Potency Assay Considerations 
for Monoclonal Antibodies and 
Other Therapeutic Proteins 
Targeting Viral Pathogens 
Guidance for Industry 
 
 
 
DRAFT GUIDANCE 
 
  This guidance document is being distributed for comment purposes only. 
 
Comments and suggestions regarding this draft document should be submitted within 60 days of 
publication in the Federal Register of the notice announcing the availability of the draft 
guidance.  Submit electronic comments to https://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.  All comments should be identified with the 
docket number listed in the notice of availability that publishes in the Federal Register. 
 
For questions regarding this draft document, contact Natalia Comella at 301-796-6226.  
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
March 2023 
Pharmaceutical Quality/CMC 
 
46610036dft.docx 
02/24/23   
Potency Assay Considerations 
for Monoclonal Antibodies and 
Other Therapeutic Proteins 
Targeting Viral Pathogens 
Guidance for Industry 
 
 
 
Additional copies are available from: 
 
Office of Communications, Division of Drug Information 
Center for Drug Evaluation and Research 
Food and Drug Administration 
10001 New Hampshire Ave., Hillandale Bldg., 4th Floor  
Silver Spring, MD  20993-0002  
Phone: 855-543-3784 or 301-796-3400; Fax: 301-431-6353; Email: druginfo@fda.hhs.gov 
https://www.fda.gov/drugs/guidance-compliance-regulatory-information/guidances-drugs 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
March 2023 
Pharmaceutical Quality/CMC 
   
Table of Contents 
 
I.  INTRODUCTION ............................................................................................................. 1 
II.  BACKGROUND ................................................................................................................ 3 
III.  POTENCY ASSAY CONSIDERATIONS ...................................................................... 3 
IV.  MEASURING AND MONITORING POTENCY CONSIDERATIONS .................... 4 
A.  Methods .......................................................................................................................................... 5 
1. Binding Assays ................................................................................................................................. 5 
2. Viral Neutralization Assays ............................................................................................................. 6 
3. Fc-effector Function Assays ............................................................................................................ 9 
B.  Additional Considerations ............................................................................................................ 9 
REFERENCES ............................................................................................................................ 11 
 
 
  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
 
1  Potency Assay Considerations for Monoclonal Antibodies and Other 
2  Therapeutic Proteins Targeting Viral Pathogens 
3  Guidance for Industry1 
4   
5   
6   
7   
8  This draft guidance, when finalized, will represent the current thinking of the Food and Drug 
9  Administration (FDA or Agency) on this topic.  It does not establish any rights for any person and is not 
10  binding on FDA or the public.  You can use an alternative approach if it satisfies the requirements of the 
11  applicable statutes and regulations.  To discuss an alternative approach, contact the FDA staff responsible 
12  for this guidance as listed on the title page. 
13   
14   
15   
16   
17  I.  INTRODUCTION 
18   
19  The purpose of this guidance is to provide to sponsors recommendations that assist in the 
20  development of monoclonal antibodies (mAbs) and other therapeutic proteins2 that directly 
21  target viral proteins or host cell proteins mediating pathogenic mechanisms of infection.3  A 
22  critical quality control measure for these products is the development and implementation of a 
23  potency assay(s)4 adequate to ensure that each lot is produced consistently with the potency5 
24  necessary to achieve clinical efficacy and that such potency is maintained over the shelf life of 
25  the product.  This guidance provides detailed recommendations to drug developers with the goal 
 
1 This guidance has been prepared by the Office of Pharmaceutical Quality in the Center for Drug Evaluation and 
Research at the Food and Drug Administration.  
 
2 The term protein is one of the statutory categories of biological products (section 351(i)(1) of the Public Health 
Service Act (42 U.S.C. 262(i)(1))).  Under 21 CFR 600.3(h)(6), a protein is any alpha amino acid polymer with a 
specific, defined sequence that is greater than 40 amino acids in size. 
 
