Manufacturing Changes and 
Comparability for Human Cellular 
and Gene Therapy Products 
 
 
Draft Guidance for Industry 
 
 
 
 
This guidance document is for comment purposes only. 
 
 
 
 
 
 
Submit one set of either electronic or written comments on this draft guidance by the date 
provided in the Federal Register notice announcing the availability of the draft guidance.  
Submit electronic comments to http://www.regulations.gov.  Submit written comments to the 
Dockets Management Staff (HFA-305), Food and Drug Administration, 5630 Fishers Lane, Rm. 
1061, Rockville, MD 20852.  You should identify all comments with the docket number listed in 
the notice of availability that publishes in the Federal Register. 
 
Additional copies of this guidance are available from the Office of Communication, Outreach 
and Development (OCOD), 10903 New Hampshire Ave., Bldg. 71, Rm. 3128, Silver Spring, 
MD 20993-0002, or by calling 1-800-835-4709 or 240-402-8010, or email ocod@fda.hhs.gov, or 
from the Internet at https://www.fda.gov/vaccines-blood-biologics/guidance-compliance-
regulatory-information-biologics/biologics-guidances. 
 
For questions on the content of this guidance, contact OCOD at the phone numbers or email 
address listed above. 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Biologics Evaluation and Research 
July 2023 
     
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Table of Contents 
 
 
I.  INTRODUCTION............................................................................................................. 1 
II.  BACKGROUND ............................................................................................................... 2 
III.  CONSIDERATIONS FOR THE MANAGEMENT OF MANUFACTURING 
CHANGES ......................................................................................................................... 3 
A.  Risk Management ................................................................................................. 3 
B.  Stability and Delivery Device Compatibility ...................................................... 4 
C.  Nonclinical studies ................................................................................................ 5 
D.  Clinical studies ...................................................................................................... 5 
IV.  REGULATORY REPORTING OF MANUFACTURING CHANGES ...................... 7 
A.  CMC Changes Requiring a New IND Submission............................................. 7 
B.  Reporting Manufacturing Changes to an IND ................................................... 8 
C.  Reporting Manufacturing Changes to a BLA .................................................... 9 
V.  COMPARABILITY ASSESSMENT AND REPORT ................................................... 9 
A.  Risk Assessment .................................................................................................. 10 
B.  Analytical Comparability Study Design ........................................................... 12 
C.  Analytical Methods ............................................................................................. 16 
D.  Results .................................................................................................................. 18 
E.  Statistics ............................................................................................................... 18 
VI.  SPECIAL CONSIDERATIONS FOR TISSUE-ENGINEERED MEDICAL 
PRODUCTS..................................................................................................................... 20 
VII.  COMMUNICATION WITH FDA ................................................................................ 21 
VIII.  REFERENCES ................................................................................................................ 22 
 
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1  Manufacturing Changes and Comparability for Human Cellular 
2  and Gene Therapy Products 
3   
4   
5  Draft Guidance for Industry 
6   
7  This draft guidance, when finalized, will represent the current thinking of the Food and Drug 
8  Administration (FDA or Agency) on this topic.  It does not establish any rights for any person 
9  and is not binding on FDA or the public.  You can use an alternative approach if it satisfies the 
10  requirements of the applicable statutes and regulations.  To discuss an alternative approach, 
11  contact the FDA staff responsible for this guidance as listed on the title page.  
12   
13   
14  I.  INTRODUCTION 
15   
16  The management of manufacturing changes presents many challenges for human cellular 
17  therapy1 or gene therapy2 (CGT) products due to the complexity of these products.  We, FDA, 
18  are providing you, sponsors of Investigational New Drug Applications (INDs) and applicants of 
19  Biologics License Applications (BLAs) for CGT products, with recommendations regarding 
20  product comparability and the management of manufacturing changes for investigational and 
21  licensed CGT products.3  The purpose of this guidance is to provide FDAs current thinking on 
22  1) management and reporting of manufacturing changes for CGT products based on a lifecycle 
23  approach, and 2) comparability studies to assess the effect of manufacturing changes on product 
24  quality.4, 5  
25   
26  In general, FDAs guidance documents do not establish legally enforceable responsibilities. 
27  Instead, guidances describe the Agencys current thinking on a topic and should be viewed only 
 
1 For the purposes of this guidance cellular therapy products include certain tissue-engineered medical products 
(referred to in this guidance as TEMPs) that contain living cells (see section VI of this guidance) and are regulated 
under section 351 of the Public Health Service (PHS) Act (42 U.S.C. 262). 
2 Human gene therapy seeks to modify or manipulate the expression of a gene or to alter the biological properties of 
living cells for therapeutic use.  FDA generally considers human gene therapy products to include all products that 
mediate their effects by transcription or translation of transferred genetic material, or by specifically altering host 
(human) genetic sequences.  Some examples of gene therapy products include nucleic acids, genetically modified 
microorganisms (e.g., viruses, bacteria, fungi), engineered site-specific nucleases used for human genome editing, 
and ex vivo genetically modified human cells.  
3 Cellular and gene therapy products meet the definition of biological product in section 351(i) of the PHS Act (42 
U.S.C. 262(i)) when such products are applicable to the prevention, treatment, or cure of a disease or condition of 
human beings (see Federal Register Notice:  Application of Current Statutory Authorities to Human Somatic Cell 
Therapy Products and Gene Therapy Products (58 FR 53248, October 14, 1993), 
https://www.fda.gov/media/76647/download). 
4 This guidance does not apply to vaccines for infectious disease indications, bacteriophage products, live 
biotherapeutic products, fecal microbiota for transplantation (FMT) products and allergenic products. 
5 For the purposes of this guidance, the term product quality refers to identity, strength, quality, purity, and 
potency of a product, as these factors may relate to the safety or effectiveness of the product. 
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28  as recommendations, unless specific regulatory or statutory requirements are cited.  The use of 
29  the word should in Agency guidances means that something is suggested or recommended, but 
30  not required. 
31   
32   
33  II.  BACKGROUND 
34   
35  CGT products are regulated under the existing framework for biological products.  
36  Manufacturing and control of CGT products can often be affected by unique factors, including 
37  limited knowledge of product quality attributes, limited manufacturing experience, limited and 
38  variable starting materials, limited amount of product, complex manufacturing processes, and 
39  limited product shelf life.  These aspects of CGT products may make the management of 
40  manufacturing changes more challenging than for other biological products. 
41   
42  A CGT product manufacturer may seek to implement a manufacturing change for a variety of 
43  reasons, including improving product quality, expanding product supply, or improving 
44  manufacturing efficiency.  The risk that a manufacturing change may adversely impact product 
45  quality should be prospectively assessed under the manufacturers quality risk management 
46  processes (Refs. 1, 2).  We note that while improvement of product quality is always desirable 
47  and encouraged, if the results of comparability studies indicate an improved product quality 
48  suggesting a significant benefit in effectiveness and/or safety, the pre- and post-change products 
49  may be different products and, therefore, not comparable. 
50   
51  Risk assessment should be performed for all types of manufacturing changes, regardless of the 
52  stage of product development.  If a risk assessment indicates that a manufacturing change has the 
53  potential to adversely affect product quality, comparability studies should be performed to 
54  evaluate the impact of the proposed manufacturing change.  It can be difficult to fully 
55  characterize CGT products using analytical methods, and in some cases analytical studies alone 
56  may not be sufficient to reach a conclusion regarding comparability.  In such cases, additional 
57  data from nonclinical studies may help to support comparability. Otherwise, additional clinical 
58  studies may be warranted.  
59   
60  The extent of analytical evaluation needed to adequately evaluate a manufacturing change in 
61  comparability studies generally increases with the stage of clinical and product development and 
62  should be supported by knowledge of critical quality attributes (CQAs) (Ref. 3), accumulated 
63  manufacturing experience, and further understanding of the mechanism of action (MOA).  For 
64  both licensed and investigational products, assessing the risks of manufacturing changes is 
65  essential before designing comparability studies.  For licensed products, applicants are required 
66  to assess the effects of each change in the product, production process, quality controls, 
67  equipment, facilities, responsible personnel, or labeling established in the approved license 
68  application(s) (Title 21 of the Code of Federal Regulations (CFR) 601.12(a)(1)-(2)).6 
69  Applicants must also demonstrate through appropriate validation and/or other clinical and/or 
 
