Guidance for Industry 
 
Immunogenicity Assessment for 
Therapeutic Protein Products 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
Center for Biologics Evaluation and Research (CBER) 
 
 
 
 
 
 
 
 
August 2014 
Clinical/Medical 
 
  Guidance for Industry 
 
Immunogenicity Assessment for 
Therapeutic Protein Products 
 
 
Additional copies are available from: 
 
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Division of Drug Information, WO51, Room 2201 
Center for Drug Evaluation and Research 
Food and Drug Administration 
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druginfo@fda.hhs.gov  
http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm 
 
and/or 
 
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 Food and Drug Administration 
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 ocod@fda.hhs.gov 
http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/default.htm   
( 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
Center for Biologics Evaluation and Research (CBER) 
 
August 2014 
Clinical/Medical 
  Contains Nonbinding Recommendations 
 
TABLE OF CONTENTS 
 
 
I.  INTRODUCTION............................................................................................................. 1 
II.  BACKGROUND ............................................................................................................... 2 
III.  CLINICAL CONSEQUENCES ...................................................................................... 3 
A.  Consequences for Efficacy ............................................................................................................ 3 
B.  Consequences for Safety ................................................................................................................ 3 
IV.  RECOMMENDATIONS FOR IMMUNOGENICITY RISK MITIGATION IN 
THE CLINICAL PHASE OF DEVELOPMENT OF THERAPEUTIC PROTEIN 
PRODUCTS................................................................................................................................... 6 
V.  PATIENT- AND PRODUCT-SPECIFIC FACTORS THAT AFFECT 
IMMUNOGENICITY .................................................................................................................. 9 
A.  Patient-Specific Factors That Affect Immunogenicity ............................................................... 9 
1.  Immunologic Status and Competence of the Patient ...................................................................... 9 
2. Prior Sensitization/History of Allergy ........................................................................................... 10 
3. Route of Administration, Dose, and Frequency of Administration ................................................ 10 
4. Genetic Status ................................................................................................................................ 11 
5. Status of Immune Tolerance to Endogenous Protein ..................................................................... 11 
B.  Product-Specific Factors That Affect Immunogenicity ............................................................ 12 
1. Product Origin (foreign or human) ............................................................................................... 13 
2. Primary Molecular Structure/Posttranslational Modifications ..................................................... 14 
3. Quaternary Structure: Product Aggregates and Measurement of Aggregates .............................. 15 
4. Glycosylation/Pegylation ............................................................................................................... 17 
5. Impurities with Adjuvant Activity ................................................................................................... 18 
6. Immunomodulatory Properties of the Therapeutic Protein Product ............................................. 18 
7. Formulation ................................................................................................................................... 19 
8. Container Closure Considerations ................................................................................................ 19 
9. Product Custody ............................................................................................................................ 21 
VI.  CONCLUSION ............................................................................................................... 21 
REFERENCES ............................................................................................................................ 23 
APPENDIX A .............................................................................................................................. 31 
1.  Diagnosis of Anaphylaxis ............................................................................................................ 31 
2.  Cytokine Release Syndrome ........................................................................................................ 32 
3.  Non-Acute Immune Responses ................................................................................................... 33 
4.  Antibody Responses to Therapeutic Protein Products ............................................................. 33 
5.  Utility of Animal Studies ............................................................................................................. 35 
6.  Comparative Immunogenicity Studies ....................................................................................... 35 
i 
  Contains Nonbinding Recommendations 
 
1
Guidance for Industry  
Immunogenicity Assessment for Therapeutic Protein Products 
 
 
This guidance represents the Food and Drug Administrations (FDAs) current thinking on this topic.  It 
does not create or confer any rights for or on any person and does not operate to bind FDA or the public.  
You can use an alternative approach if the approach satisfies the requirements of the applicable statutes 
and regulations.  If you want to discuss an alternative approach, contact the FDA staff responsible for 
implementing this guidance.  If you cannot identify the appropriate FDA staff, call the appropriate 
number listed on the title page of this guidance.  
 