3 In January 2021, FDA published the guidance for industry COVID-19:  Potency Assay Considerations for 
Monoclonal Antibodies and Other Therapeutic Proteins Targeting SARS-CoV-2 Infectivity, which focuses solely on 
addressing potency assays as they relate to mAbs and other therapeutic proteins that directly target SARS-CoV-2.  
That guidance states it is intended to remain in effect only for the duration of the public health emergency related to 
Coronavirus Disease 2019 declared by the Secretary of Health and Human Services under section 319 of the Public 
Health Service Act (section 319 public health emergency).  FDA is issuing this draft guidance because many of the 
recommendations set forth in the 2021 guidance are applicable outside the context of the section 319 public health 
emergency and are applicable to mAbs and other therapeutic proteins directly targeting any viral surface 
(glyco)proteins mediating pathogenic mechanisms of infection, not just SARS-CoV-2. 
 
4 See 21 CFR 610.10. 
 
5 Under 21 CFR 600.3(s), potency is interpreted to mean the specific ability or capacity of the product, as indicated 
by appropriate laboratory tests or by adequately controlled clinical data obtained through the administration of the 
product in the manner intended, to effect a given result. 
  1  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
 
26  of helping to ensure that drug developers provide adequate information to assess potency at each 
27  stage of a products life cycle. 
28   
29  This guidance applies only to mAbs and other therapeutic proteins regulated by the Center for 
30  Drug Evaluation and Research that are designed to bind to viral proteins or their receptors on 
31  host cells, inhibit viral entry, and/or elicit Fc-mediated effector function, and are subject to 
32  licensure under section 351(a) or section 351(k) of the Public Health Service Act (42 U.S.C. 
33  262(a) or (k)).  This guidance does not apply to other biological products such as 
34  immunomodulatory drugs (e.g., cytokines or cytokine antagonists), vaccines, hyperimmune 
35  globulins, gene therapies, cell therapies, and convalescent plasma.6 
36   
37  The guidance describes approaches that sponsors should use to develop potency assay methods 
38  for release and stability that assess comprehensively known or potential mechanism(s) of action 
39  of the product.  The sensitivity of such methods must be established,7 for example, to conduct 
40  the appropriate laboratory determination of satisfactory conformance to final specifications for 
41  the drug product (i.e., to demonstrate lot-to-lot consistency).  In addition to release and stability 
42  methods, other methods that demonstrate the biological function(s) of the product may be needed 
43  for characterization and comparability studies.  The guidance describes methods that sponsors 
44  should use to ensure the potency of mAbs and other therapeutic proteins intended to prevent or 
45  treat a viral infection. 
46   
47  Although assays and model systems vary with different viruses, the principles in this guidance, 
48  where applicable, are relevant to mAbs and other therapeutic proteins under development for the 
49  prevention or treatment of viral infections.8  Because this field is dynamic and continually 
50  evolving, the principles are also intended to be flexible to accommodate new assays or 
51  technologies.  If circumstances warrant, this guidance may be revised to inform sponsors of 
52  additional methodologies to consider. 
53   
54  In general, FDAs guidance documents do not establish legally enforceable responsibilities.  
55  Instead, guidances describe the Agencys current thinking on a topic and should be viewed only 
56  as recommendations, unless specific regulatory or statutory requirements are cited.  The use of 
57  the word should in Agency guidances means that something is suggested or recommended, but 
58  not required. 
59   
60   
 
6 Manufacturers of vaccines and certain medical devices (e.g., in vitro diagnostics) should consult the center review 
offices regarding appropriate assays and methods for products regulated by the Center for Biologics Evaluation and 
Research and the Center for Devices and Radiological Health. 
 
7 See 21 CFR 211.165(a) and (e), 21 CFR 601.2(d), and 21 CFR 610.10. 
 
8As an example, some mAbs do not demonstrate both virus neutralization and Fc-mediated effector functions as 
their mechanisms of action; therefore, only the principles relevant to the particular mAb would apply under those 
circumstances. 
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Draft  Not for Implementation 
 