6 For purposes of this guidance, the term manufacturing change in the context of a licensed product, refers to a 
change (other than a labeling change) that would fall within the types of changes described in 21 CFR 601.12(a)(1).  
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70  nonclinical laboratory studies that each manufacturing change does not adversely affect product 
71  quality before distributing a product manufactured using the change (21 CFR 601.12(a)(2)).  For 
72  investigational products, sponsors must provide sufficient chemistry, manufacturing, and control 
73  (CMC) information to assure product safety, identity, quality, purity, and strength (including 
74  potency) of the product (21 CFR 312.23(a)(7)(i)), and some manufacturing changes without 
75  adequate comparability data may result in a clinical hold (21 CFR 312.42(b)). 
76   
77  The guidance entitled Demonstration of Comparability of Human Biological Products, 
78  Including Therapeutic Biotechnology-derived Products dated April 1996 (Ref. 4) contains 
79  general recommendations applicable to biological products, but it does not address the specific 
80  challenges of performing comparability studies with CGT products.  The guidance entitled Q5E 
81  Comparability of Biotechnological/Biological Products Subject to Changes in Their 
82  Manufacturing Process dated June 2005 (Ref. 5) contains principles that may be useful for 
83  comparability studies of CGT products.  However, its scope is limited to certain proteins and 
84  polypeptides that can be highly purified and characterized, which are typically less complex, 
85  better characterized, and manufactured to more stringent tolerances than CGT products.  Other 
86  FDA guidance documents related to management of manufacturing changes and risk 
87  management for biological products generally do not address specific CGT product challenges 
88  (e.g., Refs. 1, 2, 6).  The purpose of this guidance is to provide recommendations for managing 
89  manufacturing changes and assessing comparability for both investigational and licensed human 
90  CGT products while considering the unique challenges that apply to these products.  
91   
92   
93  III.  CONSIDERATIONS FOR THE MANAGEMENT OF MANUFACTURING 
94  CHANGES 
95   
96  An effective quality system maintains consistency in drug product (DP) quality throughout the 
97  product lifecycle, including by adequately managing manufacturing changes.  In general, 
98  manufacturing changes should be thoroughly assessed and documented using effective change 
99  control procedures.  For investigational products, maintaining product quality by control of 
100  CQAs and critical process parameters (CPPs) during manufacturing changes is important for 
101  obtaining interpretable clinical study data that can support licensure.  A robust framework for 
102  managing manufacturing changes is especially valuable for CGT products because of the 
103  complexity of these products and their manufacturing processes. 
104   
105  A.  Risk Management 
106   
107  Managing manufacturing changes can be challenging for CGT products due to difficulty 
108  in identifying risks to product quality and uncertainty about how to mitigate risk.  
109  Therefore, we recommend that you apply a systematic approach to quality risk 
110  management designed to identify, assess, analyze, and mitigate potential risks.  Such an 
111  approach can facilitate science-based decision-making and enable a risk-based evaluation 
112  of manufacturing changes (Ref. 1).  
113   
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114  Defining acceptable ranges for CQAs and establishing operating ranges for CPPs prior to 
115  making a manufacturing change facilitates conducting a risk assessment and evaluating 
116  the change.  For example, for a cellular product that has a manual wash step, it would 
117  generally be easier to transition to an automated wash process if the acceptable operating 
118  range for the duration of the cell washes has already been established, because this 
119  parameter can impact product CQAs and process performance. 
120  Factors such as product and process knowledge, qualification/validation of methods, and 
121  the stage of clinical development should be considered when assessing the risk of the 
122  manufacturing change.  In particular, you should carefully assess risks to product quality 
123  if extensive manufacturing changes are introduced shortly before BLA submission.  In 
124  such a situation, a comparability study should be comprehensive and should provide high 
125  confidence that the change does not adversely impact product quality (section V of this 
126  guidance).  Additionally, introducing a manufacturing change at this late stage of 
127  development or after licensure could require additional process performance qualification 
128  studies if the existing qualification study is not representative of the intended commercial 
129  process (e.g., 21 CFR 211.22, 211.100, 211.110(a) and 211.165).  For a process that has 
130  already been validated, you should also determine whether there is a need for any 
131  changes to the plans for continued process verification as a result of the manufacturing 
132  change (Ref. 7).  For these reasons, we recommend that any extensive manufacturing 
133  changes be introduced prior to initiating clinical studies that are intended to provide 
134  evidence of safety and effectiveness in support of a BLA. 
135   
136  To facilitate manufacturing changes during rapid clinical development, CGT product 
137  manufacturers should ensure that the pace of product development is aligned with the 
138  stage of clinical development.  For example, if you initiate clinical studies using product 
139  generated by a manufacturing process designed with a potential for scalability, this will 
140  help decrease the likelihood of delays later in clinical development when the 
141  manufacturing process is scaled up. 
142   
143  For both investigational products subject to 21 CFR part 211 and licensed products, you 
144  must evaluate data at least once a year to determine if changes in product specifications 
145  or manufacturing or control procedures are needed to maintain the quality standards of 
146  the product, even when no manufacturing changes are undertaken (21 CFR 210.2, 
147  211.180(e) and 601.2(d)).  Data trend analysis throughout product development can also 
148  be useful for verifying that manufacturing changes do not lead to shifts in manufacturing 
149  consistency over time. 
150   
151  B.  Stability and Delivery Device Compatibility 
152   
153  Product stability may be adversely affected by manufacturing changes, including changes 
154  made during processing, holding steps for intermediates, and shipping or storing the drug 
155  substance (DS) or DP.  CGT products are often sensitive to storage and handling 
156  conditions. DP stability should be thoroughly assessed after changes to the container 
157  closure system, formulation, product concentration, or shipping conditions. 
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158  Manufacturing changes to CGT products may also have the potential to affect 
159  compatibility of the DP with delivery devices.  
160   
161  When evaluating the risk of a manufacturing change, we recommend that you determine 
162  if there is a need to perform stability and/or delivery device compatibility studies to 
163  assess the effect of the change on product quality, and whether any such studies should 
164  evaluate in-process material, DS, or DP.  Stability studies should focus on the evaluation 
165  of stability-indicating quality attributes.  The stability testing plan should define 
166  appropriate acceptance criteria, which may be different from the acceptance criteria for 
167  release of the product.  
168   
169  Many CGT products are stored frozen for a significant length of time.  Accelerated stability 
170  studies performed under stress conditions may be useful for identifying stability-indicating 
171  attributes, but shelf life should be based on real-time stability data obtained at the long-term 
172  storage condition.  Generating real-time long-term stability data can delay product 
173  development, especially when manufacturing changes that have the potential to adversely 
174  affect stability are implemented during late stages of product development.  For post-licensure 
