this Annexure is 413 suggestive but not limited to the specified method and the requirements may vary on case by 414 case. 415

9.3.1 Product Characterization 416 Characterization studies for similar biologics include physicochemical properties, biological 417 activity, immunological properties, functional assays, purity (process and product-related 418 impurities etc.), strength and content. Principles outlined in the ICH Q6B guideline should be 419 followed. 420 18

1. Structural and Physicochemical Properties: The analysis of physicochemical characteristic should include determination of primary and higher order structure (secondary/tertiary/quaternary) and product variants of the drug substance and the product along with other significant physicochemical properties.
   The amino acid sequence of a similar biologic should be confirmed to be the same as that of 425 its RBP. It is, however, further recommended that manufacturers should pay special attention 426 to any sequence variants present in the similar biologic. Although an identical primary 427 sequence between the similar biologic and the RBP is expected, low-level sequence variants 428 may occur due to transcription and translation errors, especially through amino acid 429 misincorporation during high-level expression, and should be identified if present. The 430 presence of such variants could be acceptable if properly described and controlled to a 431 reasonable level. An assessment of the potential clinical impact of such variants would also 432 need to be considered. 433 An inherent degree of structural heterogeneity occurs in proteins as a result of biosynthesis 434 processes. These include C-terminal processing, N-terminal pyroglutamation, deamidation, 435 oxidation, isomerization, fragmentation, disulfide bond mismatch and free sulfhydryl groups, N-436 linked and O-linked oligosaccharide, glycation and aggregation. The structural heterogeneity 437 present in the similar biologic should be evaluated relative to the RBP. Experimentally 438 determined disulfide bonding patterns should be compared to the predicted structure based on 439 well-established structural data on the molecule. In cases, where post translational 440 modifications are taking place, these modifications need to be identified and quantified. In case 441 any significant differences are found, these should be scientifically justified and critically 442 examined in preclinical studies and clinical trials. 443

2. Biological Activity: Biological activity is the specific ability or capacity of the product to 444 achieve a defined biological effect. It serves multiple purposes in the assessment of product 445 quality and is required for characterization and for batch analysis. Ideally, the biological assay 446 used will reflect the understood mechanism of action of the drug substance of the RBP and will 447 thus serve as a link to clinical activity. A biological assay is a quality measure of the activity of 448 the drug substance and can be used to determine whether a product variant is active (that is, a 449 product-related substance) or inactive (and therefore defined as an impurity). Biological assays 450 can also be used to confirm that small differences observed in the higher-order structure of a 451 molecule have no influence on its biological activity. Thus, the use of relevant biological 452 assay(s) of appropriate precision, accuracy and sensitivity provides an important means of 453 confirming that there is no significant functional difference between the similar biologic and the 454 RBP. 455
   For a product with multiple biological activities, manufacturers should perform, as part of 456 product characterization, a set of relevant functional assays designed to evaluate the range of 457 activities of the product. For example, certain proteins possess multiple functional domains 458 that express enzymatic and receptor-binding activities. In such situations, manufacturers 459 should evaluate and compare all relevant functional activities of the similar biologic and the 460 RBP. 461 19

Potency is the measure of the biological activity. The potency assay should be used together with an in-house qualified reference material that is representative of the similar biologic material. The use of the international standards for determining potency depends on the prevailing practice for the product. Where appropriate, international or national standards and reference reagents should be used to determine product potency and to express results in 466 International Units (IU)  for other products, a suitable in-house reference material should be 467 used. In-house reference materials should be quantitatively calibrated against either an 468 international or national standard or reference reagent, where available and appropriate. 469 Depending on the purpose of the method (batch release assay or characterization), the 470 functional assays used may or may not be fully validated, but they must be scientifically sound 471 and produce consistent and reliable results. The available information on these assays 472 (including extent of validation, assessed parameters and available validation data) should be 473 confirmed before they are applied to the testing and establishing of biosimilarity between a 474 similar biologic and its RBP. It should be noted that many biological assays may have 475 relatively high variability that might preclude detection of small but significant differences 476 between the similar biologic and RBP. Therefore, it is recommended that assays are 477 developed that are more precise and can detect changes in the intended biological activities of 478 the product to be evaluated with adequate accuracy. Such assays can include target-binding 479 assays (which are usually less variable) in addition to cell-based assays. Adopting automated 480 laboratory equipment to help minimize manual operations, applying good analytical practices 481 and appropriate control sampling, and using critical reagents calibrated against WHO or 482 national reference standards where available (for example, tumour necrosis factor alpha (TNF-483   tency assays for anti-TNF products) may help to reduce the variability of biological 484 assays. For a given method variability, the number of RBP batches tested should be high 485 enough to allow for a reliable assessment of similarity. 486 Biological assays should be validated against an international or national reference standard, 487 where available and appropriate. If no such standards are available, an internal reference 488 standard must be established as per the ICH guidelines. If the methods of bioassay(s) are 489 documented in the specification, test(s) can be conducted accordingly 490

1. Immunological Properties: The manufacturing process of similar biologics is known to 491 affect the level of process related impurities and post translational modifications of the product. 492 These characteristics may affect the immunogenicity of the product. Hence evaluation by 493 characterization (antibody or antibody-derived product); comparison to reference biologic with 494 respect to specificity, affinity, binding strength and Fc function; and evaluation by animal 495 studies if required should be performed. When immunochemical properties are part of the 496 activity attributed to the product (for example, antibodies or antibody-based products) 497 analytical tests should be performed to characterize these properties and used in the 498 comparative studies. 499 For mAbs, the specificity, affinity and binding kinetics of the product to relevant fragment 500 crystallizable (Fc) receptors (for example, neonatal Fc receptor, complement component 1q 501     should be compared using suitable methods such as surface 502 20

plasmon resonance and biolayer interferometry. In addition, appropriate assays should be used to provide information on Fc mediated functions  for example, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement dependent cytotoxicity (CDC), where relevant.

The correlation between Fc-        507 physicochemical characteristics (for example, glycan pattern) should be considered and, 508 whenever possible, established. Such analyses will facilitate the interpretation of subtle 509 differences between the similar biologic and the RBP and inform prediction of their clinical 510 impact. 511

1. Purity and Impurities: Characterization of a similar biologic requires evaluation of the 512 following using orthogonal and state-of-the-art technologies: 513  Product related variants (e.g., glycoforms, isomers, aggregated, oxidized or deamidated 514 product) 515

 Process related impurities (residual media components, resin leachates etc., Host cell 516 related impurities (e.g., host cell protein, host cell DNA etc. 517 Product-related substances and impurities, such as those caused by protein degradation, 518 oxidation, deamidation, aggregation or potential post translational modification of the protein, 519 should be compared for the similar biologic and RBP. If comparison reveals differences in 520 product-related substances and impurities between the similar biologic and RBP, the impact of 521 the differences on the clinical performance of the drug product (including its biological activity) 522 should be evaluated. 523

Specifically, if the manufacturing process used to produce the proposed similar biologic 524 introduces different impurities or higher levels of impurities than those present in the RBP then 525 additional functional assays to evaluate the impact of the differences may be necessary. To 526 obtain sufficient information of the product-related substances and impurities it is 527 recommended that comparative stability studies under accelerated and/or stress conditions 528 are conducted Process-related impurities such as host cell proteins, host cell DNA, cell culture 529 residues and downstream processing residues may be quantitatively and/or qualitatively 530 different between the similar biologic and RBP due to the different manufacturing processes 531 used for their drug products. Nevertheless, process related impurities should be kept to a 532 minimum through the use of state-of-the-art manufacturing technologies. The risk related to 533 any newly identified impurities in the similar biologic should be evaluated. 534 Differences observed in the purity and impurity profiles of the similar biologic relative to the 535 RBP should be evaluated to assess their potential impact on safety and efficacy. Where the 536 similar biologic exhibits different impurities, those impurities should be identified and 537 characterized when possible. Depending on type and amount of the impurity, conduct of 538 preclinical and/or clinical studies can help to confirm that there is no adverse impact on safety 539 and efficacy of the similar biologic. 540

9.3.2 Quantity 541 21

In general, a similar biologic is expected to have the same concentration or strength e.g. fill volume of the drug substance as the RBP. However, concentration deviations not affecting the posology might be permissible, if justified. The quantity of the similar biologic drug substance should be expressed using the same measurement system as that used for the RBP (that is, mass units or units of activity). A description with appropriate justification should also be 546 included to describe how the quantity was calculated (including, for example, the selection of 547 the extinction coefficient). 548

9.4 Comparative analytical assessment 549

9.4.1 Considerations for the RBP and the similar biologic 550 The number of RBP batches needed for the comparative analytical assessment will be 551 influenced by the criticality of the quality attribute(s) under investigation and the approach 552 chosen for demonstrating similarity. The manufacturer of the similar biologic should include an 553 appropriate and scientifically supportable number of batches of the RBP in the comparability 554 assessment. In order to characterize independent RBP batches, it is recommended that the 555 RBP batches are sourced over an extended time period. 556 These batches should also include the RBP batches used in the clinical comparison studies of 557 the similar biologic. In general, adequate number of RBP batches will provide a better estimate 558 of the true batch-to-batch variability of the RBP and allow for a more robust statistical 559 comparison with the similar biologic. Random sampling of RBP batches is desirable but may be 560 difficult to achieve in practice depending on the availability of such batches. However, the 561 sourcing of RBP batches should be carefully managed to generate a sample that captures the 562 inherent variability of the RBP (for example, collected over a sufficient timeframe with the aim of 563 covering different manufacturing campaigns). 564 The RBP batches should be transported and stored under the recommended conditions and 565 tested within their approved shelf-life. Any exception to this would have to be fully substantiated 566 with experimental data. The shelf-life of the RBP at time of characterization should be 567 considered and it is expected that RBP batches of different ages will be included in the similarity 568 assessment. The similar biologic batches included in the comparability assessment should be 569 manufactured using the intended commercial manufacturing process and should preferably 570 originate from different drug substance batches. Generally, each value for an attribute being 571 assessed for a similar biologic should be contributed by an independent batch. 572 For example, a single drug product batch produced from a single drug substance batch would 573 be considered to be an independent batch while different drug product batches produced from 574 the same drug substance batch cannot be considered to be independent. In addition, small- or 575 pilot-scale batches can be included if comparability between the small- and commercial scale 576 batches has been properly demonstrated. 577 Usually all commercial-scale batches produced  including process performance qualification 578 batches and batches applied in the clinical trial(s)  should be included in the similarity 579 assessment. As with the RBP, the exact number of similar biologic batches required will be 580 22

influenced by several factors, such as the criticality of the quality attribute(s) under investigation and the approach applied for similarity evaluation. In general, the risk of a false-positive conclusion on similarity will decrease with increasing number of batches. A robust manufacturing control system and demonstrated batch-to-batch consistency of the similar biologic are prerequisites for a successful similarity assessment. 585

9.4.2 Considerations for similarity assessment 586 The quality comparison between Similar Biologic and Reference Biological Product is essential. 587 The applicant should submit a full quality dossier as per CDSCO guidance for industry, 2024 588 including the results of comparability exercise for the similar biologic with the RBP before the 589 applicant proposes to take the similar biologic to clinical development. All manufactured batches 590 (including developmental and clinical batches) used in the similarity assessment should be 591 presented at the time of MA application. 592 Three consecutive standardized batches which have been used to demonstrate consistency of 593 the manufacturing process should be used. 594 The quality comparison between the similar biologic and the reference biologic should be 595 governed by Quality Attributes (QA), which employ state-of-the-art high resolution analytical 596 techniques and methods that are sensitive enough to detect the possibilities of changes to the 597 product. 598 Quality attributes are those quality attributes which have direct impact on the clinical safety or 599 efficacy. QAs must be controlled within limits that need to be established based on the 600 Reference Biologic. 601 The most frequently used approach for similarity assessment relies on demonstrating that the 602 quality attributes of the similar biologic batches lie within the predetermined similarity ranges 603 established based on characterization data from multiple batches of the RBP. Other 604 approaches (such as equivalence testing of means) can also be used for similarity 605 assessment. 606 Each statistical approach has, however, specific strengths and weaknesses which should be 607 appropriately discussed in the submission and considered in the similarity conclusion. In order 608 to mitigate the risks inherent in employing statistical tests on limited samples (false-positive 609 and false-negative conclusions), a comprehensive control strategy must be established for the 610 similar biologic to ensure consistent manufacturing. 611