61  II.  BACKGROUND 
62   
63  The Agency receives investigational new drug applications (INDs) and biologics license 
64  applications (BLAs) for mAbs and other therapeutic proteins designed to bind to viral surface 
65  (glyco)proteins or their receptors on host cells, inhibit viral entry, and/or elicit Fc-mediated 
66  effector function.  Monoclonal antibodies and mAb cocktails9 being developed to target viral 
67  pathogens, including newly emerging viruses, generally use mechanisms of action that may 
68  include virus neutralization, Fc-mediated effector functions, or both.  Monoclonal antibodies 
69  may neutralize the virus by binding the viral attachment protein or the host cell receptor (e.g., 
70  angiotensin-converting enzyme 2 for coronaviruses, sialic acid for influenza), thereby blocking 
71  attachment to host cell receptors; may prevent the fusion of the viral and host cell membranes 
72  (e.g., respiratory syncytial virus F protein); and/or may inhibit other viral or host factors 
73  necessary for entry.  These products are referred to as neutralizing mAbs.  Some mAbs directed 
74  against viruses mediate Fc-effector functions in addition to or instead of neutralizing virus entry.  
75  Other mAbs may target alternative cellular receptors or cellular proteins that facilitate virus 
76  infection.   
77   
78  In addition to mAbs, other protein therapeutics intended to target viral entry may be developed to 
79  prevent or treat viral infection.  These include scaffold proteins, which are engineered to have 
80  similar mechanisms as neutralizing mAbs, bifunctional molecules engineered to interfere with 
81  different steps of viral entry, or recombinant virus receptor proteins, which serve as decoy 
82  receptors to inhibit viral infectivity.  As the science evolves, other novel proteins with other 
83  mechanism(s) of action may also be developed and submitted to the Agency. 
84   
85   
86  III.  POTENCY ASSAY CONSIDERATIONS 
87   
88  To ensure mAbs and therapeutic proteins clinical effectiveness through product expiry, a 
89  sponsor must develop a method or methods to monitor the potency of the biological product.10  
90  Potency assays for mAbs and therapeutic proteins should be designed to measure the binding to 
91  viral receptors on host cells, the inhibition of viral entry, and/or to elicit Fc-mediated effector 
92  function.  Potency assays also should be designed to reflect the biological activity of the mAb or 
93  therapeutic protein in vivo.  Potency measurements from these methods should be used to 
94  demonstrate that only product lots that meet defined specifications or acceptance criteria are 
95  administered during all phases of clinical investigation and after approval. 
96   
 
9 In general, the term mAb cocktails refers to two or more mAbs administered at a fixed ratio.  They may be filled in 
a single vial or separate vials.  For the purposes of this guidance, the term refers to two or more mAbs filled in a 
single vial. 
 
10 See 21 CFR 610.10. 
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97  All potency assays used for release and stability testing must comply with applicable biologics 
98  and current good manufacturing practice regulations.11,12  When evaluating the appropriateness 
99  of a potency assay for a specific mAb or other therapeutic proteins for treating or preventing a 
100  viral infection, FDA may consider various factors, including:  (1) product characteristics; (2) 
101  manufacturing processes; (3) the stage of development in which the assay will be used; (4) the 
102  strength of the sponsors risk-based quality assessment; and (5) the totality of the information 
103  provided by the sponsor. 
104   
105   
106  IV.  MEASURING AND MONITORING POTENCY CONSIDERATIONS 
107   
108  Sponsors developing mAbs or other therapeutic proteins for the treatment and/or prevention of a 
109  viral infection should implement potency assays for release and stability testing specifically 
110  designed to demonstrate the biological function(s) of the product13 and provide justification for 
111  how assays used for release and stability testing measure comprehensively known or potential 
112  mechanism(s) of action of the product.  Sponsors should develop a manufacturing control 
113  strategy that will identify potential shifts in the products critical quality attributes known to 
114  affect each known and/or potential mechanism of action and detect changes in the performance 
115  of the manufacturing process before beginning phase 3 trials.    
116   
117  Depending on the proposed mechanism(s) of action, one or more assays should be developed to 
118  support the control strategy for confirming the biologics potency.14  If the mAb or other 
119  therapeutic protein has multiple mechanisms of action (e.g., neutralization and Fc-effector 
120  function), multiple potency assays should be used.  All quality-control release test methods, 
121  including potency assays for demonstrating the mechanism(s) of action, should be shown to be 
122  suitable for their intended purposes during development and must be validated by the time of a 
 
11 See, for example, 21 CFR 610.10 (regarding tests for potency for biological products); 21 CFR 211.165(e) 
(regarding the establishment and documentation of the accuracy, sensitivity, specificity, and reproducibility of test 
methods); 21 CFR 211.160(b) (regarding laboratory controls); and 21 CFR 211.194(a)(2) (regarding laboratory 
records of testing methods). 
 