175  manufacturing changes, there may be a need to generate real-time stability data with the post-
176  change product to demonstrate a lack of adverse effect on product quality, and generating these 
177  data could severely delay the implementation of the manufacturing change. 
178   
179  C.  Nonclinical studies 
180   
181  Nonclinical studies may be needed to support manufacturing changes for an 
182  investigational product after clinical studies have been initiated (Ref. 8), or for a licensed 
183  product (21 CFR 601.12(a)(2)).  If analytical studies alone are insufficient to determine 
184  the impact of the manufacturing changes on CGT product quality, then nonclinical 
185  studies may contribute to a demonstration of comparability.  
186   
187  D.  Clinical studies 
188   
189  We recommend that comparability of investigational or licensed CGT products be 
190  evaluated through analytical assessment and, if appropriate, nonclinical studies.  When 
191  applicable and feasible, studies evaluating pharmacokinetic/pharmacodynamic (PK/PD) 
192  parameters may be used to contribute evidence in support of comparability between the 
193  pre- and post-change products.  When comparability cannot be established through 
194  analytical, nonclinical, and/or PK/PD studies, the evidence of safety and effectiveness 
195  accumulated during clinical investigation with the pre-change product will be insufficient 
196  to support a BLA for the post-change product, and the sponsor should contact FDA to 
197  discuss plans for additional clinical investigations of the safety and/or effectiveness of the 
198  post-change product. 
199   
200  Investigational Products  
201   
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202  If analytical and/or nonclinical comparability studies are insufficient to assure that a 
203  manufacturing change will not adversely affect safety, then the sponsor should discuss 
204  with the FDA (section VII of this guidance) their plans for safety evaluation of the post-
205  change product, which may include conducting new clinical studies and/or incorporating 
206  additional safeguard measures and safety evaluations in ongoing clinical studies.  For 
207  example, it may be appropriate to consider broadening the scope of the adverse events of 
208  special interest, staggering enrollment of subjects, modifying study stopping rules, and 
209  conducting additional dose-finding studies. 
210   
211  If comparability studies demonstrate that the manufacturing change does not adversely 
212  affect product safety but are insufficient to exclude an adverse impact on product 
213  effectiveness, then the sponsor will need to evaluate the effectiveness of the post-change 
214  product in clinical studies to support a BLA for the post-change product. 
215   
216  It is important to critically evaluate any manufacturing change that has the potential to 
217  affect product effectiveness when the change is proposed after initiation of studies 
218  intended to provide substantial evidence of effectiveness in support of a BLA.  In 
219  addition, evidence demonstrating a prospect of direct benefit of a pre-change 
220  investigational CGT product to pediatric subjects, as required for studies conducted in 
221  accordance with 21 CFR 50.52, may not be adequate to demonstrate prospect of direct 
222  benefit with respect to the post-change product.  If comparability cannot be established 
223  between the pre- and post-change product, the sponsor should discuss with the FDA 
224  (section VII of this guidance) any proposed modifications to the clinical development 
225  program for the post-change product.  Such modifications could include an increase in 
226  the number of subjects exposed to the post-change product and initiation of new clinical 
227  studies with the post-change product.  In the case of pediatric studies for which a prospect 
228  of direct benefit is required, nonclinical data demonstrating prospect of benefit may be 
229  sufficient during early-stage clinical development. 
230   
231  If you wish to pool clinical data from subjects treated with the post-change product and 
232  subjects treated with the pre-change product, you should demonstrate that the products 
233  are comparable and justify that the clinical study designs are appropriate for pooling.  We 
234  also recommend that you seek FDAs advice (section VII of this guidance) on the design 
235  of the pooled data analysis, preferably before conducting late-phase studies intended to 
236  demonstrate product effectiveness in support of a BLA.  
237   
238  Licensed Products 
239   
240  If analytical and/or nonclinical comparability studies are unable to demonstrate that a 
241  manufacturing change to a licensed product has no adverse effect on product quality, 
242  FDA will not be able to approve the manufacturing change based on those studies (21 
243  CFR 601.12).  In such cases, we recommend that you discuss alternative approaches with 
244  the FDA (section VII of this guidance), which will be evaluated on a case-by-case basis. 
245  For example, you may consider initiating new clinical studies with the post-change 
246  product under an IND to obtain evidence of its safety and effectiveness.  
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247  IV.  REGULATORY REPORTING OF MANUFACTURING CHANGES  
248   
249  IND sponsors must notify FDA of manufacturing changes through an amendment if 
250  manufacturing information previously submitted no longer accurately reflects the current state of 
251  manufacturing because essential information is missing (21 CFR 312.31(a)(1)).  Applicants must 
252  notify FDA of manufacturing changes through a BLA supplement or annual report in accordance 
253  with 21 CFR 601.12 (Ref. 6).  When submitting an IND amendment or a BLA supplement for a 
254  manufacturing change, your cover letter should clearly describe the purpose of the amendment 
255  and highlight proposed changes (Ref. 9).  For amendments containing extensive changes, we 
256  recommend that you provide a Reviewers Guide or a comprehensive summary of the changes 
257  in Common Technical Document (CTD) sections 1.2 or 1.11.1, respectively.7  Module 3 and any 
258  other relevant sections of the IND or BLA should be modified to include the change, and the 
259  developmental history of the manufacturing process should be updated in the pharmaceutical 
260  development sections (3.2.S.2.6 and 3.2.P.2.3) of your IND or BLA.  The type of submission, 
261  timing of submission, and amount of information required in the submission will vary depending 
262  on the stage of product and clinical development and the nature of the manufacturing changes, as 
263  described further below.  
264   
265  A.  CMC Changes Requiring a New IND Submission 
266   
267  Some changes can fundamentally alter the design or nature of the product, resulting in a 
268  new product.  Initiation of clinical studies with the new investigational product generally 
269  requires the submission of a separate IND (21 CFR 312.20).  We recommend that you 
270  seek FDA advice (section VII of this guidance) regarding any manufacturing changes that 
271  could alter the product and require a new IND.  Some examples of changes that may 
272  require a new IND include: 
273    Change in the cellular starting material of a cellular product (e.g., allogeneic vs. 
274  autologous donor; adipose-derived cells vs. umbilical cord-derived cells) 
275    Change to the types of cells in a cellular product (e.g., mixture of CD4+ and CD8+ 
276  T cells instead of solely CD4+ T cells) 
277    Change to the scaffold or matrix component of the final construct in a TEMP 
278  (e.g., changes to chemical or physical properties) causing significant modification 
279  to the product characteristics 
280    Change in a viral vector capsid or envelope that changes the tropism or serotype 
281  of a viral vector used for in vivo gene therapy 
282    Change to the sequence of a transgene or addition of a transgene (e.g., changes to 
283  the intracellular signaling domain of a chimeric antigen receptor) 
284    Change in expression control elements of a viral vector (e.g., change from a 
285  tissue-specific to a ubiquitous promoter) 
286    Change of target gene for genome editing products, including addition of a target 
287  gene 
 