1. Statistical intervals for the establishment of similarity ranges 612 Where possible, quantitative similarity ranges should be established for the similar 613 biologic comparability exercise. As the allowable differences in quality attributes between the 614 similar biologic and the RBP are usually difficult to establish based on clinical considerations 615 alone, the batch-to-batch variability of the RBP is typically used to further inform acceptable 616 differences in quality attributes. The established similarity range should therefore tightly reflect 617 the quality profile of the marketed RBP batches. The ranges should normally not be wider than 618 23

the batch-to-batch variability present in the RBP unless it can be determined which differences would be acceptable (for example, less impurities is usually acceptable). Wide similarity ranges based on inappropriate use of statistical methods should not be used. Different statistical intervals can be used to establish similarity ranges. Commonly used approaches include mean ± x SD, the min-max range and tolerance intervals: 623 The most commonly applied approach for establishing similarity ranges is the x-sigma interval, 624 that is, mean ± x SD of the RBP batch data. The multiplier used (x) should be scientifically 625 justified and could be linked to the criticality of the quality attribute tested, with a smaller 626 multiplier applied for high criticality quality attributes. 627

• A conservative approach would be used to establish the similarity ranges directly based on 628 the min-max quality attribute data obtained from the characterization studies of RBP 629 batches. Such similarity ranges could be viewed as clinically qualified (since the RBP 630 batches are on the market and taken by patients). However, compared to other approaches 631 the min-max approach is often associated with high risk of a false-negative conclusion (that 632 is, a high risk of concluding non-similarity even though the underlying data distributions for 633 the RBP and similar biologic would support a similarity claim). 634
• Similarity ranges based on tolerance intervals would usually require a high number of RBP 635 batches for establishing meaningful ranges. With a limited number of RBP batches 636 characterized and/or inappropriate parameterization, the tolerance interval approach can 637 result in an estimated range that is much wider than the actual minmax quality attribute 638 ranges of the RBP. The risk of a false-positive conclusion of similarity (that is, the risk of 639 concluding similarity where the underlying data distributions do not support such a claim) 640 may therefore be unreasonably high when the similarity ranges are based on 641 inappropriately applied tolerance intervals. 642 The most frequently applied overall similarity criteria require that a certain percentage of the 643 similar biologic batches (usually between 90% and 100%) fall within the similarity range. This 644 figure should be determined prior to the initiation of the similarity assessment. 645

1. Analytical similarity evaluation 646 647 It is up to the manufacturer to justify the relevance of the established similarity ranges and 648 criteria. Ideally, the data analyses should be robust and should as far as possibly minimize 649 the risk of a false-positive conclusion. Although decreasing the risk of a false-positive 650 conclusion is of primary importance from a patient and regulatory point of view, the risk of a 651 false-negative conclusion also needs to be managed by the manufacturer and should be 652 thoroughly considered during the planning of the similarity exercise. 653 654 Differences between the Similar Biologic and the RBP should be evaluated for their potential 655 impact on safety and efficacy of the Similar Biologic and additional characterization studies 656 may be necessary. 657 24

Some minor differences between the RBP and the similar biologic are expected. Nevertheless, any quality attributes not fulfilling the established similarity criteria should be considered as a potential signal for non-similarity and should be assessed for possible impact on clinical safety and efficacy. 662 Confirmed differences in low criticality quality attributes also need to be adequately 663 considered, but in the case of such differences reference to available information (which 664 could, for example, originate from scientific publications) is usually sufficient. 665 666 Lower impurity levels in the similar biologic (for example, of aggregates) or differences in 667 quality attributes present at very low levels in both the RBP and the similar biologic would in 668 most cases be predicted to have no clinical relevance, and could therefore be accepted 669 without further assessment. 670 671 For differences in quality attributes with higher criticality, functional assays to thoroughly 672 address their possible clinical impact are generally expected. Where there are confirmed 673 differences in the most critical quality attributes it will be more challenging to justify the 674 conclusion that the product is a true similar biologic. For example, if differences are found in 675 quality attributes that alter the PK of the product and thereby change the dosing scheme then 676 the product cannot be considered to be a similar biologic. 677

9.5 Specifications 678 Specifications of Similar Biologics (for drug substance and drug product) are established around 679 quality attributes (QAs) with the intent of ensuring consistency in product quality and 680 comparability to Reference Biologic according to relevant guideline (ICH Q6B). Methods used 681 for setting specifications may or may not be the same as the analytical methods used for 682 product characterization and for establishing product comparability. Acceptance limits should be 683 set based on Reference Biological product data and candidate similar biologic data including 684 data from developmental or clinical batches, which must be in line with international norms. 685 Furthermore, a similar biologic should show the same level of compliance with a pharmacopeial 686 monograph as that required for the RBP  however, compliance with a pharmacopeial 687 monograph is not sufficient to establish biosimilarity. 688 Reference to the analytical methods used and acceptance limits for each test parameter of the 689 similar biologic should be provided and justified. All analytical methods referenced in the 690 specification should be validated and the corresponding validation documented. Specifications 691 for a similar biologic may not be the same as for the RBP since the manufacturing processes 692 will be different, and different analytical procedures and laboratories will be used for the assays. 693 Nonetheless, the specifications should capture and control important known product quality 694 attributes. 695

             the 696 similar biologic (for example, with regard to its manufacturing history, assay capability and the 697 25

quality profile of batches used for establishing similarity); (b) the experimental results obtained by testing and comparing the similar biologic and RBP; and (c) attributes with potential impact on product performance. The manufacturer should take into consideration that the limits set for a given specification should not, unless properly justified, be significantly wider than the range of variability of the RBP over the shelf-life of the product. 702 For release specifications, Indian Pharmacopoeia Monograph should be followed, if available as 703 per the provisions of Drugs and Cosmetics Act and Rules made thereunder. 704

9.6 Stability 705 The shelf-life and storage condition of drug substance and drug product should be assigned 706 based on real-time stability studies. Stability studies on drug substance and drug product should 707 be carried out using containers and conditions that are representative of the actual storage 708 containers and conditions, according to relevant guidelines (e.g. ICH Q1 A(R2), ICH Q5C, WHO 709 TRS 822 and WHO TRS 953). Side-by side accelerated and stressed stability studies 710 comparing the Similar Biologic to the Reference Biologic will be of value in determining the 711 Similarity of the products by showing comparable degradation profiles. Stability studies should 712 be carried out to show which release and characterization methods are stability-indicating for 713 the product. 714 Stability studies should be summarized in an appropriate format (such as tables) and should 715 include results from accelerated degradation studies and studies under various stress 716 conditions (for example, high temperature, oxidation, freeze-thaw, light exposure, humidity and 717 mechanical agitation). 718

1. Data Requirements for Preclinical Studies 719 This section addresses the pharmaco-toxicological assessment of the similar biologic. It is 720 important to note that in order to design an appropriate nonclinical study programme a clear 721 understanding of the characteristics of the RBP is required. The nature and complexity of the 722 RBP will have an impact on the extent of the nonclinical studies needed to confirm similarity. In 723 addition, any differences observed between the similar biologic and RBP in the physicochemical 724 and biological analyses will also guide the planning of the nonclinical studies. Other factors that 725 need to be taken into consideration include the mechanism(s) of action of the drug substance 726 (for example, the receptor(s) involved) in all authorized indications of the RBP, and the 727 pathogenic mechanisms involved in the disorders included in the therapeutic indications. 728 A stepwise approach should be applied during nonclinical development to evaluate the similarity 729 of the similar biologic and its selected RBP. At first, in vitro studies should be conducted and 730 then a decision made on whether or not additional in vivo animal studies are required. 731 The following approach to nonclinical evaluation may be considered and should be tailored on a 732 case-by-case basis to the similar biologic concerned. In all cases, the approach chosen should 733 be scientifically justified in the application dossier. 734 26

10.1 In vitro studies In order to assess any relevant difference in pharmaco-toxicological activity between the similar biologic and chosen RBP, data from a number of comparative in vitro studies  some of which may already be available from the quality-related assays  should be provided. In light of this data overlap, it is suggested that the in vitro nonclinical studies related to 739 characterization of the biological activity of the similar biologic be addressed alongside the 740 related quality data in the corresponding quality module. Any other nonclinical in vitro studies 741 should then be addressed in the relevant nonclinical modules of the dossier where they 742 should be reviewed and discussed from the point of view of potential impact on the efficacy 743 and safety of the similar biologic. 744 Since experience has shown that in vitro assays are in general more specific and sensitive 745 than in vivo studies in animals for detecting differences between the similar biologic and 746 RBP, the use of in vitro assays is of paramount importance in the nonclinical similar biologic 747 comparability exercise. 748 For such in vitro studies, the following general principles apply: 749

• Typically, a battery of interaction studies addressing the primary binding events should be 750 performed, along with cell-based or isolated-tissue-based functional assays (see below) in 751 order to assess if any (clinically) relevant differences in reactivity exist between the similar 752 biologic and RBP and, if so, to determine the likely causative factor(s). 753 • Together, these assays should cover the whole spectrum of pharmaco-toxicological 754 aspects with potential clinical relevance for the RBP and for the product class. In the 755 dossier, the manufacturer should discuss to what degree the in vitro assays used can be 756 considered representative/predictive of the clinical situation according to current scientific 757 knowledge. 758 • The studies should be comparative and designed to be sufficiently sensitive, specific and 759 discriminatory to allow for the detection of (clinically) relevant differences in pharmaco-760 toxicological activity between the similar biologic and RBP  or, conversely, to provide 761 evidence that any observed differences in quality attributes are not clinically relevant. 762 • The studies should compare the concentrationactivity/binding relationship of the similar 763 biologic and the RBP at the pharmacological target(s), covering a concentration range 764 within which potential differences are most accurately detectable (that is, the ascending 765 part of the concentrationactivity/binding curve). 766 • A sufficient number of RBP batches and similar biologic batches (preferably 767 representative of the material intended for commercial use) should be evaluated. Assay 768 and batch-to-batch variability will affect the number of batches needed. The number 769 tested should be sufficient to draw meaningful conclusions on the variability of a given 770 parameter for both the similar biologic and the RBP and on the similarity of both products. 771 • Where available, international reference standards can be used to support assay 772 characterization, calibration and performance. When no such reference standard exists, 773 an inhouse reference material should be established. 774 775 The nonclinical in vitro programme for similar biologics should usually include relevant assays for the following: Binding studies- Evaluation of the primary binding events  that is, binding of the similar biologic to cell membrane receptors or to other membrane-bound or soluble targets that are known/assumed to be involved in the pharmaco-toxicological effects of the RBP in the 780 clinically approved indications  for example, for immunoglobulin G (IgG)-based mAbs, 781 antigen-binding fragment (Fab)-associated binding to the antigen and Fc-associated binding 782 to representative isoforms of the relevant Fc receptors and to C1q . 783 Functional studies/determination of biological activities- Studies should evaluate signal 784 transduction and/or functional activity/viability of cells or isolated tissues known to be of 785 relevance for the pharmaco-toxicological effects of the RBP. Together these assays should 786 broadly cover all the known mechanisms of action of the RP in the clinically authorized 787 indications  for example, for IgG-based mAbs directed against membrane-bound antigens, 788 evaluation of Fab-associated functions and of Fc-associated functions such as ADCC, ADCP 789 and CDC 790 Such assays are often technically demanding and the experimental approach chosen should 791 be appropriately justified by the manufacturer. 792

10.2 Determination of the need for in vivo animal studies 793 794 On the basis of the totality of quality and nonclinical in vitro data available and the extent to 795 which there is residual uncertainty about the similarity of a similar biologic and its RBP, it is at 796 the discretion of Licensing Authority to waive or not to waive a requirement for additional 797 nonclinical in vivo animal studies. The decision of Licensing Authority on whether or not to 798 require such studies should take into account the following: 799