12 Sponsors should consider potency assay development in their overall drug development program.  The IND 
regulations at 21 CFR 312.23(a)(7)(i) require that, in each phase of the investigation, sponsors submit sufficient 
information to assure the proper identification, quality, purity, and strength of the investigational drug, and 
indicate that the amount of information needed to make that assurance will vary with the phase of the investigation, 
the proposed duration of the investigation, the dosage form, and the amount of information otherwise available.  
For additional information, see the guidances for industry Content and Format of Investigational New Drug 
Applications (INDs) for Phase 1 Studies of Drugs, Including Well-Characterized, Therapeutic, Biotechnology-
Derived Products (November 1995) and INDs for Phase 2 and Phase 3 Studies; Chemistry, Manufacturing, and 
Controls Information (May 2003).  We update guidances periodically.  For the most recent version of a guidance, 
check the FDA guidance web page at https://www.fda.gov/regulatory-information/search-fda-guidance-documents. 
 
13 See the ICH guidance for industry Q6B Specifications:  Test Procedures and Acceptance Criteria for 
Biotechnological/Biological Products (August 1999).   
 
14 Ibid. 
 
  4  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
 
123  BLA submission.15  The development of quality-control release methods should be informed by 
124  the product characterization.  Special consideration should be given for emerging or highly 
125  pathogenic viruses.  Additional parameters may need to be incorporated into the development of 
126  the potency assay(s).  Sponsors should consult FDA for further guidance.16 
127   
128  This section provides examples of assays, depending on the mechanism(s) of action, that can be 
129  included as part of the overall control strategy.  
130   
131  A.  Methods  
132   
133  Because a binding assay demonstrates binding between the mAb or therapeutic protein and its 
134  target, it is generally sufficient to serve as a potency assay at the early stages of drug 
135  development.  However, a binding assay assesses only one aspect of the potency of a product.  
136  Therefore, sponsors should subsequently develop methods that more comprehensively monitor 
137  the proposed mechanism(s) of action of the products.  These methods should be incorporated 
138  into drug substance and drug product release testing and stability protocols.  Potency assays 
139  should be described, justified, qualified, and validated to support a BLA.   
140   
141  This section addresses considerations for possible assays. 
142   
143  1.  Binding Assays  
144   
145  For the purposes of this guidance, binding assays are defined as assays that quantify the binding 
146  between the mAb or other therapeutic protein and its target.  These assays are established early 
147  in product development, typically in the form of a direct binding assay such as an enzyme-linked 
148  immunosorbent assay (ELISA) or a surface plasmon resonance (SPR) assay.  Product lots should 
149  be compared to an appropriately qualified in-house reference material and activity should be 
150  expressed as a percentage of the reference material value.  Although helpful in the initial phases 
151  of development, these assays do not directly confirm the products ability to inhibit the target 
152  proteins activity and should not be used in lieu of methods that confirm potency.  
153   
154  For products intended to inhibit viral protein binding to a host cell receptor, FDA recommends a 
155  potency assay that is a better reflection of the intended mechanism of action instead of a direct 
156  binding assay; for example, an inhibition assay, such as an inhibition ELISA,17 or SPR.  An 
 
15 See 21 CFR 211.165(e) and as described in the guidance for industry Analytical Procedures and Methods 
Validation for Drugs and Biologics (July 2015).  See also the ICH draft guidance for industry Q2(R2) Validation of 
Analytical Procedures (August 2022); when final, this guidance will represent the FDAs current thinking on this 
topic. 
 
16 Although this guidance covers in vitro approaches for potency assays, there may be situations in which an in vivo 
assay is the only approach that can ensure product quality; however, these situations are rare and should be 
discussed with the Agency. 
 