7 For information on electronic CTD (eCTD) submission requirements, please see the FDA website 
https://www.fda.gov/drugs/electronic-regulatory-submission-and-review/electronic-common-technical-document-
ectd. 
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288  B.  Reporting Manufacturing Changes to an IND 
289   
290  FDA regulations require all sponsors of investigational new drug products, including 
291  investigational CGT products, to describe the CMC information for the DS (21 CFR 
292  312.23(a)(7)(iv)(a)) and the DP (21 CFR 312.23(a)(7)(iv)(b)).  The CMC information in 
293  your IND must be sufficient to assure the safety, identity, quality, purity, and strength 
294  (including potency) of the investigational product (21 CFR 312.23(a)(7)(i)).  The CMC 
295  information in an IND describes a sponsors commitment to perform manufacturing and 
296  testing of the investigational product as stated in the IND or in a cross-referenced IND or 
297  master file.  If a manufacturing change could affect product quality, we consider the 
298  manufacturing change essential information that must be submitted in an information 
299  amendment to the IND (21 CFR 312.31(a)(1)).  The sponsor should submit such 
300  amendments for FDA review prior to use of the changed product in clinical 
301  investigations.  The FDA will review data or study reports submitted to support the 
302  change, and may provide comments (section V of this guidance).  In addition, each year 
303  you must submit an annual report that provides a summary of any significant 
304  manufacturing changes made during the past year (21 CFR 312.33(b)(7)).  
305   
306  If a manufacturing change has the potential to adversely affect safety, and if you do not 
307  submit evidence to your IND demonstrating that the post-change product has an 
308  acceptable safety profile, then your IND may be placed on clinical hold at any phase of 
309  clinical development (21 CFR 312.42(b)(1)(i), 21 CFR 312.42(b)(1)(iv), and 21 CFR 
310  312.42(b)(2)(i)).  Evidence may be provided as an amendment to the IND in the form of 
311  analytical comparability data or other analytical data relevant to safety.  If these data do 
312  not allow for a conclusive determination that the manufacturing change has no adverse 
313  effect on product quality as it relates to safety, then you should consider performing a 
314  toxicology study to evaluate whether the post-change product has an acceptable safety 
315  profile. 
316   
317  If you make a manufacturing change that has the potential to adversely impact the 
318  effectiveness of the product without submitting evidence to your IND demonstrating that 
319  the post-change product is comparable to the pre-change product, this may also result in a 
320  clinical hold for certain clinical studies (21 CFR 312.42(b)).  FDAs review of an IND 
321  submission for a phase 2 or 3 clinical study includes assessing the likelihood that the 
322  study will yield data capable of meeting statutory standards for marketing approval (21 
323  CFR 312.22(a)), and a phase 2 or 3 study may be placed on clinical hold if the plan or 
324  protocol for the study is clearly deficient in design to meet its stated objectives (21 CFR 
325  312.42(b)(2)(ii)).  If, for example, a phase 3 study intended to provide substantial 
326  evidence of effectiveness to support a BLA for a post-change product uses lots of both 
327  pre- and post-change product, but those products are not comparable, then the study may 
328  lack statistical power to demonstrate effectiveness of the post-change product.  Such a 
329  study may be considered clearly deficient in design to meet its stated objectives and 
330  placed on clinical hold if the IND submission does not provide evidence demonstrating 
331  comparability of the pre- and post-change products.  
332   
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333  In addition, FDA may place studies on clinical hold if subjects would be exposed to an 
334  unreasonable and significant risk of illness or injury (21 CFR 312.42(b)(1)(i) and 
335  312.42(b)(2)(i)).  If you make a manufacturing change that could adversely affect the 
336  effectiveness of the investigational product without demonstrating comparability, then the 
337  capacity of the post-change product to provide a potential benefit to subjects may be in 
338  doubt.  This may lead to a conclusion that a significant risk of illness or injury involved 
339  in a clinical investigation is unreasonable, and the study may be placed on clinical hold.   
340   
341  C.  Reporting Manufacturing Changes to a BLA 
342   
343  For licensed products, you must report each change in the product, production process, 
344  quality controls, equipment, facilities, responsible personnel, or labeling established in 
345  the approved license application, in accordance with the requirements in 21 CFR 601.12. 
346  When reporting these changes, your supplement or annual report should include a risk 
347  assessment report and must include data from appropriate studies performed to evaluate 
348  the effect of the changes on product quality as required under 21 CFR 601.12(b)(3)(iv)-
349  (v), 21 CFR 601.12(c)(3), or 21 CFR 601.12(d)(3)(ii) (Ref. 6).   
350   
351  To facilitate management of post-approval manufacturing changes, you may submit one 
352  or more comparability protocols to your BLA for FDA review, as described in 21 CFR 
353  601.12(e).  These protocols may be submitted either in the original BLA or, if the 
354  application is already approved, in a prior approval supplement (Ref. 10).  Comparability 
355  protocols should be located in section 3.2.R of your BLA.  Upon approval, this protocol 
356  becomes an agreed-upon plan for implementation of the manufacturing change using the 
357  reporting category specified in the approved comparability protocol submitted under 21 
358  CFR 601.12(e), provided that there is successful completion of the plan for 
359  implementation of the change(s) as described in the comparability protocol (including 
360  achievement of all of the predefined acceptance criteria for success in the approved 
361  comparability protocol) (Ref. 10). 
362   
363   
364  V.  COMPARABILITY ASSESSMENT AND REPORT 
365   
366  Comparability between the pre-change and post-change products is generally demonstrated by 
367  evidence that the change does not adversely affect product quality for the licensed (21 CFR 
368  601.12(a)(2)) or investigational product.  However, if the change is intended to improve product 
369  quality, such that there is a significant benefit in effectiveness and/or safety, then the post-change 
370  product may be considered a different product, and therefore not comparable to the pre-change 
371  product.  We recommend that you seek FDA advice (section VII of this guidance) when planning 
372  significant manufacturing changes and when designing study protocols for comparability studies. 
373  Section V of this guidance describes considerations for designing a comparability study, 
374  analyzing comparability data, and submitting a comparability study report.  For information on 
375  reporting manufacturing changes to FDA, please refer to sections IV.B of this guidance for 
376  reporting changes to an IND and section IV.C of this guidance for reporting changes to a BLA. 
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377  When submitting a comparability study report to an IND or BLA, you should include a cover 
378  letter or reviewers guide outlining the submission contents to streamline the FDA review 
379  process.  In the cover letter or reviewers guide, you should provide a description of the proposed 
380  change, rationale for the proposed change, proposed timeline for implementing the change, and 
381  justification for the design of the comparability study.  Further, to aid FDA review of your study, 
382  we recommend that you provide a short summary of your current relevant manufacturing and 
383  clinical experience.  When submitting a comparability study report to your IND, for example, it 
384  is helpful to describe the stage of clinical development, the number of subjects to whom the pre-
385  change product will be administered, and the number of subjects expected to receive the post-
386  change product.  You should provide a summary of relevant previous manufacturing changes and 
387  their effect on process consistency and product quality.  You should also note any previous 
388  changes made to product specifications (for DP, DS, and key intermediates) and provide a 
389  description of any CQAs for which an analytical method is still under development.  
390   
391  Comparability study reports should be submitted to CTD sections 3.2.S.2.6 or 3.2.P.2.3 of the 
392  BLA or IND, as appropriate.  Your comparability study report should evaluate the totality of the 
393  comparability data, including historical manufacturing data, to determine if the pre- and post-
394  change products are comparable.  We recommend that you summarize the findings of the 
395  comparability study and discuss how the data and analyses support your conclusion from the 
396  study.  You should also include a discussion of any potential limitations of the study.  If a 
397  product quality attribute does not meet the pre-defined acceptance criterion for comparability, 
398  but you still consider the pre- and post-change products to be comparable, you should provide 
399  justification and/or additional scientific information to support your conclusion for FDA review.  
400   
401  A.  Risk Assessment  
402   
403  Manufacturing changes that can present potential risk to product quality include, but are 
404  not limited to, changes to the manufacturing site, manufacturing process, materials, 
405  container closure, testing, storage, and shipping conditions.  To evaluate whether the 
406  proposed manufacturing change may impact product quality, you should conduct a 
407  detailed risk assessment as recommended in International Council for Harmonisation 
408  (ICH) Q9 dated June 2006 (Ref. 1).  The process of evaluating the risk of a 
409  manufacturing change for a CGT product is similar to risk evaluation for other types of 
410  drugs, and the same tools can generally be applied.  
411   
412  We recognize that risk assessment for changes to the manufacturing of CGT products 
413  may be more challenging than for other product types because the effects of 
414  manufacturing changes are often difficult to predict for these complex products.  For 
415  example, manufacturing changes may unexpectedly alter product purity (increase 
416  process-related impurities, cellular impurities, aggregates, or particulates), reduce product 
417  stability, or change product potency.  
418   
419  Transferring a manufacturing process to a new manufacturing facility is generally 
420  considered a major change that may require extensive comparability evaluation in 
421  addition to technology transfer, because it may involve changes to the manufacturing 
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422  process, shipping, manufacturing equipment, testing equipment, and operators. 
423  Performing a thorough risk assessment, including consideration of method equivalence 
424  and CPPs, is essential when transferring a manufacturing process to a new facility. 
425    
426  Your risk assessment should consider potential impacts of the change on the 
427  manufacturing steps and in-process parameters that are downstream of the manufacturing 
428  change, as well as the impact on the product.  We recommend that you take a stepwise 
429  approach to select all quality attributes and process parameters to be evaluated in a 
430  comparability study; first, you should determine which attributes might be affected by the 
431  particular change, and then you should assign a score to each attribute based on the 
432  probability, severity, and detectability of the risk.  The assigned score can be used to 
433  determine the overall risk for each attribute.  Manufacturing changes that are determined 
434  to have a high risk to product quality should be supported by an extensive analytical 
435  comparability study, while it may be possible to evaluate low-risk changes using a more 
436  focused approach. 
437   
438  You should consider whether your risk assessment is constrained by gaps in product 