• If the quality comparability exercise and the nonclinical in vitro studies have shown high 800 similarity and the level of residual uncertainty is considered acceptable to move to the 801 clinical phase of the similarity exercise then an additional in vivo animal study is not 802 considered necessary. 803
• If a need is identified to reduce remaining uncertainties concerning the similarity (including 804 drug safety) of a similar biologic and its RBP before the initiation of clinical evaluations 805 then additional in vivo animal studies may be considered, if a relevant animal model is 806 available  however this should only occur: (a) when it is expected that such studies 807 would provide relevant additional information; and (b) if the needed additional information 808 cannot be obtained using an alternative approach that does not involve in vivo animal 809 studies. In this respect, the factors to be considered could include:  qualitative and/or 810 quantitative differences in potentially or known relevant quality attributes between the 811 similar biologic and its RBP (for example, qualitative and/or quantitative differences in the 812 post-translational glycosylation of proteins); and  relevant differences in formulation (for 813 example, use of excipients in the similar biologic not widely used in medicinal products). 814

• On the basis of regulatory experience gained to date in marketing authorization 815 applications for similar biologics, the need for additional in vivo animal studies would be 816 expected to represent a rare scenario. 817

• If the quality and nonclinical in vitro comparability exercises indicate relevant differences between the similar biologic and the RBP (thus making it unlikely that similarity would eventually be established), then standalone development to support a full marketing authorization application should be considered.
  822 Animal toxicity studies waiver for a similar biologic product may be considered if the following 823 conditions/criteria are met: 824

1. Candidate similar biologic is expressed in an established expression system. 825
2. The amino acid sequence of the similar biologics is identical to that of the RBP. 826
3. The strength, route of administration, human dose, and indications proposed for 827 similar biologics are the same as the RBP. 828
4. Applicant should use appropriate analytical methodologies with adequate sensitivity 829 and specificity to detect and characterize differences between the proposed similar 830 biologic and the RBP. 831
5. For all the product-related variants, identification and determination of the relative 832 levels of these variants should be included in the comparative analytical 833 characterization studies. 834
6. For all the product-related impurities, applicants should characterize, identify and 835 quantify product-related impurities (as defined in ICHQ6B) in the proposed similar 836 biologic and the RBP, to the extent feasible. Further, if the manufacturing process 837 used to produce the proposed similar biologic introduces different impurities or higher 838 levels of impurities than those present in the RBP, additional pharmacological/ 839 toxicological studies may be necessary. 840
7.              Quality Attributes 841 (QA). Further, based on the potential impact on the mechanism of action and function 842 of the product, the applicant to identify the other QAs. 843
8. Acceptance limits should be set based on Reference Biological product data and 844 accordingly sufficient number of batches of RBP to be used (Minimum of n=3). 845 Further, for the quantitative data analysis, statistical methods such as Min-Max 846 approach is the most recommended for establishing the similarity acceptance criteria 847 because a very large number of RBP batches would not be required to establish 848 meaningful intervals. For the similar biologic data, falling beyond the Min-Max range, if 849 not supported by other orthogonal techniques, then additional pharmacological/ 850 toxicological studies may be necessary. Further, the applicants may propose other 851 methods of data analysis, including equivalence testing. The data generated using 852 qualitative methods, which is not amenable to statistical evaluation, may be analyzed 853 by visual comparison of the data for similarity. 854

9. To the extent possible, RBP batches to be selected with a range of expiration dates 855 spread across the product`s shelf-life to provide a representation of the data from 856 different time points for obtaining marketing authorization. 857

10. Applicant to conduct analytical similarity with state-of-the-art techniques as per tests mentioned in Annexure II. For example, secondary structure analysis can be performed either by FAR UV CD or FTIR, as applicable. Applicant to submit the summary sheet of the generated CMC data.
    In case, the proposed dosage form and formulation of a similar biologic is different from the 862 Reference biologics, the applicant needs to provide the rationale for this difference. 863

Toxicity waiver for a similar biologic product may not be granted in any of the 864 following scenarios: 865

1. If there are differences that cannot be ruled out as having no safety impact. 866
2. When a novel excipient is being used for the first time for biological products specific 867 to the claimed route of administration. 868
3. If the applicant plans to do a clinical study using a route of administration that is not 869 tested/approved by regulatory authorities for the Reference biologics. 870
4. If the planned human dose of the drug is higher than approved for the Reference 871 biologics. 872 If the toxicity study is requested by the Licensing Authority, the applicant shall refer to 873 relevant application requirement which is detailed in Annexure IV. 874

10.3 In vivo studies 875 10.3.1 General aspects to be considered 876 The 3Rs principles for animal experiments (Replace, Reduce, Refine) should always be 877 followed to minimize the use of animals in testing in accordance to New Drugs and Clinical 878 Trial Rules 2019. 879 To address the residual uncertainties, the use of relevant/suitable animal species and/or of 880 specific animal models (for example, transgenic animals or transplant models) may be 881 considered. 882 Animal models are often not sensitive enough to detect small differences. If a relevant and 883 sufficiently sensitive in vivo animal model cannot be identified, the manufacturer may choose to 884 proceed directly to clinical studies, taking into account strict principles to mitigate any potential 885 risk. 886 The effects of RBPs are often species specific. In accordance with ICH S6(R1) and the WHO 887 Guidelines on the quality, safety and efficacy of biotherapeutic protein products prepared by 888 recombinant DNA technology, in vivo studies should be performed only in relevant species  889 that is, species which are known to be pharmacologically and/or toxicologically responsive to 890 the RBP. 891

The duration of the study/studies should be justified, taking into consideration the PK behaviour of the RBP, the time to onset of formation of anti-drug antibodies (ADAs) in the test species and the clinical use of the RBP. 10.3.2 Specific aspects 896

PK and/or PD studies 897 In cases where such studies are considered necessary, the PK and/or PD of the similar 898 biologic and the RBP should be compared quantitatively, when the model allows, using a 899 doseresponse assessment that includes the intended exposure in humans. 900 The studies may include animal models of disease to evaluate functional effects on disease-901 related PD markers or efficacy measures. 902

Safety studies 903 Where in vivo safety studies are deemed necessary, a flexible approach that follows the 3R 904 principles to maximize the readout of relevant data and minimize the use of animals in testing 905 should always be followed. If appropriately justified, a repeated dose toxicity study with refined 906 design  for example, using just one dose level of similar biologic and RBP, and/or just one 907 gender and/or no recovery animals, and/or only in-life safety evaluations such as clinical signs, 908 body weight and vital functions  may be considered. Depending on the chosen end-points, it 909 may not be necessary to sacrifice the animals at the end of the study. 910 Repeated dose toxicity studies in non-human primates are not recommended and nor are 911 toxicity studies in non-relevant species (for example, to assess unspecific toxicity due to 912 impurities). 913

Immunogenicity studies 914 Qualitative or quantitative difference(s) in product-related variants (for example, in 915 glycosylation patterns, charge, aggregates, and impurities such as host-cell proteins) may 916 have an effect on immunogenic potential and on the potential to cause hypersensitivity. 917 Antibody response to the Similar Biologic should be compared to that generated by the 918 reference Biologic in suitable animal model. The test serum samples should be tested for 919 reaction to host cell proteins. For evaluating immune toxicity of the Similar Biologic under 920 study, the results of local tolerance (part of repeat dose or standalone test) should be analyzed 921 with the observations regarding immunogenicity in sub-chronic study. Therefore, the 922 immunogenicity testing should be included as part of the sub-chronic repeated-dose study 923 while developing the protocols. 924 The other parameters for evaluating immune toxicity include immune complexes in targeted 925 tissues may be considered while evaluating histopathology observations, etc. 926

Local tolerance studies 927 31

Studies on local tolerance are usually not required. However, if excipients are introduced for which there is little or no experience with the intended clinical route of application, local tolerance may need to be evaluated. If other in vivo animal studies are to be conducted, the evaluation of local tolerance may be integrated into the design of those studies.

Other studies 932 In general, safety pharmacology and reproductive and development toxicity studies  as well 933 as genotoxicity and carcinogenicity studies are not warranted during the nonclinical testing of 934 similar biologics. 935 936

1. Data Requirements for Clinical Trial Application 937 The applicant has to submit application for conduct of clinical trial as per the CDSCO guidance 938 for Industry, 2024. The quality data submitted should indicate that there are no differences in 939 Quality Attributes (QAs), and all quality attributes are well controlled in order to allow the 940 initiation of clinical evaluation. 941 Clinical studies play an important role in validating similarity by confirming that there are no 942 clinically significant differences between the proposed similar biologic and the RBP. These 943 studies should be designed to demonstrate confirmatory evidence of similar clinical 944 performance of the similar biologic and RBP and therefore need to use sensitive testing 945 strategies that are sufficiently sensitive to detect any clinically relevant differences between the 946 similar biologic and the RBP. 947 Clinical data should be generated using the similar biologic produced from the final 948 manufacturing process, representing the product intended for marketing authorization. Any 949 deviation from this recommendation needs to be justified and additional data may be required. 950 For manufacturing process changes, the appropriate guidelines should be followed. Ideally, 951 reference biologic product (RBP) from a single marketing authorization holder should be used 952 as the comparator throughout quality and clinical comparability studies, to ensure consistency in 953 data and conclusions. 954 If relevant differences between the similar biologic and the reference biological product (RBP) 955 are identified at any stage of development, these differences must be thoroughly investigated 956 and justified. If a justification cannot be provided, the product may not meet the criteria for a 957 similar biologic, and a standalone licensing application should be considered. 958 For clinical evaluation, a comparative bioequivalence study assessing pharmacokinetic (PK) 959 and/or pharmacodynamic (PD) similarity is generally required. An adequately powered 960 comparative efficacy and safety trial will not be necessary if sufficient evidence of similarity can 961 be drawn from other parts of the comparability exercise. The need for a comparative clinical 962 efficacy and safety trial for the proposed similar biologic (and type of trial if required) will be 963 influenced by factors such as: 964 32

• the ability to thoroughly characterize the similar biologic;
• the availability of suitable sensitive, orthogonal assays for robust analytical and functional characterization; the extent of analytical and functional similarity with the reference biological product (RBP);
• the existence of a relevant pharmacodynamic (PD) marker; 969
• the degree of         970 different indications, and the extent to which these can be explored in binding and functional 971 in vitro assays, the contribution of each mechanism of action to the observed clinical effect is 972 not relevant as long as it can be measured. 973
• understanding of any potential unwanted immunogenicity concerns, such as ADA incidence, 974 ADA response magnitude, levels of neutralizing antibodies, and antibodies against 975 endogenous substances (e.g., erythropoietin, coagulation factors); and clinical concerns 976 related to the similar biologic 977 Current examples of biologics that can be well-characterized and have established mechanisms 978 of action include, but are not limited to, teriparatide, insulin, G-CSF, and somatropin. Current 979 data also suggest that more complex products, such as monoclonal antibodies, can be 980 effectively characterized with advanced analytical methods, as structurefunction relationships 981 are well-defined and measurable through sensitive, orthogonal functional assays. 982