17 See, for example, Tan, CW, WN Chia, X Qin, P Liu, MI-C Chen, C Tiu, Z Hu, VC-W Chen, BE Young, WR Sia, 
Y-J Tan, R Foo, Y Yi, DC Lye, and DE Anderson, 2020, A SARS-CoV-2 Surrogate Virus Neutralization Test 
Based on Antibody-Mediated Blockage of ACE2Spike ProteinProtein Interaction, Nat Biotechnol. 
  5  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
 
157  inhibition assay should be designed to evaluate the inhibition of virus-receptor interactions and 
158  may be appropriate to conduct in, for example, either a biosafety level (BSL)-1 or BSL-2 lab.18  
159   
160  2.  Viral Neutralization Assays  
161   
162  In comparison to binding assays, in vitro viral neutralization assays more comprehensively 
163  confirm a mAbs or therapeutic proteins mechanism of action and potency in blocking infection 
164  of susceptible cells.  Because of the potential importance to evaluating these products, the 
165  Agency recommends establishing an in vitro viral neutralization assay early in development.  
166  This type of assay can be useful for advancing development, quality control, and characterization 
167  of neutralizing mAbs and other products targeting viral attachment and entry.  Given the 
168  diversity of mechanisms for viral attachment and entry into host cells, the assay should reflect 
169  that viruss mechanisms for attachment and entry. 
170   
171  Assays that assess the ability of the mAbs or other therapeutic proteins to inhibit any of the 
172  binding or entry steps are predominantly cell-based assays and typically involve the use of wild-
173  type (wt) virus,19 pseudotyped virus, or pseudotyped virus-like particles (VLP).  When 
174  considering which method to use, sponsors should select a method that best monitors the 
175  binding/entry step the product is expected to target in the virus replication cycle.  Although wt 
176  virus neutralization assays are considered the gold standard for in vitro potency assays, 
177  alternative methods may be acceptable.  For example, a potency assay could be designed to 
178  characterize the effect of the product on a specific entry step (e.g., virus-cell fusion).  
179  Additionally, accessibility to appropriate BSL laboratories, as well as challenges to qualifying 
180  critical reagents and validating the overall assay performance, should be considered in assay 
181  selection.  For methods using transfected cell lines, sponsors should also address target cell 
182  viability and variability.  Whichever method is ultimately used, sponsors should observe all 
183  provisions of the select agent regulations20 (if applicable) and other applicable governmental and 
184  institutional biosafety and biosecurity provisions.   
185   
 
18 Relevant biosafety considerations may be found in the National Institutes of Healths guidelines NIH Guidelines 
for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (April 2019) (available at 
https://osp.od.nih.gov/wp-content/uploads/NIH_Guidelines.pdf) and on the FAQ web page Interim Laboratory 
Biosafety Guidance for Research with SARS-CoV-2 and IBC Requirements under the NIH Guidelines 
(https://osp.od.nih.gov/policies/biosafety-and-biosecurity-policy/interim-laboratory-biosafety-guidance-for-research-
with-sars-cov-2-and-ibc-requirements-under-the-nih-guidelines/) and the Centers for Disease Control and 
Preventions guideline Biosafety in Microbiological and Biomedical Laboratories (available at 
https://www.cdc.gov/labs/BMBL.html). 
 
19 For the purposes of potency assays discussed in this guidance, references to wt virus indicate a clinical isolate, 
which has not been intentionally modified, except for mutations that may occur naturally during in vitro virus 
passaging and expansion, compared to the initial isolate.  Use of lab-adapted viruses should be appropriately 
justified. 
 