439  knowledge related to the type of change being proposed.  Gaps in knowledge typically 
440  raise the level of risk and may necessitate a more extensive comparability study.  Please 
441  note that relying solely on established release tests and in-process controls is generally 
442  insufficient to assess the impact of manufacturing changes.  Therefore, we recommend 
443  that you consider the potential impact of manufacturing changes on quality attributes that 
444  are not routinely evaluated by established release tests and process controls, and consider 
445  additional characterization studies as appropriate.  Additionally, your risk assessment 
446  should evaluate whether more than one analytical method should be used to evaluate a 
447  particular attribute.  Such an approach could be useful for high-risk attributes, particularly 
448  with respect to assessment of potency, as described in section V.B of this guidance.  In 
449  your risk assessment, you should justify how the selected quality attributes and process 
450  parameters can be used to comprehensively evaluate the potential effect of the change on 
451  product quality. 
452   
453  Your risk assessment should also inform the statistical approach to comparability.  
454  Higher risk attributes typically warrant a more stringent statistical analysis than lower 
455  risk attributes.  Side-by-side or graphical presentations (such as dot plot) to allow visual 
456  comparison, in lieu of statistical analysis, may be sufficient for characterization of 
457  attributes at low risk of being impacted by a manufacturing change. 
458   
459  It is important to note that a manufacturing change may affect product stability even if 
460  the change has no other effect on product quality or process performance.  As discussed 
461  in section III.B, you should assess the potential risk to product stability and delivery 
462  device compatibility. 
463   
464  Finally, if multiple changes are to be implemented simultaneously, we recommend that 
465  you assess the risk of each individual change and the potential cumulative effect of the 
466  changes on product quality.  It may be possible to evaluate these multiple changes under 
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467  a single comparability study.  However, if you fail to demonstrate comparability in this 
468  single study, it will likely be difficult to identify which of the changes caused an adverse 
469  effect on product quality.  
470   
471  B.  Analytical Comparability Study Design 
472   
473  It is essential that a comparability study be sufficiently robust to reach a definitive 
474  conclusion regarding comparability.  Therefore, it is important to carefully select relevant 
475  quality attributes, analytical methods, acceptance criteria, and statistical methods.  Prior 
476  to conducting a comparability study for a CGT product that is licensed or being studied 
477  under an IND, we recommend that you submit a detailed study protocol (comparability 
478  protocol) and request feedback from the FDA (section VII of this guidance) on the study 
479  design and statistical approach.  As noted above, the regulations also provide for 
480  applicants to submit and seek FDA approval of a comprehensive, prospectively written 
481  plan for assessing the effect of a proposed post-approval manufacturing change(s) on 
482  product quality (21 CFR 601.12(e) and Ref. 10).  These comparability protocols can be 
483  submitted in an original BLA or in a prior approval supplement (21 CFR 601.12(e)).  
484   
485  The extent of a comparability study should be driven by the conclusions from the risk 
486  assessment, which should inform your selection of:  1) a relevant set of quality attributes 
487  to measure the effect of the manufacturing change on product quality, 2) appropriate test 
488  methods, and 3) comparability acceptance criteria that are adequate to demonstrate a lack 
489  of adverse effect of the manufacturing change on product quality, as discussed later in 
490  this section.  To adequately evaluate the impact of the manufacturing change on product 
491  quality, a comparability study will frequently need to include measurement of attributes 
492  that are not routinely used for product release. 
493   
494  We recommend that you consider the following factors when designing a comparability 
495  study: 
496   
497  Selection of product lots for the study 
498   
499  A comparability study should generally be performed using lots that have been 
500  manufactured at full scale.  Experience with smaller scale lots can be used to identify 
501  potential risks to product quality and process controls and to aid the design of a 
502  comparability study.  If it is not feasible to manufacture full-scale lots for the 
503  comparability study, you should perform data-driven risk assessment of CPPs, CQAs 
504  (including potency), and other relevant product characteristics to justify that scaling down 
505  the manufacturing process provides for an adequate evaluation of the effects of the 
506  manufacturing change on product quality.     
507   
508  A comparability study may be designed as a comparison of historical pre-change testing 
509  data to newer data from post-change lots.  Such a study design requires that the analytical 
510  test methods are equivalent across product lots to provide interpretable data.  If analytical 
511  methods have changed over time, retained samples from pre-change lots may need to be 
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512  reanalyzed using the current analytical methods.  You should avoid biased selection of 
513  historical data.  Ideally, the only differences between the historical pre-change lots and 
514  the post-change lots should be the manufacturing changes that are being evaluated in the 
515  comparability study.  If the pre-change product was manufactured using multiple 
516  processes or facilities, comparability should be demonstrated across the pre-change lots 
517  before they are included in a comparability study evaluating a newly proposed change.    
518   
519  For some CGT products, the number of lots may be very small due to, for example, 
520  limited manufacturing for rare disease indications, rapid development timelines during 
521  clinical studies, or difficulty obtaining cellular starting materials from an adequate 
522  number of donors.  An insufficient number of lots could compromise statistical power 
523  and be insufficient to demonstrate comparability, particularly if there is high lot-to-lot 
524  variability, as discussed later in section V.E of this guidance.  
525   
526  Special considerations for products derived from a variable cellular starting material  
527   
528  Cell-based products where each product lot is derived from a different donor often have 
529  product characteristics with very wide ranges due to the inherent variability of the 
530  cellular source materials.  The number of lots that might be used for such products to 
531  perform a statistically valid comparability study could be quite large, or even unfeasible 
532  in some cases.  However, there are study design considerations that may be useful for 
533  decreasing the number of lots included for the comparability study.  We recommend that 
534  you use a split-source study design, whenever possible.  A split-source design limits the 
535  impact of cellular variability by splitting individual cellular source materials into two 
536  equal portions.  One portion of each source material is then subjected to the pre-change 
537  manufacturing conditions, and the other portion is subjected to the post-change 
538  manufacturing conditions.  As described in Comparability acceptance criteria later in 
539  this section, the results obtained from the split runs should meet the in-process and 
540  release specifications and be representative of relevant historical data.  Paired difference 
541  analysis is typically performed.  If a split-source study design is not possible, and it is 
542  already known that CQAs for a specific product and clinical indication can vary within a 
543  wide range without any adverse impact on product quality, then accordingly, it may be 
544  acceptable to set wide acceptance criteria for comparability studies, which would reduce 
545  the number of lots for the study. 
546   
547  When manufacturing cell-based product lots for use in comparability studies, we 
548  recommend using the same type of cellular source material that would normally be used 
549  to manufacture your product.  If this is not feasible due to limited source material or other 
550  justified reasons, it may be appropriate to use small-scale manufacturing runs or 
551  alternative cellular source material.  For example, if patient cells are not available, using 
552  cells from healthy donors could be considered.  If the number of cells from a single donor 
553  is not sufficient to manufacture a large enough lot for the comparability study, it may be 
554  possible to use cells pooled from multiple cell collections from the same or multiple 
555  donors.  In your comparability study report, you should explain why the alternative 
556  cellular source material is relevant, including:  1) whether there are differences in process 
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557  parameters that might occur when using the alternative material, and 2) whether product 
558  quality can effectively be evaluated using the alternative source material.  For example, 
559  for a product consisting of genetically modified cells, healthy donor cells may not be an 
560  appropriate alternative for patient cells, if transduction efficiency is different. 
561  Additionally, in the case of product intended to treat a genetic disease, the lack of the 
562  genetic defect in healthy donor cells may interfere with measurement of potency.  
563   
564  Special consideration for vectors used for ex vivo cell modification  
565   
566  GT vectors8 used for ex vivo cell modification must be manufactured in compliance with 
567  current good manufacturing practices (cGMP) under section 501(a)(2)(B) of the Federal 
568  Food, Drug, and Cosmetic Act (FD&C Act), as appropriate for the stage of development 
569  (Ref. 11).  This should include effective quality risk management and change control 
570  activities (Ref. 1).  Changes to the manufacturing of GT vectors should be carefully 
571  evaluated not only for risks to the quality of the vector and the performance of the vector 
572  manufacturing process, but also for risks to the quality and manufacturing process 
573  performance for the ex vivo gene-modified cells.  
574   
575  Analytical comparability of the vector should typically be evaluated using the vector 
576  release assays (including an assay that measures the biological activity of the vector), as 
577  well as any relevant characterization assays, if appropriate.  In addition, the effect of the 
578  vector manufacturing change on the quality of the ex vivo gene-modified cells (DS 
579  and/or DP) should be evaluated in an analytical comparability study using an adequate 
580  number of vector, DS and/or DP lots.  
581   
582  The number of vector lots available for comparability studies may be small in situations 
583  where each lot of vector is sufficient for the manufacture of large numbers of DP lots.  In 
584  such cases, it may be appropriate for comparability studies to include vector lots that 
585  were manufactured during process development or engineering runs, if manufacture of 
586  these vector lots is similar to the manufacture of the vector lots used to manufacture DP 
587  for clinical studies.  Your risk management strategy should ensure that sufficient vector 
588  lots will be available for future comparability studies because difficulties in 
589  implementing vector manufacturing changes can cause delays in clinical studies or 
590  shortages in licensed products. 
591   
592  Assessment of potency 
593   
594  The biological activity of CGT products can be highly sensitive to manufacturing 
595  changes.  Therefore, we recommend that a quantitative potency assay (Ref. 12) be 
596  included when performing analytical comparability studies.  You may wish to consider 
597  using several analytical methods to evaluate potency if the routinely used analytical 
 