11.1 Pharmacokinetic (PK) Studies 983 984 The clinical comparability assessment should typically include a comparative pharmacokinetic 985 (PK) study if the drug can be measured in blood, along with pharmacodynamic (PD) marker 986 measurements (if available) and immunogenicity data. 987 The PK study should be designed to confirm similar PK profiles between the similar biologic and 988 reference biological product (RBP). When the RBP and its proposed similar biologic have more 989 than one route of administration (most commonly intravenous and subcutaneous) then carrying 990 out the study/studies using the non-intravenous route of administration is preferred as this is 991 usually the more immunogenic route and will provide more meaningful information for the 992 comparability exercise. 993 The omission of a PK study of other approved routes of administration needs to be justified for 994 approval of all available options  for example, in cases when the molecule has an absorption 995 constant that is much lower than the elimination constant (flip flop kinetics). 996 The study should have an adequate sample size, considering PK variability in the population 997 studied, statistical rationale (i.e. statistically justified) and comparability limits should be defined 998 and justified prior to conducting the study and consideration should be given to whether a cross-999 over or parallel group design would be the most adequate. If existing population PK or PK-PD 1000 models for the RBP are available in the literature, modeling and simulation may be used to 1001 refine the study design, such as by determining the appropriate dose and selecting the most 1002 sensitive population to detect PK differences, as well as optimizing sample size. When ethically 1003 33

acceptable, PK studies should be performed in healthy volunteers with a standardized population regarding factors that may influence PK variability (e.g., ethnicity, body weight, and gender). If safety or tolerability concerns make PK studies in healthy volunteers unsuitable, PK study should be a part of Efficacy and safety study in patients The preferred design is a randomized, two-period, two-sequence, single dose cross-over PK 1008 study using a dose within the therapeutic range at which the ability to detect differences is 1009 sufficient to observe meaningful differences. A cross-over design eliminates inter-subject 1010 variability, thus reduces the sample size required to demonstrate PK equivalence between the 1011 similar biologic and RBP. The treatment periods should be separated by a wash out phase that 1012 is sufficiently long to ensure that drug concentrations are below the lower limit of bioanalytical 1013 quantification in all subjects at the beginning of the second period  that is, at least 5 times the 1014 terminal half-life. 1015 If a cross-over design is unsuitable (e.g., for biologics with long half-lives or those associated 1016 with immunogenicity impacting PK), a parallel group design should be used. In parallel group 1017 studies, attention should be given to maintaining balance between groups to prevent factors 1018 such as ethnicity, body weight, and gender from affecting PK results. 1019 A multiple-dose study in patients is acceptable as a pivotal PK study if a single-dose study 1020 cannot be conducted in healthy volunteers due to risks or tolerability reasons or if a single-dose 1021 study is not feasible in patients. 1022 Multiple-dose studies may also be allowed in rare cases where limitations in the sensitivity of 1023 analytical methods prevent precise measurement of plasma or serum concentrations after a 1024 single dose. However, since a multiple-dose study is less sensitive to differences in Cmax 1025 compared to a single-dose study, this approach should be justified with valid reasoning. 1026 PK comparisons between the similar biologic and the reference biological product (RBP) 1027 should consider not only the rate and extent of absorption but also include a descriptive 1028 analysis of elimination characteristics, such as clearance and/or elimination half-life, as these 1029 may differ between the two products. Both linear (nonspecific) and nonlinear (target-mediated) 1030 clearance should be evaluated through partial  1031 Acceptance criteria for the demonstration of PK similarity between the similar biologic and the 1032 RBP must be predefined and appropriately justified. It should be noted that the criteria used in 1033 standard clinical PK comparability studies (bioequivalence studies) may not necessarily be 1034 applicable to all biotherapeutic products. However, the traditional 80125% equivalence range 1035 will in most cases be sufficiently conservative to establish similar PK profiles Correction for 1036 protein content may be acceptable on a case-by-case basis if pre-specified and adequately 1037 justified, with the assay results for the similar biologic and RBP being included in the protocol. 1038 If adjustments for covariates are intended for parallel group studies (for example, in the case of 1039 adalimumab, stratification for body weight and gender), they should be predefined in the 1040 statistical analysis plan rather than being included in post hoc analyses. 1041 34

Additional PK studies, such as interaction studies with commonly co-administered drugs or studies in special populations (e.g., children, elderly, or patients with renal or hepatic impairment), are not required for a similar biologic.              protein over time in a complex biological matrix with other proteins. The method should be 1046 optimized to offer satisfactory specificity, sensitivity, and quantification accuracy, and the same 1047 assay should measure serum concentrations of both the similar biologic and RBP. A single PK 1048 assay (using the same binding reagents and a single analytical standard, typically a similar 1049 biologic) may be used to assess similar biologic and RBP concentrations, provided that 1050 bioanalytical comparability is verified with supporting data. 1051 In cases where measurable endogenous protein affects the concentration-time profile of the 1052 administered exogenous protein, manufacturers should describe and justify their method to 1053 account for this (e.g., using baseline correction). 1054 Establishing PK similarity may be challenging or impractical for certain substances (e.g., 1055 heparin fractions that cannot be measured in blood), specific administration routes (e.g., 1056 intraocular injections of aflibercept or ranibizumab), or products with high PK variability (e.g., 1057 romiplostim). In such cases, clinical similarity should be demonstrated through 1058 pharmacodynamics (PD), immunogenicity, or other clinical parameters. 1059

11.2 Pharmacodynamic Studies 1060 1061 It is preferable to investigate PD parameters alongside comparative PK studies. However, when 1062 conducting PK studies is not feasible, PD markers may become more critical. For instance, with 1063 heparins, where serum concentrations are unmeasurable, similarity should be established 1064 based on key PD endpoints, specifically anti-FXa and anti-FIIa activity. 1065 1066 PD effects should be evaluated in an appropriate population, using doses within the steep 1067 portion of the dose-response curve to improve the likelihood of identifying any differences 1068 between the similar biologic and the reference biologic. PD markers should be selected on the 1069 basis of their clinical relevance. 1070

11.3 Confirmatory PK and/or PD studies 1071 If an adequately powered comparative efficacy trial is not necessary, comparative PK and/or PD 1072 studies may be sufficient for establishing confirmative evidence of the similar clinical 1073 performance of a similar biologic and its RBP, provided that: 1074

• the acceptance ranges for confirmatory PK and/or PD end-points are predefined and 1075 appropriately justified; 1076 • the PD biomarker reflects the mechanism of action of the biological product; 1077 • the PD biomarker is sensitive to potential differences between the proposed similar biologic 1078 and the RBP; and 1079 • the PD biomarker assay is validated. 1080 The applicant should consider the option of using additional PD measures (usually as secondary end-points) to assess the comparability of the PD properties of the RBP and proposed similar biologic. Furthermore, even if relevant PD measures are not available, sensitive PD end-points may be assessed if such assessment may help to reduce residual uncertainty about similar biosimilarity. 1085 An example of acceptable confirmatory PK/PD studies would be the use of euglycaemic clamp 1086 studies to compare the efficacy of two insulins. In addition, absolute neutrophil count and 1087 CD34+ cell count are the relevant PD markers for assessing the activity of G-CSF and could be 1088 used in PK/PD studies in healthy volunteers to demonstrate the similar efficacy of two medicinal 1089 products containing G-CSF. 1090 The study population and dosage should represent a test system that is known to be sensitive 1091 in detecting potential differences between a similar biologic and the RBP. In the case of insulin, 1092 for example, the study population should consist of non-obese healthy volunteers or patients 1093 with type 1 diabetes rather than insulin-resistant obese patients with type 2 diabetes. Otherwise, 1094 it may be necessary to investigate more than one dose to demonstrate that the test system is 1095 discriminatory. 1096 The acceptance ranges for confirmatory PK and/or PD parameters (that is, for primary end-1097 points) should be predefined and appropriately justified. If PD comparison is not essential for a 1098 conclusion of similar biosimilarity but the results are still expected to reasonably support similar 1099 biosimilarity then a purely descriptive analysis of the PD results may be justified. This may be 1100 the case for biological substances that have been extensively characterized and for which 1101 similar biosimilarity can already be concluded from the analytical, functional and PK 1102 comparisons. If appropriately designed and performed, such PK/PD studies are usually more 1103 sensitive in detecting potential differences in efficacy than trials using hard clinical end-points. 1104 However, PD markers may also be used as end-points in clinical efficacy studies in patients. 1105 Examples of appropriate markers include haemoglobin for measuring the efficacy of an epoetin, 1106 and lactate dehydrogenase (which is a sensitive biochemical marker of intravascular 1107 haemolysis) for evaluating the efficacy of a complex drug such as eculizumab. For denosumab, 1108 investigation of bone formation and resorption markers as part of the PK study may be useful or 1109 possibly sufficient. This would involve measurement of bone mineral density and bone turnover 1110 markers such as serum C-terminal telopeptide of type 1 collagen (CTX-1) and procollagen type 1111 1 N-terminal propeptide (P1NP) after denosumab administration. 1112 In certain cases (for example, when analytical similarity of the active ingredient in the similar 1113 biologic and the RBP can be demonstrated to such a degree that clinical differences can be 1114 excluded) a comparative PK study may provide sufficient clinical evidence to support similar 1115 biosimilarity. However, a risk assessment (including for example, the impurity profile) should be 1116 conducted to determine the need for additional safety/immunogenicity data on the similar 1117 biologic. 1118 36

11.4 Efficacy studies A comparative efficacy trial may not be necessary if sufficient evidence of biosimilarity can be inferred from other parts of the comparability exercise. A comparative clinical trial, if necessary, should confirm that the clinical performance of the similar biologic and the RBP is comparable. Demonstration of comparable potency, PK and/or PD profiles provide the basis for use of the 1123 RBP posology in the comparative clinical trial. If a comparative clinical trial of the similar biologic 1124 and RBP is deemed necessary then it is expected that it will be an adequately powered, 1125 randomized and controlled clinical trial performed in a patient population that allows for 1126 sensitive measurement of the intended clinical parameters. 1127 In principle, equivalence trial designs (requiring lower and upper comparability margins) are 1128 preferred for comparing the efficacy and safety of the similar biologic and RBP. Non-inferiority 1129 designs (requiring only one margin) or trials with asymmetrical margins may be considered if 1130 appropriately justified. Regardless of which design is selected in a particular case, the 1131 comparability margin(s) must be pre-specified and justified on the basis of clinical relevance  1132 that is, the selected margin should represent the largest difference in efficacy that would not 1133 matter in clinical practice. Treatment differences within this margin would therefore be 1134 acceptable as they would have no clinical relevance. 1135 Similar efficacy implies that similar treatment effects can be achieved when using the same 1136 posology, and the same dosage(s) and treatment schedule should be used in clinical trials 1137 comparing the similar biologic and RBP. In this regard, equivalence trials are again preferable 1138 to ensure that the similar biologic is not clinically less or more effective than the RBP when used 1139 at the same dosage(s). 1140 A non-inferiority design could be acceptable, if justified by the applicant, for example: 1141

 for biological products with high efficacy (for example, a response rate of over 90%), making it 1142 difficult to set an upper margin; or 1143

 in the presence of a wide safety margin. 1144 When using asymmetrical margins, the narrower limit should rule out inferior efficacy and the 1145 broader limit should rule out superior efficacy. The use of asymmetrical margins should be fully 1146 justified by the sponsor of the proposed similar biologic. Factors that would allow for the use of 1147 such margins in a clinical trial include: 1148

 if the dose used in the clinical study is near the plateau of the dose response curve; and 1149

 there is little likelihood of dose-related adverse effects (for example, toxicity). 1150 Careful consideration should be given to the design of the comparative study/studies, including 1151 the choice of primary efficacy end-point(s). Studies should be conducted using a clinically 1152 relevant and sensitive end-point within a homogenous population that responds well to the 1153 pharmacological effects of the biological product of interest to show that there are no clinically 1154 meaningful differences between the similar biologic and RBP. Clinical outcomes, surrogate 1155 37

outcomes (PD markers) or a combination of both can be used as primary end-points in similar biologic trials. The same study end-points used to establish the efficacy of the RBP may be used because a large body of historical data would generally be available in the public domain for setting the comparability margin(s) and calculating the sample size. However, the primary end-point could be different from the original study end-point for the RBP if it is well justified and 1160 relevant data are available to support its use as a sensitive end-point and its suitability for the 1161 determination of the comparability margin(s). A relevant PD end-point can be used as the 1162 primary end-point  for example, when it is a known surrogate of efficacy or when it can be 1163 linked to the mechanism of action of the product. The primary or secondary end-points can also 1164 be analyzed at different time points compared to those used in clinical trials with the RBP if 1165 these are considered to be more sensitive in capturing the pharmacological action(s) of the 1166 biological product  for example, adalimumab efficacy could be measured by responses at 1167 week 12 or 16 in addition to week 24. 1168 The sample size and duration of the comparative clinical study should both be adequate to allow 1169 for the detection of clinically meaningful differences between the similar biologic and RBP. 1170 When a comparative clinical trial is determined to be necessary then adequate scientific 1171 justification for the choice of study design, study population, study end-point(s), estimated effect 1172 size for the RP and comparability margin(s) should be provided and may be discussed with 1173 regulators in order to obtain agreement at least in principle prior to trial initiation. 1174