20 See 7 CFR part 331, 9 CFR part 121, and 42 CFR part 73. 
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186  Below is a list of assays that may be suitable for use as a potency assay, along with key 
187  considerations for each assay: 
188   
189    Virus neutralization assays.  Depending on the virus, these assays (such as plaque 
190  reduction, TCID , and microneutralization assay) involve working in the appropriate 
50
191  BSL laboratory.  If using minimally passaged wt virus or a lab-adapted isolate(s), 
192  sponsors should determine virus titer and develop, aliquot, and store master and working 
193  virus stocks appropriately.  If using a lab-adapted isolate(s), a sponsor should indicate the 
194  isolate used and justify the use of that isolate(s) in applicable submissions to FDA.  
195  Sponsors should provide details on the production, storage condition, and stability of 
196  virus stocks used in the assays as discussed in existing guidance on these topics.21  The 
197  qualification of any new working stock should include the sequencing of the epitope. 
198   
199    Pseudotyped virus- or VLP-based assay.  An alternative to working with a wt virus is 
200  the implementation of pseudotyped virus-based methods.  These methods should be 
201  performed under appropriate biosafety conditions.22  Pseudotyped virions can be 
202  generated by replacing the surface protein(s) expressing gene of a less pathogenic virus 
203  (e.g., vesicular stomatitis virus) with the gene encoding the viral surface 
204  (glyco)protein(s)23 of the pathogen of interest, creating engineered, replication-competent 
205  virions.  Another approach is to generate fusion-competent, but replication-incompetent 
206  VLPs by co-transfecting producer cells (usually 293T cells) with a set of plasmids 
207  encoding the viral surface (glyco)protein(s) and a matrix protein(s) driving the VLP 
208  budding in trans.  Retrovirus-based VLP packaging systems have already been adapted 
209  for viral surface (glyco)protein(s) pseudotyping.  
210   
211  During method development of a neutralization assay using a pseudovirus or VLP, 
212  sponsors should generate data demonstrating viral surface (glyco)protein-mediated cell 
213  entry and describe the generation, isolation, purification, and concentration steps used to 
214  minimize lot-to-lot variability of the pseudovirus or VLP.  Additionally, sponsors should 
215  address the following: 
216   
217    The critical reagents (i.e., producer and target cell lines, and virion constructs, as well 
218  as controls (negative and positive)) used in the assay should be described.  When 
 
21 See the References section for a list of guidances regarding analytical procedures, method validation, and 
documentation. 
 
22 Relevant biosafety considerations may be found in the National Institutes of Healths guidelines NIH Guidelines 
for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (April 2019) (available at 
https://osp.od.nih.gov/wp-content/uploads/NIH_Guidelines.pdf) and on the FAQ web page Interim Laboratory 
Biosafety Guidance for Research with SARS-CoV-2 and IBC Requirements under the NIH Guidelines 
(https://osp.od.nih.gov/policies/biosafety-and-biosecurity-policy/interim-laboratory-biosafety-guidance-for-research-
with-sars-cov-2-and-ibc-requirements-under-the-nih-guidelines/) and the Centers for Disease Control and 
Preventions guideline Biosafety in Microbiological and Biomedical Laboratories (available at 
https://www.cdc.gov/labs/BMBL.html). 
 