8 For the purposes of this guidance, a vector is defined as a vehicle consisting of, or derived from, biological 
material that is designed to deliver genetic material. Examples include plasmids, viruses, and bacteria that have been 
modified to transfer genetic material.  (Long Term Follow-Up After Administration of Human Gene Therapy 
Products; Guidance for Industry; January 2020, at 29, available at https://www.fda.gov/media/113768/download).  
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598  method is imprecise or unable to assess all aspects of the products MOA that might be 
599  affected by the manufacturing change.  For some products, animal models may be used to 
600  supplement a relevant quantitative assay(s) to demonstrate that the product has the 
601  desired biological effect and to provide supportive evidence for comparable biological 
602  activity of the pre-change and post-change product.  
603   
604  CGT products may have multifaceted mechanisms of action due to, for example, product 
605  complexity, the presence of multiple active ingredients, and complex PK/PD profiles. 
606  Assays that measure relevant biological activities of CGT products are challenging to 
607  develop, and these assays are often inherently variable.  These difficulties can delay 
608  establishing a potency assay and release acceptance criteria until later-stage clinical 
609  studies because the relationship between the products MOA and safety and effectiveness 
610  may not be well understood.  Yet, exclusion of potency analysis from a comparability 
611  evaluation compromises the conclusions drawn from a comparability study.  To avoid 
612  this situation, we recommend that samples be retained from all lots to facilitate future 
613  analysis of potency to support comparability.  
614   
615  When establishing an acceptance criterion for potency in comparability studies, you 
616  should consider that product quality may be adversely affected not only by a significant 
617  decrease in potency, but also if there is a significant increase in potency.  A 
618  manufacturing change that significantly increases potency, even if intentional, may raise 
619  safety concerns.  In such cases, if you are unable to demonstrate that the change will not 
620  adversely affect safety, the post-change product will not be considered comparable to the 
621  pre-change product. 
622   
623  Comparability acceptance criteria 
624   
625  It is not necessary for the measurements of pre- and post-change CQAs to be identical to 
626  reach a conclusion of comparability if there is evidence demonstrating that there is no 
627  adverse impact of the change on product quality.  A comparability acceptance criterion 
628  should be defined prior to initiating the comparability study for each CQA determined, 
629  through risk assessment, to have a potential to be impacted by the change.  For 
630  quantitative attributes, a comparability acceptance criterion may be defined as the largest 
631  acceptable difference between the pre-change and post-change attribute (an equivalence 
632  margin) or as an acceptable range for the post-change attribute (a quality range).  In 
633  addition to meeting the comparability acceptance criteria, lots used in comparability 
634  studies should also meet the established in-process and release acceptance criteria, and, 
635  unless otherwise justified, the results should be representative of data (e.g., mean, 
636  standard deviations, median) from relevant pre-change historical lots.  
637   
638  An equivalence approach is often appropriate for evaluating comparability of CQAs 
639  when it is important to directly compare the pre- and post-change values and determine 
640  whether they are sufficiently similar.  For normally distributed data, the equivalence 
641  margin should be defined as the maximum acceptable difference in population means. 
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642  Exceeding this margin would be interpreted as an adverse effect of the post-change 
643  manufacturing process on product quality. 
644   
645  A quality range approach evaluates whether the post-change quality results fall within a 
646  defined range.  This range should often be narrower than the release acceptance criteria 
647  for those same quality attributes.  The quality range approach can potentially be used for 
648  attributes with various risk levels, but higher-risk attributes should be evaluated using the 
649  more rigorous equivalence approach.  The number of post-change lots sufficient for a 
650  comparability study when using the quality range approach will depend on the totality of 
651  evidence supporting the lack of adverse effect of the change on product quality.  For 
652  example, if additional relevant data from other studies (such as impurity clearance studies 
653  or other process characterization studies) provide evidence that the manufacturing change 
654  does not have an adverse effect on a particular quality attribute, then this may justify the 
655  use of a smaller number of post-change lots in the comparability study.  Otherwise, you 
656  should ensure that the comparability study is designed with sufficient power by 
657  calculating the number of post-change lots needed to demonstrate with high confidence 
658  that an appropriate proportion of future lots will fall within the quality range. 
659   
660  Regardless of the approach used, comparability acceptance criteria should ideally be 
661  based on understanding the potential effect of the attribute on the safety and effectiveness 
662  of the product, and not based solely on statistical analysis of historical data from the pre-
663  change product.  If there is clinical or manufacturing experience supporting the 
664  differences in CQAs that negatively and/or positively impact product quality, you should 
665  use this information to select appropriate quality ranges or equivalence margins for your 
666  comparability study.  If instead you are using statistical analysis of historical data to 
667  define comparability acceptance criteria (e.g., based on standard deviation), you should 
668  justify how your statistical-based acceptance criteria are adequate to ensure the safety and 
669  effectiveness of the post-change product (i.e., justify how your statistical-based parameter 
670  is relevant to a biologically meaningful difference).  
671   
672  Please refer to section V.E of this guidance regarding statistical analysis of comparability 
673  study results.  
674   
675  C.  Analytical Methods 
676   
677  Interpretation of comparability test results depends on the suitability of the analytical 
678  methods used.  For example, using an imprecise, insensitive, or inconsistent method in a 
679  comparability study can invalidate the conclusions of the study.  We recommend that you 
680  provide a tabular listing of the analytical methods and testing sites used in the 
681  comparability study.  If method descriptions, qualification studies, or validation studies 
682  are provided elsewhere in your application, you may refer to them.  For comparability 
683  studies of investigational products, all release assays used to demonstrate comparability 
684  should be qualified or validated, depending on the phase of clinical study.  Assays used 
685  for extended characterization do not necessarily need to be qualified, but they should be 
686  scientifically sound and fit for their intended use, be sufficiently precise to detect 
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687  meaningful differences in product quality, and provide results that are reliable.  If not 
688  described elsewhere, you should describe sample acquisition (e.g., process step, sample 
689  volume, storage temperature) and justify any differences in acquiring samples from the 
690  pre-change and post-change manufacturing processes.  
691   
692  FDA has issued guidance providing general guiding principles to assist applicants with 
693  assay validation (Refs. 13, 14). Some of the challenges with validation of assays for CGT 
694  products are highlighted below: 
695   
696  Analytical Method Precision 
697   
698  Small changes in an attribute can sometimes have a profound impact on the quality of 
699  CGT products.  However, measuring such small changes can be challenging when the 
700  analytical methods are not precise.  Therefore, it is especially important that the 
701  analytical methods used to assess the effect of manufacturing changes on product quality 
702  and process control are sufficiently precise.  For example, if a 5% change in a particular 
703  cell marker represents a meaningful difference in product quality, then a flow cytometry 
704  assay with an intermediate precision of 20% coefficient of variation would not be 
705  adequate for evaluating whether there is a meaningful difference in that attribute between 
706  the pre-change and post-change products. 
707   
708  Consistent Method Performance 
709   
710  Analytical methods are often changed, added, or transferred to a new facility over the 
711  course of a CGT product lifecycle because of advancing technology and/or increasing 
712  understanding of MOA.  To provide the most readily interpretable data for a 
713  comparability study, we recommend that you perform side-by-side testing9 of pre-change 
714  and post-change product attributes or analyze all samples using the same analytical 
715  method performed at the same testing facility.  Reference material should also be used, if 
716  available.  
717   
718  At all stages of the product lifecycle, when changing an assay or transferring an assay to 
719  a new testing facility, you should perform a risk assessment for the assay change to 
720  determine if there is a potential impact on evaluation of product quality, including 
721  evaluations conducted in comparability studies.  For example, a change to an ELISA kit 
722  from a manual to an automated method could result in meaningful differences in 
723  sensitivity or precision.  The equivalence of the old and new assays should be evaluated 
724  by testing identical samples with each assay.  Similarly, when using multiple facilities to 
725  perform the same assay, a method transfer study should be performed to ensure 
726  reproducibility, and the assays should include identical samples or common reference 
727  materials to ensure consistent assay readouts.  Additional assay qualification or validation 
728  may also be warranted after transferring an assay to a new facility (Ref. 13). 
 