11.5 Safety 1175 Safety data should be collected throughout clinical development, including from PK/PD studies 1176 and clinical efficacy trials, when conducted. Key factors informing the data needed to 1177 characterize the similar biologicrequency, and severity of 1178 adverse events compared to the RBP; (b) whether these events result from enhanced 1179 pharmacological effects; (c) the level of analytical and functional similarity between the similar 1180 biologic and RBP; and (d) any novel impurities or excipients present in the similar biologic. 1181 If the clinical program for the similar biologic is limited to confirmatory PK/PD studies, a clear 1182 justification and risk assessment are required to evaluate the need for additional safety data. 1183 For example, in the case of insulin, hypoglycemiaan effect of its pharmacological actionis 1184 the primary safety concern. Highly similar physicochemical properties and PK/PD profiles 1185 between the similar biologic and RBP could sufficiently ensure a comparable hypoglycemia risk, 1186 potentially eliminating the need for further safety data. Similar cases include teriparatide, 1187 filgrastim, or somatropin. Emerging data also suggest that more complex products, such as 1188 mAbs, may be characterized effectively and could fit into this category. 1189 If the similar biologic contains impurities not found in the RBP (e.g., due to the use of a novel 1190 expression system), additional safety data may be required, or scientific justification should be 1191 provided to explain why such data are unnecessary. Manufacturers should consult with 1192 regulators when proposing a clinical program that relies exclusively on PK/PD studies. 1193 As for all medicinal products, further monitoring of the safety of the similar biologic will be 1194 necessary in the post-marketing phase. 1195 38

11.6 Immunogenicity Immunogenicity should be evaluated as part of the clinical development of the similar biologic in comparison to the RBP, unless the manufacturer provides a scientific justification for not including human immunogenicity data. This justification should be based on the extent of physicochemical similarity between the similar biologic and RBP, as well as a comprehensive 1200 risk assessment of potential immunogenicity and its known clinical consequences for the RBP. 1201 While published data can help assess the immunogenicity risk of the RBP and guide the 1202 immunogenicity strategy, it is typically insufficient on its own to support similar biologic approval. 1203 The goal of the immunogenicity programme is to exclude an unacceptable/marked increase in 1204 the immunogenicity of the similar biologic when compared with the immunogenicity of the RBP 1205 and to generate descriptive data in support of similar biologic approval and its clinical use. If 1206 conducted, the immunogenicity study report should include data on antibody incidence, 1207 magnitude of ADA response and neutralization ability, whether antibodies are transient or 1208 persistent, and their impact on PK and clinical correlates. 1209 The marketing authorization application should include a comprehensive immunogenicity 1210 summary, which should cover a risk assessment and, if applicable, the results of testing using 1211 appropriately validated assays. It should also provide details on the clinical study duration, 1212 sampling schedules, dosing regimen, and the clinical immunogenicity assessment. 1213 Immunogenicity studies should be specifically designed for each product and require a 1214 multidisciplinary approach that considers both quality and clinical factors. The risk assessment 1215 should include: 1216

• Information on the immunogenicity of the RBP, such as the nature, frequency, and clinical 1217 significance of the immune response. 1218 • Evaluation of quality aspects, including the complexity of the drug substance, glycosylation 1219 status, expression system, product and process-related impurities, and aggregates. 1220 • Consideration of excipients, the container closure system, product stability, route of 1221 administration, and dosing regimen. 1222 • Consideration of patient- and disease-related factors, such as immune status (immune-1223 competent or compromised) and any concurrent immunomodulatory treatments. 1224 Focusing on differences in product-related factors, such as impurities from novel expression 1225 systems or new excipients, is essential in the immunogenicity risk assessment of a similar 1226 biologic. It is also important to consider the type of product, as the risk is higher for products 1227 with an endogenous non-redundant counterpart (e.g., epoetin). In these cases, particular 1228 attention should be given to the potential for an immune response to adversely affect the 1229 endogenous protein and its unique biological function, leading to serious side effects. Real-1230 time testing for neutralizing ADAs is recommended for high-risk products like epoetins, enzyme 1231 replacement therapies, and coagulation factors. On the other hand, for well-characterized 1232 biologics, such as insulin, somatropin, filgrastim, and teriparatide, where extensive literature 1233 and clinical experience show that immunogenicity does not impact safety or efficacy, 1234 immunogenicity studies may not be required, provided the similar biologic is highly similar to 1235 the reference biologic and the risk assessment indicates a low risk. This approach may also 1236 39

apply to other products, including monoclonal antibodies (mAbs). In such cases, manufacturers should engage with regulatory authorities and provide a valid scientific justification for not conducting a safety or immunogenicity study. 11.6.1 Immunogenicity testing A comprehensive, multi-tiered approach that includes screening and confirmatory 1241 immunoassays to detect binding ADAs, followed by assays to assess ADA magnitude and 1242 neutralization potential, is typically required. Any deviation from this approach must be justified. 1243 Information on the current assays, their formats, benefits, limitations, and result interpretations 1244 has been thoroughly reviewed. The manufacturer must justify the antibody-testing strategy and 1245 the selection of assays. Special attention should be paid to choosing appropriate controls for 1246 assay validation and determining cut-off points to differentiate antibody-positive from antibody-1247 negative samples. Consideration should also be given to potential interference from matrix 1248 components, such as the pharmacological target or residual drug in the sample. To minimize 1249 such interference, corrective measures should be taken. For example, drug interference, often 1250 seen in samples from patients treated with monoclonal antibodies, can be managed by 1251 allowing time for drug clearance before sampling or incorporating steps to dissociate immune 1252 complexes or remove the drug. Care must be taken to ensure these measures do not interfere 1253 with ADA detection or affect patient treatment. 1254 When required, comparative immunogenicity testing should use the same assay format and 1255 sampling schedule. In new drug development, antibody testing typically uses the therapeutic 1256 administered to the patient. However, in the similar biologic context, developing screening 1257 assays with comparable sensitivity for both the similar biologic and reference biological product 1258 (RBP) within the same study is challenging. As such, relative immunogenicity is often 1259 assessed using a single assay that uses the similar biologic1260 for both patient groups. This approach ensures the detection of all antibodies against the 1261 similar biologic. The manufacturer must demonstrate the suitability of the methods used and 1262 provide data showing that the methods detect ADAs to both the RBP and similar biologic 1263 similarly. 1264 hanism of action, are typically based on 1265     -cell ligand-based assays are appropriate when the 1266 therapeutic binds to a soluble ligand and inhibits its biological action. For high-risk products 1267 (e.g., those with non-redundant endogenous counterparts) and those where effector functions 1268 are crucial, functional cell-based bioassays are recommended. If necessary, guidance on the 1269 need for a neutralization assay and the appropriate assay format (cell-based, ligand-based, or 1270 enzyme activity-based) can be sought from regulatory authorities. 1271 Additional characterization of antibodies, such as isotype determination, should be performed if 1272 clinically relevant or in specific circumstances (e.g., the occurrence of anaphylaxis or the use of 1273 certain assay formats), considering the immunogenicity profile of the reference biologic (RBP). 1274 For instance, if the RBP does not trigger an IgE response, it is unlikely that the similar biologic 1275 40

will do so if the same expression system is used. Patient samples should be stored under suitable conditions to allow for retesting in cases where issues arise with the original assay 11.6.2 Clinical evaluation Clinical evaluation can impact the pharmacokinetics (PK), pharmacodynamics (PD), safety, 1280 and/or efficacy of the administered product. The immunogenic risk of a biological product is 1281 influenced by the incidence of ADAs in the treated population and the extent of any adverse 1282 clinical effects, which in turn affects the benefit-risk profile of the therapy. 1283 If human immunogenicity data are necessary, they should be generated in a comparative 1284 manner throughout the clinical program. The preferred patient population for immunogenicity 1285 studies is typically the one most likely to mount an immune response. For instance, if epoetin is 1286 approved for treating both renal anemia and chemotherapy-induced anemia, it is 1287 recommended to select patients with renal anemia. Comparative PK and/or PD studies should 1288 also collect immunogenicity data, regardless of the population being studied (e.g., healthy 1289 volunteers or patients). A PK/PD crossover design can be used for immunogenicity testing, but 1290 if the exposure time before switching is insufficient to gather enough immunogenicity data, the 1291 sponsor must ensure a sufficient number of patients are treated without crossovereither by 1292 extending the crossover study with two parallel treatment arms or by proposing a separate 1293 immunogenicity study. 1294 1295 If ADAs are known to influence the pharmacokinetics (PK) of the reference biologic (RBP), 1296 assessments of ADA rates and kinetics should be conducted, along with an analysis of their 1297 impact on PK through pre-specified subgroup comparisons of ADA-negative and ADA-positive 1298 subjects. 1299 1300 The duration of the observation period for immunogenicity testing should be based on the 1301 expected time for antibody development and must be justified by the manufacturer. Sampling 1302 during immunogenicity testing should include baseline samples (taken before treatment) to 1303 detect pre-existing antibodies, as well as samples during treatment and, in some cases, post-1304 treatment, especially if ADAs persist or are undetectable at earlier time points (due to the 1305 product's immunosuppressive effects or technical issues like drug interference). The sampling 1306 schedule should align with PK evaluations, as well as safety and efficacy assessments, to 1307 understand how antibodies may affect clinical outcomes. 1308 1309 Significant differences in immunogenicity between the similar biologic and reference biologic 1310 (RBP) would require further investigation to identify the underlying cause. Data and a clear 1311 justification must be provided to support any claim that the observed difference is not clinically 1312 relevant. The clinical impact of ADAs on pharmacokinetics (PK), efficacy, and/or safety should 1313 be analyzed through a stratified comparison of ADA-negative and ADA-positive subjects. 1314 If there is a potential for the development of neutralizing antibodies against critical endogenous 1315 factors (e.g., after epoetin administration), clinical studies in patients will be required. 1316 As with the RBP, the similar biologic must undergo thorough post-marketing surveillance, 1317 including the monitoring of any serious adverse events related to immunogenicity. 1318 41

11.7 Waiver of safety and efficacy study The confirmatory clinical safety and efficacy study can be waived if all the below mentioned conditions are met: i. Structural and functional comparability of Similar Biologic and Reference Biologic can be characterized to a high degree of confidence by physicochemical and in vitro 1323 techniques. 1324 ii. The Similar Biologic is comparable to Reference Biologic in all preclinical evaluations 1325 conducted. 1326 iii. PK / PD study has demonstrated comparability of PD markers validated for clinical 1327 outcome and has preferentially been done in an in-patient setting with safety 1328 measurement (including meaningful immunogenicity assessment) for adequate period 1329 justified by the applicant and efficacy/PD measurements. 1330 iv. A comprehensive post-marketing risk management plan has been presented that will 1331 gather additional safety data with a specific emphasis on gathering immunogenicity 1332 data. 1333 1334 The confirmatory clinical safety and efficacy study cannot be waived especially for large 1335 molecular weight biologics like Monoclonal antibodies if validated PD marker is not 1336 available. 1337 1338 In case, the safety and efficacy study is waived all the indications approved for 1339 reference product may be granted based on comparable quality, non-clinical as well as 1340 convincing PK/PD data. 1341 Wherever the phase III trial is waived, the immunogenicity should have been gathered in 1342 the PK/PD study and will also need to be generated during post- approval Phase IV 1343 study. 1344 The confirmatory clinical safety and efficacy study cannot be waived if there is no 1345 reliable PD marker validated for clinical outcome. For a product which is found Similar in 1346 pre-clinical, in-vitro characterization having established PK methods and a PD marker 1347 that is surrogate of efficacy, the residual risk is significantly reduced in the Phase I study 1348 if equivalence is demonstrated for both PK and PD. In such cases clinical trials may be 1349 waived. 1350

11.8 Extrapolation of Efficacy and Safety Data to Other Indications 1351 Extrapolation of the safety and efficacy data of a particular clinical indication (for which clinical 1352 studies has been done) of a Similar Biologic to other clinical indications may be possible if 1353 following conditions are met: 1354

• Similarity with respect to quality has been proven to Reference Biologic. 1355 • Similarity with respect to non-clinical assessment has been proven to Reference Biologic. 1356 • Clinical safety and efficacy is proven in one indication which covers the most sensitive 1357 population. 1358 • Mechanism of action is same for other clinical indications. 1359 • Involved receptor(s) are same for other clinical indications. 1360