23 In the context of this guidance, the term viral surface (glyco)protein(s) refers to virus components (molecules) 
involved in the virus binding and/or entry into susceptible cells. 
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219  qualifying these critical reagents, sponsors should demonstrate long-term stability and 
220  address possible variability associated with the transfected cells lines. 
221   
222    Attention should be given to the manufacturing, stability, and qualification of the 
223  pseudovirus or VLP, which is a critical reagent for the assay.  Lot-to-lot variability of 
224  VLPs can be observed in the quantity of the VLPs and the activity of the VLP stock.  
225  Sponsors should demonstrate VLP lot-to-lot consistency is appropriately controlled.  
226   
227    Viral surface (glyco)protein(s)-mediated cell-cell fusion-based assays.  These assays 
228  can be used as an alternative to the pseudotyped virus- or VLP-based assays.  Typically, 
229  the level of fusion between the viral surface (glyco)protein(s)-expressing cells and the 
230  virus receptor-expressing cells should be assessed using a reporter gene.  The expression 
231  of the reporter construct is dependent on the successful fusion between the two cell 
232  populations.  Alternatively, the level of syncytia formation or dye transfer also can be 
233  used to quantify cell-cell fusion.  As with the pseudotyped virus-based assays, the 
234  following should be addressed:  
235   
236    The controls (negative and positive), cell lines, constructs, and reporter constructs 
237  used in the method should be described.  When qualifying these critical reagents, 
238  sponsors should demonstrate their long-term stability and address possible variability 
239  associated with the transfected cells lines. 
240   
241    How well the assay mimics viral-cell fusion should be described.  For example, if 
242  certain conformational or environmental (e.g., pH) changes are needed for fusion to 
243  occur, the assay should monitor those changes.  
244   
245    The testing platform to be used with the assay should be indicated. 
246   
247    Defined quantitation criteria should be used regardless of whether a reporter 
248  construct, syncytia formation, or dye transfer is used to measure cell-cell fusion. 
249   
250  Given possible differences between wt virus neutralization assay(s) and the alternative methods 
251  mentioned above (pseudotyped virus neutralization methods, VLP-based methods, or viral 
252  surface (glyco)protein(s)-mediated cell-cell fusion-based assays), FDA recommends that 
253  sponsors provide information addressing how (or whether) the assays results correlate with wt 
254  virus neutralization. 
255   
256  Although this section of the guidance focuses on neutralizing assays for products that block viral 
257  surface (glyco)protein(s)-mediated virus entry, the concepts described herein are applicable to 
258  the development of mAbs or other therapeutic proteins that block other steps in the virus 
259  replication cycle.  
260   
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261  3.  Fc-effector Function Assays 
262   
263  In general, sponsors should assess mAbs for their ability to demonstrate Fc-mediated effector 
264  functions.24  This can include complement activity and activities mediated through binding to 
265  Fc receptors, such as antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-
266  dependent cellular phagocytosis.  For mAbs demonstrating Fc-effector functions, appropriate 
267  methods should be included as part of the specifications to ensure consistent mAb potency and 
268  functions.  Fc glycosylation relevant to the mechanism of action should also be monitored and 
269  controlled throughout the product life cycle.  FDA recommends including an FcRIIIa-
270  mediated/natural killer cell ADCC assay because that appears to be the most sensitive to changes 
271  in glycosylation.   
272   
273  For mAbs engineered to alter binding to Fc receptors and complement components, 
274  characterization studies should be conducted on a one-time basis to demonstrate the engineered 
275  mAb performs as designed.  Fc-fusion proteins may also engage Fc receptors and complement 
276  components and thus should also be characterized for these potential effects or to confirm the 
277  intended effect of any Fc modifications.  For example, the Agency recommends that virus 
278  receptor-Fc fusion proteins be characterized for their ability to carry out Fc-effector functions 
279  using methods similar to those for the characterization of neutralizing mAbs.  
280   
281  B.  Additional Considerations 
282   
283  Sponsors should address the following additional considerations when developing methods to 
284  monitor potency. 
285   
286    When describing a potency assay, sponsors should ensure that the virus isolate, or viral 
287  surface (glyco)protein(s), used reflects common isolates prevalent in the United States.  
288  Sponsors should discuss how the isolates or proteins were selected and whether they 
289  reflect the viruses currently in circulation.  Sponsors also should provide either the full 
290  genome sequence(s) of the isolate(s) or GenBank ID(s). 
291   
292    Whether using wt virus, pseudotyped virus, or VLPs, a master cell bank of producer cells 
293  should be appropriately qualified and used to generate a working bank of virus producer 
294  cells.   
295   
296    Information and data submitted to support BLAs should describe any differences in the 
297  methods used for release testing and stability program compared to those used to initially 
298  characterize the potency of the mAb or other therapeutic protein during earlier 
299  development.  That includes, but is not limited to, reagents, testing site(s), and testing 
300  platform(s), if applicable, to conduct the assay in question. 
301   
 
24 For more information on Fc-mediated effector functions, see, for example, Jiang, X, A Song, S Bergelson, 
T Arroll, B Parekh, K May, S Chung, R Strouse, A Mire-Sluis, and M Schenerman, 2011, Advances in the 
Assessment and Control of the Effector Functions of Therapeutic Antibodies, Nature Reviews; Drug Discovery 
10:101-110. 
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302    Relevant positive and negative controls should be included as part of the potency assay.   
303   
304    For mAb cocktails, release testing methods should include an identity method that 
305  demonstrates the presence of each individual mAb and a quantitative method verifying 
306  the ratio of the individual mAbs.  The sponsor should ensure the ratio is consistent from 
307  lot to lot.    
308   
309  Novel mechanisms of action that are not addressed in this guidance may be identified in the 
310  future.  For mAbs or other therapeutic proteins with such novel mechanisms of action, sponsors 
311  should consult the Agency for further recommendations regarding compliance with requirements 
312  for potency assays for release and stability testing.25  
 