9 In this guidance, side-by-side testing, also often referred to as head-to-head testing, means testing of the pre- and 
post-change samples in the same experiment. 
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729   
730  D.  Results  
731   
732  For each product attribute and process parameter assessed, we recommend that the results 
733  for each lot and the corresponding lot numbers be provided in a tabular format, together 
734  with tables that list summary statistics for the data alongside the predefined study 
735  acceptance criteria.  When appropriate, we recommend that you also display data in a 
736  graphical format.  We recommend that you describe and analyze any differences in the 
737  study data between the pre-change and post-change products.  Any deviations from pre-
738  established procedures should be described and justified. 
739   
740  E.  Statistics 
741   
742  When designing comparability studies for CGT products, appropriate statistical methods 
743  should be used to determine if the pre- and post-change products are comparable.  The 
744  statistical methods should be defined in the comparability protocol before executing the 
745  comparability study.  Selection of a statistical approach to demonstrate comparability of 
746  pre- and post-change products can be challenging when there are only a limited number 
747  of samples, when quality attributes are highly variable, or when the data is not normally 
748  distributed.  
749   
750  We recommend that you consult with a statistician before discussing the study design and 
751  statistical approach with FDA.  In general, there could be multiple appropriate statistical 
752  methods that may be used to evaluate whether data from the post-change product are 
753  within predetermined acceptable limits.  To avoid errors in the design and analysis of 
754  comparability studies, a careful consideration of fundamental statistical concepts is 
755  important.  For example: 
756   
757    Some statistical methods may be inappropriate for a given comparison due to 
758  invalid assumptions, a need for a very large number of samples, high variability in 
759  sample data, or limited information about the population distribution.  For 
760  example, parametric tests that assume a normal population distribution should not 
761  be used if the data are not normally distributed.  When justified, data 
762  transformation could be useful to meet the assumption of data normality.  You 
763  should describe the statistical method, justify the assumptions of the statistical 
764  approach, justify the acceptance criteria selected, and discuss limitations. 
765  Different statistical methods may be used within the same study to analyze 
766  different CQAs, if the CQAs differ in their underlying distribution (e.g., normal 
767  vs. binomial).  
768   
769    The variability of a statistic is determined by the spread of its sampling 
770  distribution.  Having only a small number of lots can lead to greater sampling 
771  variability, which can significantly reduce the statistical power.  Therefore, the 
772  appropriate number of lots should be considered early, as the lack of sufficient 
773  numbers of samples may impede comparability analysis and implementation of 
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774  manufacturing changes, especially during late-stage development and after 
775  licensure.  
776   
777    As described in section V.C of this guidance, it can be difficult to evaluate the 
778  comparability of an attribute when using an assay that has poor precision.  In such 
779  situations, an alternative to improving the precision of the assay would be to 
780  reduce measurement uncertainty by performing the assay multiple times 
781  independently for each lot and reporting the mean value.  Such an approach will 
782  improve the statistical power of the comparability analysis for that attribute.  It is 
783  important to note that the mean of the assay results for each lot should be treated 
784  as a single data point when analyzing comparability; it is inappropriate to treat 
785  each individual assay result as an independent data point in the comparability 
786  analysis. 
787   
788    For studies that compare two cellular manufacturing processes using split-donor 
789  starting material, the product data from each donor are paired.  In such cases, you 
790  should select a statistical test suitable for analysis of the difference between paired 
791  data, rather than a test that assumes an independent data structure. 
792    The absence of a statistically significant difference between the pre- and post-
793  change products (e.g., p-value > 0.05) does not demonstrate comparability.  For 
794  example, using a two-sample t-test is not appropriate for comparability claims 
795  when the null hypothesis is that the means of CQAs of pre- and post-change 
796  products are equal, and the alternative hypothesis is that they are different.  In 
797  other words, failing to reject this null hypothesis is not the same as showing 
798  equivalence.  
799   
800    To evaluate equivalence, you may consider calculating an appropriate confidence 
801  interval for the difference between the pre- and post-change data, and conclude 
802  equivalence if this confidence interval is within the equivalence margin.  When 
803  the CQA of interest is a mean value, you may consider using the Two-One-Sided 
804  Tests procedure (TOST) or other appropriate statistical method to establish 
805  comparability.  For some attributes (e.g., impurity, viability), it may be possible to 
806  demonstrate that the manufacturing change has no adverse effect on product 
807  quality using a one-sided statistical comparison, such as non-inferiority testing or 
808  other appropriate method.  
809   
810    If the lots selected for the comparability study are not representative of your 
811  typical manufacturing process, the corresponding results will have limited 
812  meaningful interpretation, regardless of the particular statistical methodology 
813  applied.  You should justify your selection of comparability lots and (if 
814  applicable) the cellular source material used to produce those lots. 
815   
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816  VI.  SPECIAL CONSIDERATIONS FOR TISSUE-ENGINEERED MEDICAL 
817  PRODUCTS 
818   
819  Tissue-engineered medical products (TEMPs)10 commonly incorporate viable cells and 
820  scaffolds, with cells either seeded onto the scaffolds surface or embedded within the scaffold.  
821  Oftentimes, TEMPs are intended to mimic the in vivo cellular microenvironment.  Although 
822  manufacturers are gaining experience with these products, there is generally still limited 
823  understanding regarding product quality, interactions between the cells and scaffolds in vitro 
824  (e.g., maturation), interactions of the DP with the host environment (e.g., remodeling), and 
825  sensitivity of TEMPs to manufacturing changes.  For these reasons, manufacturing changes to 
826  TEMPs pose additional unique challenges, as changes may impact the cells, the scaffold and/or 
827  the combined cell-scaffold product in ways that are not readily anticipated or detectable based on 
828  current measurement technologies.  
829   
830  We recommend that you conduct a thorough risk assessment that considers the potential effects 
831  of the change on each component (e.g., cells, scaffold) and on the final cell-scaffold construct. 
832  The risk assessment should determine whether a comparability study is necessary to evaluate any 
833  potential impact of the change on product quality and whether this comparability study should 
834  evaluate the cells, scaffold, cell-scaffold intermediate(s), and/or the cell-scaffold DP.  
835   
836  When assessing manufacturing changes to TEMPs, you should consider scaffold characteristics, 
837  including but not limited to the scaffold source (e.g., natural or synthetic), density, shape, 
838  mechanical and physicochemical properties, interactions with cytokines and growth factors, and 
839  capacity for inducing cell signaling pathways (e.g., via mechanotransduction).  Similarly, you 
840  should consider relevant cell characteristics, including but not limited to cell morphology, 
841  density, aggregation, growth, viability, and the relevant biological function(s) for the proposed 
842  specific indication.  Both manufacturing changes introduced before combining the cells and 
843  scaffold and manufacturing changes introduced after combining the cells and scaffold (e.g., 
844  changes to the culture conditions, packaging, storage or shipping) may have a significant impact 
845  on the overall biological activity and/or performance of the TEMP.  Therefore, comparability 
846  studies for TEMPs should often include evaluation of the effect on DP quality even when 
847  manufacturing changes are made only to the scaffold or to the cells prior to combining these two 
848  components.  
849   
850  Furthermore, certain changes may have a significant impact on how the DP behaves after 
851  administration in terms of safety and performance, and therefore on product quality.  You 
852  should, therefore, assess the potential impact of the change on product quality 
853  post-administration (e.g., remodeling, degradation).  Depending on the outcome of the risk 
854  assessment, you may need to evaluate the performance of the TEMP in a physiologically 
855  relevant environment to demonstrate comparability.  This may involve additional nonclinical 
856  studies and/or clinical studies. 
857   
 