• Immunogenicity of the product in patient population • PK and biodistribution of the product in patient population. For example, authorization of all indications may be obtained based on highly comparable functional data  for example, for similar biologics of mAbs such as infliximab and adalimumab if they show fully comparable activity (including ADCC, CDC, reverse signaling 1365 and apoptosis) both in terms of binding to soluble TNF and membranous TNF. 1366 However, new indications not mentioned by innovator needs to be covered by separate 1367
  application. 1368

1. Data Requirements for Market Authorization Application 1369
   The applicant should submit application for market authorization as per CDSCO guidance 1370 document for Industry, 2024. For cases where commercial manufacturing is performed either at 1371 a different scale and/or with a different process as compared to that used for manufacturing 1372 phase III clinical trial batches, then information on comparability of quality needs to be 1373 additionally submitted with appropriate justification and will be dealt with on a case-to-case 1374 basis. Data from all manufactured batches (including developmental and clinical batches) used 1375 in the similarity assessment should be submitted at the time of MA application. 1376

2. Risk management plan (RMP) 1377
   The RMP for a similar biologic candidate should reflect that of the RBP in terms of safety 1378 concerns, additional pharmacovigilance activities and additional risk minimisation. If there are 1379 additional safety concerns for the similar biologic candidate these are unlikely to be due to the 1380 active molecule but rather factors such as excipient or device that are different from the RP. 1381 These should be included in the RMP. 1382 Where ongoing additional pharmacovigilance activities are required for the RBP (for example, 1383 participation in ongoing disease registries), these should also apply to the similar biologic 1384 candidate. Where possible, this would be through collaboration or participation in those studies 1385 or registries already in place for the RBP , or otherwise in other existing disease studies or 1386 registries. This will enable collection of real-world information to support characterization of risks 1387 and signal detection of potential safety signals related to the RBP and its biosimilars. 1388 Any additional risk minimisation measures that continue to be required for the RBP should also 1389 be implemented for the similar biologic candidate, for example educational materials for 1390 healthcare professionals and patients or patient alertcards. 1391

3. Post-Market Data for Similar Biologics 1392
   It is important to establish a formal Risk Management Plan to monitor and detect both known 1393 inherent safety concerns and potential unknown safety signals that may arise from the Similar 1394 Biologic since authorization is based on a reduced preclinical and clinical data package. If there 1395 are any remaining uncertainties regarding the similar biologic  due for example to the use of a 1396 43

novel excipient or device  then these should be included in the pharmacovigilance plan and followed up post-marketing. The risk management plan should consist of the following:

14.1 Pharmacovigilance Plan The clinical studies done on similar biologics prior to market authorization are limited in nature 1400 so the rare adverse events are unlikely to be encountered. Hence, a comprehensive 1401 pharmacovigilance plan should be prepared by manufacturer to further evaluate the clinical 1402 safety in all the approved indications in the post marketing phase. The pharmacovigilance plan 1403 should include the submission of periodic safety update reports (PSURs). The PSURs shall be 1404 submitted every six months for the first two years after approval of the Similar Biologic is 1405 granted to the applicant. For subsequent two years the PSURs need to be submitted annually 1406 to DCGI office as per NDCT Rules 2019. Post-marketing safety reports should include all 1407 information on product safety received by the marketing authorization holder. The safety 1408 information must be evaluated in a scientific manner and this should include evaluation of the 1409 frequency and cause of adverse events. 1410

14.2 Adverse Drug Reaction (ADR) Reporting 1411 All cases involving serious unexpected adverse reactions must be reported to the licensing 1412 authority as per NDCT Rules 2019. 1413

14.3 Post Marketing Studies (Phase IV Study) 1414 Finally, in order to further reduce the residual risk of the Similar Biologics, additional safety data 1415 may need to be collected after market approval through a pre-defined single arm study and 1416 compared to historical data of the Reference Biologic. The study should be completed 1417 preferably within 2 years of the marketing permission /manufacturing license unless otherwise 1418 justified. 1419 The primary aim of the post marketing phase IV study is safety and hence following parameters 1420 should be considered for the post marketing phase IV study protocol: 1421

 Primary endpoint: Safety 1422  Secondary endpoint: Efficacy and Immunogenicity 1423  The phase IV protocol should be submitted along with marketing authorization application for 1424 approval. 1425 • The clinical studies done on similar biologics prior to market authorization are limited in nature 1426 so post marketing studies should be conducted and the reports be submitted to DCGI. The plan 1427 of post market studies should be captured in Pharmacovigilance plan and update on the studies 1428 should be submitted to the CDSCO. 1429 • Regarding post-marketing safety and immunogenicity study at least one non- comparative post-1430 marketing clinical study with focus on safety and immunogenicity (on case-by-case basis) 1431 should be performed. This study must be designed to confirm that the Similar Biologic does not 1432 have any concerns with regard to the therapeutic consequences of unwanted immunogenicity. 1433 44

• It is not mandatory to carry out additional non-comparative immunogenicity studies in post marketing studies, if immunogenicity is evaluated in clinical studies. The immunogenicity of the Similar Biologics should be evaluated using appropriately designed studies with state-of-the-art methods, taking into consideration the potential impact on both safety and efficacy. • Rationale on the strategy for testing immunogenicity should be provided. 1438 • Assay methods should be validated and should be able to characterize antibody content 1439 (concentration or titer) as well as the type of antibodies formed. 1440 • Of most concern are those antibodies that have potentially serious impact on safety and 1441 efficacy, such as neutralizing antibodies and antibodies with cross reactivity. When neutralizing 1442 antibodies are detected in patients in clinical studies (either in pre-approval clinical studies or 1443 post-approval clinical studies), the impact of the antibodies on the PK/PD parameters of the 1444 Similar Biologics should be analyzed, where the data is available. 1445 • Furthermore, an assessment of the impact of the neutralizing antibodies and cross-reacting 1446 antibodies (if applicable) on the overall safety and efficacy of the Similar Biologics should be 1447 conducted. 1448

1. Labelling and Prescribing Information 1449
   The labelling of the similar biologic should be in accordance to Rule 96 and Rule 97 of the 1450 Drugs and Cosmetics Act 1940 and rules made thereunder and prescribing information must 1451 align the format as prescribed in Table 8 of NDCT Rules 2019. 1452 The prescribing information for a similar biologic should be as similar as possible to that of the 1453 RBP except for product-specific aspects such as use of different excipient(s) and/or 1454 presentations. This similarity is particularly important for posology and for safety-related 1455 information, including contraindications, warnings and known adverse events. However, if there 1456 are fewer indications for the similar biologic than for the RBP, the related text in various 1457 sections may be omitted unless it is considered important in informing doctors and patients of 1458 certain risks  for example, as a result of potential off-label use. In such cases it should be 1459 clearly stated in the prescribing information that the similar biologic is not intended for use in the 1460 specific indication(s) and the reasons why. 1461

2. Application Forms 1462
   Various application forms for submitting request to regulatory agencies are as 1463

45 Manufacturing permission for test, Form CT-Agency CDSCO - HQ Form CT-11/14/15 Stage Application Approval analysis and 10/12/13 Involved examination

The applicant should comply with the established pharmacopoeia requirements while testing the 1464

excipients and as well as Biological Product for which monograph is available in Indian Pharmacopoeia. 1465

Refer Drugs and Cosmetic Act,1940 and Rules 1945 for the application format. 1466

1. Archiving of Data/Retention of Samples: 1467
   The manufacturer should establish the SOP for data archival as well as sample retention. The 1468 applicant should archive all the data (quality, preclinical and clinical documentation) for a period 1469 of at least five years after marketing approval by competent authority in India. Important samples 1470 such as test substance, vehicle, plasma / serum, tissues, paraffin blocks, microscope slides, 1471 electronic material, etc., should be retained till the period of expiry. The designated authority, 1472 which will be responsible for archiving and can be approached for inspection or retrieval if 1473 required, should be indicated in the data archival and sample retention SOP. 1474

2. Glossary 1475
   Manufacturing License 46 for test, analysis and CDSCO Import and marketing CT-18 CT-19- DS CDSCO Manufacturing and CT-21 CT-22- DS CDSCO CDSCO Registration certificate Form 40 (with Form 41 Import License for Form 8 & 9 Form 10 State FDA/ Manufacturing License Form 27 D Form 28 D State FDA Form 30 Form 29 Import license for test, examination (After permission CT-20- DP (separate for marketing permission (separate for CT-23- DP for import schedule DI imported product Cell bank import / export Form IBSC / CDSCO-CDSCO Clinical Trial Permission CT-04 CT-06 analysis and CDSCO-HQ CT-16 CT-17 RCGM CDSCO permission) DS and DP) DS and DP) and DII) /transfer/received B1/B3/B5/B7 RCGM permission (countersignature) examination The definitions given below apply to the terms used in this guideline. They may have different meanings in other contexts a. Comparability/similarity exercise: direct head-to-head comparison of a biological product with a licensed reference product with the goal of establishing 1480 similarity in quality, safety and efficacy. 1481 1482 b. Comparability margin: the largest difference that can be judged as being clinically 1483 acceptable. 1484 c. Drug: Drug includes (as defined in Drugs and Cosmetics Act, 1940). 1485 1486 i. all medicines for internal or external use of human beings or animals and all 1487 substances intended to be used for or in the diagnosis, treatment, mitigation or 1488 prevention of any disease or disorder in human beings or animals, including 1489 preparations applied on human body for the purpose of repelling insects like 1490 mosquitoes; 1491 ii. such substances (other than food) intended to affect the structure or any function 1492 of human body or intended to be used for the destruction of (vermin) or insects 1493 which cause disease in human beings or animals, as may be specified from time 1494 to time by the Central Government by notification in the Official Gazette 1495 iii. All substances intended for use as components of a drug including empty gelatine 1496 capsules; and 1497 iv. Such devices intended for internal or external use in the diagnosis, treatment, 1498 mitigation or prevention of disease or disorder in human beings or animals, as 1499 may be specified from time to time by the Central Government by notification in 1500 the Official Gazette, after consultation with the Board. 1501 1502 d. Drug substance: Any substance or mixture of substances intended to be used in the 1503 manufacture of a drug (medicinal) product and that, when used in the production of a 1504 drug, becomes an active ingredient of the drug product. Such substances are intended to 1505 furnish pharmacological activity or other direct effect in the diagnosis, cure, mitigation, 1506 treatment, or prevention of disease or to affect the structure and function of the body. 1507 1508 e. Drug product: The dosage form in the final immediate packaging intended for 1509 marketing. A pharmaceutical product type that contains a drug substance, generally in 1510 association with excipients. 1511 1512 f. Efficacy study: a clinical trial to compare the efficacy of the biosimilar to the reference 1513 product. 1514 1515 g. Excipient: a constituent of a medicine other than the drug substance, added in the 1516 formulation for a specific purpose. While most excipients are considered inactive, some 1517 can have a known action or effect in certain circumstances (for example, hyaluronidase). 1518 47

The excipients may differ for a biosimilar and its reference product and need to be declared in the labelling and package leaflet of the medicine to ensure its safe use. h. Equivalent: equal or highly similar in the parameter of interest. Equivalent quality, safety and efficacy of two medicinal products denotes that they can be expected to have similar 1523 (no better and no worse) quality, safety and efficacy, and that any observed differences 1524 are of no clinical relevance. 1525 i. Generic medicine: a medicine that is structurally identical to an originator product 1526 (comparator) for which the patent and/or data protection period has expired. 1527 j. Genetic engineering: The technique by which heritable material, which does not usually 1528 occur or will not occur naturally in the organism or cell concerned, generated outside the 1529 organism or the cell is inserted into said cell or organism. It shall also mean the formation 1530 of new combinations of genetic material by incorporation of a cell into a host cell, where 1531 they occur naturally (self-cloning) as well as modification of an organism or in a cell by 1532 deletion and removal of parts of the heritable material (Rules, 1989). 1533 1534 k. Head-to-head comparison: direct comparison of the properties of a biosimilar with its 1535 corresponding reference product. Comparison based on historical data is not acceptable. 1536 1537 l. Highly Similar: Highly similar means that the characteristics of quality, biological 1538 activity, safety and efficacy of the similar biologic and its RBP have been shown to be 1539 comparable to the degree such that SBP can be called a version of the RBP. 1540 1541 m. Immunogenicity: The ability of a substance to trigger an immune response or reaction 1542 (e.g., development of specific antibodies, T cell response, allergic or anaphylactic 1543 reaction). 1544 1545 n. Impurity: Any component present in the drug substance or drug product that is not the 1546 desired product, a product-related substance, or excipient including buffer components. It 1547 may be either process- or product-related. 1548 1549 o. Manufacture:     any drug includes any process or part of a 1550 process for producing, altering, ornamenting, finishing, packing, labelling, breaking up or 1551 otherwise treating or adopting any drug with a view to its sale or distribution but does not 1552 include the compounding or 1553  1554 1555