25 See 21 CFR 610.10, 211.165(e), and 211.194(a)(2). 
  10  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
 
REFERENCES 
 
Analytical Procedures, Method Validation, and Documentation-Related Guidances for 
Industry26 
 
  Guidance for industry Analytical Procedures and Methods Validation for Drugs and 
Biologics (July 2015) 
 
  Guidance for industry Bioanalytical Method Validation (May 2018) 
 
  Guidance for industry Content and Format of Investigational New Drug Applications 
(INDs) for Phase 1 Studies of Drugs, Including Well-Characterized, Therapeutic, 
Biotechnology-Derived Products (November 1995)  
 
  Guidance for industry INDs for Phase 2 and Phase 3 Studies; Chemistry, Manufacturing, 
and Controls Information (May 2003) 
 
  Guidance for industry Points to Consider in the Manufacture and Testing of Monoclonal 
Antibody Products for Human Use (February 1997) 
 
  ICH draft guidance for industry Q2(R2) Validation of Analytical Procedures (August 
2022)27  
 
  ICH guidance for industry M4Q:  The CTD  Quality (August 2001) 
 
  ICH guidance for industry Q5C Quality of Biotechnological Products:  Stability Testing 
of Biotechnological/Biological Products (July 1996) 
 
  ICH guidance for industry Q6B Specifications:  Test Procedures and Acceptance Criteria 
for Biotechnological/Biological Products (August 1999) 
 
Centers for Disease Control and Prevention Guideline28 
 
  Biosafety in Microbiological and Biomedical Laboratories 
 
Literature 
 
  Jiang, X, A Song, S Bergelson, T Arroll, B Parekh, K May, S Chung, R Strouse, A Mire-
Sluis, and M Schenerman, 2011, Advances in the Assessment and Control of the Effector 
Functions of Therapeutic Antibodies, Nature Reviews; Drug Discovery 10:101-110. 
 
26 We update guidances periodically.  For the most recent version of a guidance, check the FDA guidance web page 
at https://www.fda.gov/regulatory-information/search-fda-guidance-documents. 
 
27 When final, this guidance will represent the FDAs current thinking on this topic. 
 
28 Available at https://www.cdc.gov/labs/BMBL.html.  
  11  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
 
 
  Tan, CW, WN Chia, X Qin, P Liu, MI-C Chen, C Tiu, Z Hu, VC-W Chen, BE Young, 
WR Sia, Y-J Tan, R Foo, Y Yi, DC Lye, and DE Anderson, 2020, A SARS-CoV-2 
Surrogate Virus Neutralization Test Based on Antibody-Mediated Blockage of ACE2
Spike ProteinProtein Interaction, Nat Biotechnol. 
 
National Institutes of Health Guidelines 
 
  NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid 
Molecules (April 2019)29   
 
  FAQ web page Interim Laboratory Biosafety Guidance for Research with SARS-CoV-2 
and IBC Requirements under the NIH Guidelines 
(https://osp.od.nih.gov/policies/biosafety-and-biosecurity-policy/interim-laboratory-
biosafety-guidance-for-research-with-sars-cov-2-and-ibc-requirements-under-the-nih-
guidelines/) 
 
Pharmaceutical Development Guidance for Industry30  
 
  ICH guidance for industry Q8(R2) Pharmaceutical Development (November 2009) 
 
 
29 Available at https://osp.od.nih.gov/wp-content/uploads/NIH_Guidelines.pdf. 
  
30 We update guidances periodically.  For the most recent version of a guidance, check the FDA guidance web page 
at https://www.fda.gov/regulatory-information/search-fda-guidance-documents. 
  12