10 For the purposes of this guidance, TEMPs are limited to products that consist of living cells combined with a 
scaffold or substrate regulated under section 351 of the PHS Act. 
20  Contains Nonbinding Recommendations 
 
Draft  Not for Implementation 
 
858  In general, the need to maintain the integrity and structure of TEMPs may make it difficult to 
859  acquire samples for testing and retention.  In addition, products that are manufactured in a closed 
860  system, such as a bioreactor, could pose additional practical challenges to acquiring samples. 
861  Further, the seeding and growth of cells on the scaffold may not be uniform, making it difficult 
862  to obtain representative samples.  Therefore, it is important to consider these unique challenges 
863  in the context of comparability study design, if relevant, surrogate11 TEMPs could be 
864  manufactured in parallel during clinical lot production or manufactured during specific 
865  production for a comparability study.  Such surrogate TEMPs could be particularly useful when 
866  destructive sampling is used for testing additional CQAs that are not routinely evaluated for lot 
867  release.  An alternate approach could include sampling of the incubation media instead of the 
868  product itself, when the incubation media can be considered a representative sample of the 
869  product for the specific CQAs. 
870   
871   
872  VII.  COMMUNICATION WITH FDA 
873   
874  We recommend that sponsors and applicants of CGT products prospectively discuss proposed 
875  significant manufacturing changes with FDAs Center for Biologics Evaluation and Research 
876  (CBER), particularly when such manufacturing changes would be implemented during later 
877  stages of the product lifecycle.  Communication with the FDA can be sought either by requesting 
878  FDA comment on relevant information submitted in an IND amendment or BLA product 
879  correspondence, or through a formal meeting request (Ref. 15).  The type of meeting used for 
880  such discussions depends on the stage of the product lifecycle and the issues to be considered.  
881   
882     
 
11 For the purposes of this guidance, surrogate refers to an additional unit of the drug product that is manufactured 
in parallel to the clinical product for characterization purposes, which may include destructive testing.  
21  Contains Nonbinding Recommendations 
 
Draft  Not for Implementation 
 
883  VIII.  REFERENCES  
884   
885  1.  Guidance for Industry:  Q9 Quality Risk Management, June 2006, 
886  https://www.fda.gov/media/71543/download. 
887  2.  Guidance for Industry:  Q10 Pharmaceutical Quality System, April 2009, 
888  https://www.fda.gov/media/71553/download. 
889  3.  Guidance for Industry:  Q8(R2) Pharmaceutical Development, November 2009, 
890  https://www.fda.gov/media/71535/download. 
891  4.  Guidance for Industry:  Demonstration of Comparability of Human Biological Products, 
892  Including Therapeutic Biotechnology-derived Products, April 1996, 
893  https://www.fda.gov/regulatory-information/search-fda-guidance-
894  documents/demonstration-comparability-human-biological-products-including-
895  therapeutic-biotechnology-derived. 
896  5.  Guidance for Industry:  Q5E Comparability of Biotechnological/Biological Products 
897  Subject to Changes in Their Manufacturing Process, June 2005, 
898  https://www.fda.gov/media/71489/download. 
899  6.  Chemistry, Manufacturing, and Controls Changes to an Approved Application:  Certain 
900  Biological Products; Guidance for Industry, June 2021, 
901  https://www.fda.gov/media/109615/download. 
902  7.  Guidance for Industry:  Process Validation: General Principles and Practices, January 
903  2011, https://www.fda.gov/media/71021/download. 
904  8.  Guidance for Industry:  Preclinical Assessment of Investigational Cellular and Gene 
905  Therapy Products, November 2013, https://www.fda.gov/media/87564/download. 
906  9.  Guidance for Industry:  M4Q:  The CTD  Quality, August 2001, 
907  https://www.fda.gov/media/71581/download. 
908  10.  FDA Guidance for Industry:  Comparability Protocols for Postapproval Changes to the 
909  Chemistry, Manufacturing, and Controls Information in an NDA, ANDA, or BLA, 
910  October 2022, https://www.fda.gov/media/162263/download. 
911  11.  Guidance for Industry:  CGMP for Phase I Investigational Drugs, July 2008, 
912  https://www.fda.gov/media/70975/download. 
913  12.  Guidance for Industry:  Potency Tests for Cellular and Gene Therapy Products, January 
914  2011, https://www.fda.gov/media/79856/download. 
915  13.  Analytical Procedures and Methods Validation for Drugs and Biologics; Guidance for 
916  Industry, July 2015, https://www.fda.gov/media/87801/download. 
917  14.  Q2(R1) Validation of Analytical Procedures:  Text and Methodology:  Guidance for 
918  Industry, September 2021, https://www.fda.gov/media/152208/download.   
919  15.  Formal Meetings Between the FDA and Sponsors or Applicants of PDUFA Products; 
920  Draft Guidance for Industry, December 2017*, 
921  https://www.fda.gov/media/109951/download.  
922   
923  *When finalized, this guidance will represent FDAs current thinking on this topic. 
924   
22