1. New Drug: New D 1556 (i) a drug, including active pharmaceutical ingredient or phytopharmaceutical drug, 1557 which has not been used in the country to any significant extent, except in 1558 accordance with the provisions of the Act and the rules made thereunder, as per 1559 conditions specified in the labelling thereof and has not been approved as safe 1560 and efficacious by the Central Licencing Authority with respect to its claims; or 1561 1562 48

(ii) a drug approved by the Central Licencing Authority for certain claims and proposed to be marketed with modified or new claims including indication, route of administration, dosage and dosage form; or (iii) a fixed dose combination of two or more drugs, approved separately for certain 1567 claims and proposed to be combined for the first time in a fixed ratio, or where the 1568 ratio of ingredients in an approved combination is proposed to be changed with 1569 certain claims including indication, route of administration, dosage and dosage 1570 form; or 1571 1572 (iv) a modified or sustained release form of a drug or novel drug delivery system of 1573 any drug approved by the Central Licencing Authority; or 1574 1575 (v) a vaccine, recombinant Deoxyribonucleic Acid (r-DNA) derived product, living 1576 modified organism, monoclonal anti-body, stem cell derived product, gene 1577 therapeutic product or xenografts, intended to be used as drug; 1578 1579 Explanation. The drugs, other than drugs referred to in sub-clauses (iv) and (v), shall 1580 continue to be new drugs for a period of four years from the date of their permission 1581 granted by the Central Licencing Authority and the drugs referred to in sub-clauses (iv) 1582 and (v) shall always be deemed to be new drugs 1583 1584 1585 q. Non-inferior: not clinically inferior to a comparator in the parameter studied. A non-1586 inferiority clinical trial is one that has the primary objective of showing that the response 1587 to the investigational product is not clinically inferior to that of a comparator within a pre-1588 specified margin. 1589 1590 r. Originator product: a medicine that has been licensed by an NRA on the basis of a full 1591 registration dossier - that is, the approved indication(s) for use were granted on the basis 1592 of full quality, efficacy and safety data. 1593 1594 s. Pharmacodynamic study: a clinical study that measures a pharmacodynamic (PD) 1595 response that effectively demonstrates the characteristics of the products target effects. 1596 PD biomarkers for biosimilars do not need to be surrogate end-points for clinical efficacy 1597 outcomes. 1598 1599 t. Pharmacovigilance: The science and activities relating to the detection, assessment, 1600 understanding and prevention of adverse effects or any other drug related problems. 1601 1602 u. Posology: dosage for each indication and each method/route of administration. 1603 Information includes dose recommendation (for example, in mg, mg/kg or mg/m2), 1604 frequency of dosing (for example, once or twice daily, or every 6 hours) and treatment 1605 duration. 1606 49

1. Reference Biological Product: A Reference Biological product is used as the comparator for comparability studies with the Similar Biologic in order to show Similarity in terms of safety, efficacy and quality. The Reference Biologic should be licensed / approved in India or ICH countries and should be the innovator's product. The Reference 1611 Biologic should be licensed based on a full safety, efficacy and quality data. Therefore, 1612 another Similar Biologic cannot be considered as a choice for Reference Biologic. 1613 1614 w. Reference standard: a measurement standard such as an international, 1615 pharmacopoeial or national standard  it should be noted that reference standards are 1616 distinct from reference products and serve a different function. 1617 1618 x. Similar Biologic: Similar biologic means a biological product which is similar in terms of 1619 quality, safety and efficacy to reference biological product licenced or approved in India, 1620 or any innovator product approved in International Council of Harmonisation (ICH) 1621 member countries. 1622 1623 y. Similarity: absence of any relevant difference in the parameter(s) of interest. 1624

2. References 1626

3. World Health Organization (WHO) TRS No. 1043 Annex 3, Guidelines on evaluation 1627 of biosimilars, 2022 (Replacement of Annex 2 of WHO Technical Report Series, No. 1628 977) 1629 II. Medicines & Healthcare products Regulatory Agency, Guidance on the licensing of 1630 biosimilar products, November 2022 1631 III. Health Canada, Guidance Document Information and Submission Requirements for 1632 Biosimilar Biologic Drugs, 2022 1633 IV. EMA Guideline on Similar Biological medicinal products containing biotechnology- 1634 derived proteins as active substance: non-clinical and clinical issues, 2014 1635 (EMEA/CHMP/BMWP/42832/2005 Rev1) 1636 V. EMA guideline on immunogenicity assessment of biotechnology-derived therapeutic 1637 proteins, 2007 (CHMP/BMWP/14327) 1638 VI. ICH guideline on preclinical safety evaluation of biotechnology-derived 1639 pharmaceuticals (S6), 1997 and addendum, 2011 1640 VII. Guideline for Safety Study of Biological Products, (KFDA, 2010) 1641 VIII. World Health Organization (WHO) Guidelines on Evaluation of Similar Biotherapeutic 1642 Products (SBP), 2009 1643 IX. World Health Organization (WHO), Guidelines on the quality, safety and efficacy of 1644 bio-therapeutic protein products prepared by recombinant DNA technology, 2013 1645 X. EMA- DNA and Host cell protein impurities routine testing versus validation studies, 1646 1997 1647 XI. ICH Q1 A(R2)- Stability Testing of New Drug Substances and Products, 2003 1648

4. The Regulations & Guidelines for Recombinant DNA Research and Biocontainment,

Annexure I: Pathway for approval to manufacture and market indigenously developed Similar Biologics

Application to CDSCO for obtaining Test License for generation of Conduct of Pre-clinical studies (PCT) Note:

1. Application for seeking waiver of Pre clinical studies/ for conduct of clinical studies is required to
   be submitted to CDSCO and decision of waiver/MA permission will be granted by Licensing Authority.

2. Firm should obtain a valid license/permission from Licensing Authority under D&C Act and Rules
   thereunder for generation of data for regulatory submission.

3. The approval of RCGM is required for experiments involving Risk Group 3 and 4 organisms.
   (Reference: The Regulations & Guidelines for Recombinant DNA Research and Biocontainment,

2017)

If applicant is seeking waiver of Pre-clinical studies, applicant needs to submit 52 application to CDSCO with CMC comparability data along with other No waiver of PCT, applicant is Evaluation of PCT results/waiver proposal by Application for Marketing justification for consideration of waiver of required to conduct pre-clinical PCT/ Waiver considered, applicant shall apply in CT -Review of CT application by Marketing Authorization approval CMC data Post marketing studies CDSCO in consultation with subject experts pre-clinical study Authorization approval studies 04 application form for Clinical Trial to CDSCO IBSC CDSCO and Subject Expert Committee (SEC)

Annexure IA: Pathway for approval to import and market Similar Biologics

clinical trial data by CDSCO in consultation with subject experts. Accord approval for Human CT and protocol Human CT conducted CDSCO grants market authorization under Drug Rules based on clinical trial data in consultation with subject experts 1652 1653 1654 1655 1656 1657 1658 1659 53 Evaluation of complete dossier including PCT results and human

Application Post marketing studies

Annexure II: Critical Quality Attributes (CQA)

Physicochemical and biological characterization of nucleic acid based recombinant products (Vector for expression of recombinant protein, siRNA/ snRNA etc.), recombinant therapeutic Proteins, recombinant mAbs, recombinant therapeutic Enzymes

Primary structure/Identity Higher order structure (Tertiary Higher order Product related structure) structure substances and Higher order Higher order 54 Quality Attributes Analytical Methodology (Conformational structure (Disulfide impurities Amino acid sequence by LC-MS/MS or Edman structure (Secondary Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-Size variants by SEC, DLS/MALLS (aggregates) Differential scanning calorimetry (DSC)/NanoDSC or Time-Fourier transform infrared spectroscopy (FTIR) stability) bridging) Protein content Absorbance Peptide mapping by LC-MS/MS (CID/ETD/HCD) degradation N-terminal and C-terminal sequence by LC-MS/MS Near UV Circular Dichroism (CD) Fluorescence spectroscopy Non-reduced LC-MS/MS Ion Mobility Mass Spectrometry (IM-MS) Sub visible particles by MFI, AUC or equivalent Intact mass (Native/deglycosylated) by LC-MS Subunit mass (Native/deglycosylated) by LC-MS structure) Far UV Circular Dichroism (CD) 1D/2D Nuclear Magnetic Resonance (NMR)* MS)* Free thiol group analysis by Ellman/LC-MS Melting temperature by DSC/DSF Correlated Single-Photon Counting (TCSPC)* Nano Differential Scanning Fluorimetry (nanoDSF)* Charge variants by CEX /cIEF/CZE-UV/LC-MS/CE-MS Size-variants by reduced and non-reduced CE-SDS /

• These next generation analytical methodologies are not mandatory and can be used if feasible. 1664 **To ensure the statistical analysis, each quantitative experiment should be done atleast three times and 1665 data should be represented in terms of mean and standard deviation. Appropriate statistical significance 1666 should be represented throughout the characterization data. 1667

1672 Process related Annexure III: Statistical tools for Biosimilarity assessment 1673 impurities

Fc effector functions DP Physical BET 55 attributes Fab-mediated Glycan characterization at intact or subunit level using LC-Fc-mediated FcRI, FcRIIa(R and H)/b, FcRIIIa(V and F)/ b, FcRn Appearance Endotoxins (if applicable) biological assays SDS-PAGE PTMs by LC-MS Major target (receptor/ligand) binding assay by BLI/SPR biological assays binding kinetics if applicable ADCC, if applicable Apoptosis, if applicable Concentration (Drug and excipient) HCD by qPCR/Picogreen Residual Protein A N-Glycan relative quantitation by HILIC (labelling methods) MS/CZE-LIF/CE-MS Cell based assay CDC, if applicable pH HCP by ELISA/2D-PAGE/CZE-MS/LC-MS Bioburden NOTE: The following text elaborates the utilities of below statistical approaches. These are meant to be illustrative and nor prescriptive. There are 3 tests recommended by regulatory agencies (World Health Organisation) for biosimilarity assessment, 1. x-sigma test, 2. min-max interval test, 3. tolerance Intervals test.

1. X-sigma interval: This tool calculates the similarity ranges based on the mean and standard 1678 deviation of the reference product batch data as shown in below equations. 1679 1680   1.1. 󰇛󰇜    1681  where, xi = lots of RBP; BS 1682 n = number of lots of RBP; BS 1683

1.2. 󰇛󰇜󰇛󰇜 1684 1.3.  󰇛 󰇜 1685 1686 2. Min-Max Range: It establishes similarity ranges using the observed minimum and maximum 1687 values of the RBP quality attribute data. 1688 1689 2.1. Min- Max Range: (󰇜 1690 1691 2.2.  1692  where, xmin = Minimum value of RBP; xmax = Maximum value of RBP; 1693 BS represents biosimilars 1694

1. Tolerance Intervals: It defines a range within which a specified percentage of future 1695 observations are expected to fall, given a certain confidence level. 1696 1697 3.1.  󰇛  󰇜 1698 1699 1700 where: 1701   󰇛󰇜 1702     = Significance level ( = 1  Confidence level) 1703  -distribution with 1704  (n-1) = degrees of freedom at /2 1705 56

Case 1: Glycosylation Table 1: Glycan attributes with their criticality, tier ranking, and data from reference product and biosimilar lots 1709

Results: 1711 Table: Summary of Mean, Standard Deviation, and Calculated Ranges for x-Sigma, Min-Max, 1712 and Tolerance Interval Tests. 1713

1714 1715

Standard Glycan Attribute RBP 57 Mean Deviation Tier Glycan Attribute RBP RBP RBP BS Lot BS Lot BS (Min  Max) X sigma Tolerance (SD) Lot 1 Lot 2 Lot 3 Lot 3 Total 9.37 0.89 (6.7, 12.04) (8.36-10.03) (8.71, 10.04) Highly Total 10.03 9.73 8.36 7.75 8.79 7.72 Highly Galactosylation GlcNAc High mannose 5.91 5.06 4.61 4.55 4.26 5.17 40.57 1.23 (36.88, 54.27 1.57 (49.56, (39.17-41.46) (39.65, 41.48) (53.06, 55.41) (Mean ± (52.63- Interval Test -2 Test -1 Test -3 High mannose 5.19 0.66 (3.21, 7.17) (4.61, 5.91) (4.70, 5.69) Low GlcNAc 52.63 55.76 54.32 53.43 55.25 53.0 Sialylation 0.87 0.31 (-0.06, 1.8) (0.6 -1.2) (0.64, 1.10) Moderate Galactosylation 41.46 39.17 41.07 44.02 40.49 41.83 Low Sialylation 1.2 0.6 0.8 0.9 0.8 1.2 Afucosylated critical Afucosylated critical 44.26) 58.98) (3. SD) 55.76)

1716 Fig 1: Illustration of biosimilarity scores for each quality attribute (glycan) assessed using three 1717 statistical methods: (1) x-sigma test, (2) min-max interval test, and (3) tolerance interval test for 1718 3 lots for reference lots. The comparison highlights the percentage of biosimilar batches falling 1719 within the similarity ranges established by each method. 1720 1721

1722 Fig 2: Illustration of biosimilarity scores for each quality attribute (glycan) assessed using three 1723 statistical methods: (1) x-sigma test, (2) min-max interval test, and (3) tolerance interval test for 1724 20 lots of reference lots. The comparison highlights the percentage of biosimilar batches falling 1725   within the similarity ranges established by each method. 1726  1727 Key Observations 1728 • For n=3 (less lots of reference) 1729   58          

      

• In this case study, X-sigma is widely accepted approach with 100% similarity for all the glycan attributes (Fig 1). For min-max approach and tolerance interval approach (highly critical and moderate attributes) showed only 33% of the similarity and batches fall within the calculated tolerance intervals, indicating tighter thresholds. • For low criticality attributes in both the tests (min-max), 100% of the BS batches fall within the tolerance intervals, reflecting good fit to the range. 1735 • For low criticality attributes in both the tests (tolerance interval), 66% of the BS batches 1736 fall within the tolerance intervals, reflecting less stringent requirements for these 1737 attributes. 1738 • For n=20 (more lots of reference): As the number of lots, there is an improvement in the 1739 similarity of both min-max approach and tolerance approach as can be seen from fig 1 1740 and 2. The increased the tolerance interval method provides a statistically robust 1741 framework for evaluating similarity but may lead to stricter conclusions when sample 1742 sizes are small. 1743 Case 2: Size Heterogeneity 1744

1745 1746 1747 1748

1749 Fig 3: Illustration of biosimilarity scores for each quality attribute (size heterogeneity) assessed 1750 using three statistical methods: (1) x-sigma test, (2) min-max interval test, and (3) tolerance 1751 59 BS Lot Criticality RBP RBP RBP BS Lot BS Lot 1.81 96.4 1.79 Lot 1 Lot 2 Lot 3 HMW moderate 1.12 2.42 0.95 1.62 2.05 Monomer moderate 97.25 95.55 96.98 96.26 95.79 LMW moderate 1.63 2.03 2.07 2.12 2.16 interval test for 3 lots of reference lots. The comparison highlights the percentage of biosimilar batches falling within the similarity ranges established by each method

Key Observations For n=3 (less lots of reference), The criticality of size attributes (monomer, high and low 1755 molecular weight species) are placed in the moderate range of criticality. X-sigma showed a 1756 good acceptance to the biosimilarity for all size attributes with 100% similarity (Fig 3). For min-1757 max approach and to tolerance interval test showed similar similarity. 1758 Case 3: Charge Variant 1759

1761 Fig 4: Illustration of biosimilarity scores for each quality attribute (Charge variant) assessed 1762 using three statistical methods: (1) x-sigma test, (2) min-max interval test, and (3) tolerance 1763 interval test for 3 lots of reference lots. The comparison highlights the percentage of biosimilar 1764 batches falling within the similarity ranges established by each method 1765

Key Observations 1766 For n=3, the criticality of size attributes (acidic, main and basic variant) are placed in the 1767 moderate range of criticality. All the 3 tests (X-sigma, min-max and tolerance interval) showed a 1768 good acceptance to the biosimilarity for all attributes with 100% similarity (Fig 4). 1769

Overall Recommendation: 1770 For Small Reference Datasets 1771 60 criticality RBP RBP RBP BS Lot BS Lot BS Lot Lot 1 Lot 2 Lot 3 Acidic moderate 6.92 6.18 8.16 7.62 7.48 6.56 Main moderate 67.46 68.9 63.93 65.83 65.9 67.95 Basic moderate 25.62 24.92 27.91 26.55 26.48 25.49 The x-sigma method is the most effective, showing high acceptance for biosimilarity with 100% similarity across all attributes. Limitations of Other Methods: The min-max approach and tolerance interval tests may yield lower similarity percentages due to stricter thresholds or overly conservative ranges, especially for highly critical and moderate attributes. 1776

For Larger Reference Datasets 1777 The tolerance interval method becomes more statistically robust and reliable as more RBP 1778 batches reduce variability-related artifacts. 1779 The min-max approach also improves in similarity acceptance, but care must be taken to 1780 prevent overly conservative conclusions. 1781

Other recommendations 1782

 Apply stricter thresholds using scientifically justified multipliers in the x-sigma method or 1783 tighter tolerance intervals. 1784  Avoid reliance on min-max ranges, as they may be overly restrictive and prone to false-1785 negative conclusions. 1786 1787

Annexure IV: Requirements of Toxicological Studies

In case of in vivo toxicity studies, at least one repeat dose toxicity study in a pharmacologically relevant species is required to be conducted with an intended route of administration. Regarding the animal models to be used, the applicant should provide the scientific justification 1791 for the choice of animal model(s) based on the data available in scientific literature. However, if 1792 the pharmacologically relevant animal species is not available and has been appropriately 1793 justified, toxicity studies need to be undertaken either in rodent or nonrodent species as per 1794 requirements of NDCT Rules 2019. 1795 Regarding route of administration either in pharmacologically relevant or pharmacologically 1796 non-relevant animal model the route of administration would include only the intended route as 1797 per NDCT Rules 2019. 1798 The duration of the study would be generally not less than 28 days with 14 days recovery 1799 period. However, the duration may vary depending on the dosage and other parameters on 1800 case-by-case basis. 1801 The dose should be calculated based on the therapeutic dose of the Reference Biologic. If 1802 required a pilot dose response study should be conducted prior to initiating the toxicity studies. 1803 Generally, there would be three levels of doses (viz. low, medium and high) used in the animal 1804 toxicology studies corresponding to 1X, 2X and 5X of human equivalent dose or higher test 1805 dose for repeated-dose toxicity studies. In the toxicity study the Similar Biologic should be 1806 compared with Reference Biologic at least at 1X of human equivalent dose (HED). Any 1807 difference in the levels of doses should be justified and approved prior to the studies. Regarding 1808 the schedule of administration, the therapeutic schedules may be used as the basis. 1809 Depending on the route of administration, local tolerance should be evaluated. This evaluation, 1810 if feasible may be performed as a part of above mentioned repeated-dose toxicity study. 1811 Accordingly, the study groups of animals in repeated-dose toxicity testing will consist of: 1812

1. Historical Control (Optional) 1813 ii. Vehicle Control 1814 iii. Vehicle Control for recovery group 1815 iv. Formulation without protein (for vaccines) if multiple adjuvants - each to be checked 1816 independently 1817 v. 1X Similar Biologic for study duration (lowest dose) 1818 vi. 1X Reference Biologic for study duration 1819 vii. 2X Medium dose Similar Biologic 1820 viii. 5X High dose Similar Biologic 1821 ix. Similar Biologic with a recovery group going beyond the end of study period for 7 to 1822 14 days 1823 The protocols and the study reports should provide complete details of various steps in the 1824 toxicity testing as indicated below: 1825  Procedures prior to euthanasia e.g. blood drawing, body weight, etc. 1826  Events immediately after euthanasia, necropsy, gross  description, organ weights and organs sampled for histopathology.

 Biochemical parameters  Equipment and methods used - units of measurement and expression.

 Haematology procedures and parameters  method to be used (automated or manual). 1831

 Statistical methods used. 1832

 Bone marrow either examined as an aspirate /smear or on histopathology section. 1833 In case of histopathological observations, the applicants should consider the following points: 1834  Every observation considered as deviation from described normal histology needs to be 1835 documented and the incidence of each of these in the different groups should be 1836 denoted. 1837

 Whether such a feature is significant or not can be decided on review of statistical 1838 significance or dose response or if it is within or outside the normal range of values in 1839 case of biochemical and haematological observations. 1840

 If all organs from all animals were not examined e.g. in 5 animals only 4 livers were 1841 examined, the reason for the 1 liver not being examined should be documented. 1842

 In case of premature death or morbidity the proposed course of action is to be included 1843 in the protocol. 1844 The final report of the study should reflect all the aspects approved in the protocol and the 1845 following additional sections/documents: 1846

 IBSC approval of report 1847

 IAEC appr 1848

 Signatures of study director and all investigators who were involved in the study 1849

 All quality analytical reports on the test material and vehicle 1850

 Animal feed and animal health certifications. 1851 Protocol deviations if any 1852  Discussion on the results. 1853

 Individual animal data, summary data and any other data like computer analysis outputs 1854 etc. 1855

 Conclusion. 1856 1857

Annexure V: Statistical consideration in sample size determination for Clinical Study

Determining the number of subjects (sample size) in a clinical trial is a critical step in the design of the study. The sample size must be large enough to reliably detect the effect of the 1861 intervention. The statistical criteria for deciding the number of subjects typically include the 1862 following key elements like Primary Objective and Endpoint, Effect Size, Statistical Power, 1863 Significance level, Variability, Equivalence / non inferiority margins, incidence rate, Dropout 1864 & Compliance Rates, Study design, Multiplicity adjustments etc. 1865 Commonly following choices are made: 1866

• Power (1 -  1867 •  1868 •  1869 • Variability estimated from previous studies or pilot data. 1870 • Dropout and Compliance Rates to increase the sample size to ensure sufficient 1871 power after adjustment 1872 • Stratification and Subgroup Analysis requires adequate numbers in each subgroup. 1873 Various statistical software packages (e.g., SAS, R, Stata, PASS, nQuery) can be used to 1874 perform sample size calculations by Biostatistician. These tools often allow for more 1875 complex designs and adjustments. 1876 Determining the number of subjects in a clinical trial involves a careful balance of statistical 1877 criteria, clinical relevance, and practical considerations. Proper sample size calculation 1878 ensures that the trial is adequately powered to detect meaningful effects while minimizing 1879 risks and resource use. The comparability Phase III clinical trials intended for seeking 1880 marketing approval of Similar Biologics falling under the category of new drugs as per Drugs 1881 and Cosmetics Rules, 1945 shall be conducted in accordance with the Indian Good Clinical 1882 Practice (GCP) guidelines and should be adequately powered to evaluate the safety, 1883 efficacy and comparability. Based on the statistical calculation of sample size, the number of 1884 subjects in test arm should not be less than 100 evaluable patients. Based on the results of 1885 such Clinical trials, the marketing approval may be considered if safety, efficacy and 1886 comparability are established. Further, Phase IV clinical trials may be required to be 1887 conducted, generally in more than two hundred patients in continuation of comparability 1888 clinical trials. In general, if the firm conducts pre approval comparative studies that included 1889 more than 100 patients on the proposed Similar Biologics drug and statistically proportionate 1890 number of patients in reference biologic arm, the number of patients in the Phase IV study 1891 can be modified accordingly so that the safety data (from both Phase III and IV) is derived 1892 from not less than 300 patients. 1893 Exceptions: 1894

In the case of Similar Biologics that can be evaluated for rare diseases, the clinical trial population size can be reduced as per the rarity and severity of the disease as well as the limitation of access to therapeutic options.

Acknowledgement