 
 
 
I.  INTRODUCTION  
 
This guidance is intended to assist manufacturers and clinical investigators involved in the 
development of therapeutic protein products for human use.2  In this document, FDA outlines 
and recommends adoption of a risk-based approach to evaluating and mitigating immune 
responses to or adverse immunologically related responses associated with therapeutic protein 
products that affect their safety and efficacy.  Any given approach to assessing and mitigating 
immunogenicity is determined on a case-by-case basis and should take into consideration the risk 
assessment we describe.  For the purposes of this guidance, immunogenicity is defined as the 
propensity of the therapeutic protein product to generate immune responses to itself and to 
related proteins or to induce immunologically related adverse clinical events.  
 
This guidance describes major clinical consequences of immune responses to therapeutic protein 
products and offers recommendations for risk mitigation in the clinical phase of development.  It 
also describes product- and patient-specific factors that can affect the immunogenicity of 
therapeutic protein products.  For each factor, recommendations are made for sponsors and 
investigators that may help them reduce the likelihood that these products will generate an 
immune response.  Appendix A provides supplemental information on the diagnosis and 
pathophysiology of particular adverse consequences of immune responses to therapeutic protein 
products and brief discussions of the uses of animal studies and the conduct of comparative 
                                                 
1 This guidance has been prepared by the Division of Medical Policy Development, Office of Medical Policy, in the 
Center for Drug Evaluation and Research (CDER) in coordination with the Center for Biologics Evaluation and 
Research (CBER) at the Food and Drug Administration.  
 
2 See the draft guidance for industry Biosimilars:  Questions and Answers Regarding Implementation of the 
Biologics Price Competition and Innovation Act of 2009 for FDAs interpretation of the category of protein (except 
any chemically synthesized polypeptide) in the amended definition of biological product in section 351(i)(1) of 
the Public Health Service Act (PHS Act).  When final, this guidance will represent the FDAs current thinking on 
this topic.  We update guidances periodically.  To make sure you have the most recent version of a guidance, check 
the FDA Drugs guidance Web page at 
http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm. 
  1  Contains Nonbinding Recommendations 
 
immunogenicity studies.  Although this guidance focuses on therapeutic protein products, the 
scientific principles may also apply to related products and biological entities, for example, 
peptides.  Although this guidance encompasses products used to modulate or modify the immune 
system, including those that are antigen specific, it does not cover products that are intended to 
induce a specific immune response to prevent or treat a disease or condition (such as vaccines to 
prevent infectious diseases) or to enhance the activity of other therapeutic interventions.  This 
guidance does not address assay development, which is covered in a separate guidance.3 
 
FDAs guidance documents, including this guidance, do not establish legally enforceable 
responsibilities.  Instead, guidances describe the Agencys current thinking on a topic and should 
be viewed only as recommendations, unless specific regulatory or statutory requirements are 
cited.  The use of the word should in Agency guidances means that something is suggested or 
recommended, but not required.  
 
II.  BACKGROUND 
 
Immune responses to therapeutic protein products may pose problems for both patient safety and 
product efficacy.  Immunologically based adverse events, such as anaphylaxis, cytokine release 
syndrome, and cross-reactive neutralization of endogenous proteins mediating critical functions 
(see Appendix A.3), have caused sponsors to terminate the development of what otherwise may 
have been efficacious therapeutic protein products.  Unwanted immune responses to therapeutic 
protein products may also neutralize their biological activities and result in adverse events not 
only by inhibiting the efficacy of the therapeutic protein product, but also by cross-reacting to an 
endogenous protein counterpart, leading to loss of its physiological function (e.g., neutralizing 
antibodies to therapeutic erythropoietin cause pure red cell aplasia by also neutralizing the 
endogenous protein) (Hermeling et al. 2004; Rosenberg and Worobec 2004; Rosenberg and 
Worobec 2005; Koren et al. 2008; Murphy 2011).  Because most of the adverse effects resulting 
from elicitation of an immune response to a therapeutic protein product appear to be mediated by 
humoral mechanisms, circulating antibody to the therapeutic protein product has been the chief 
criterion for defining an immune response to this class of products.4 
 
Both patient-related and product-related factors may affect immunogenicity of therapeutic 
protein products.  These factors are critical elements in the immunogenicity risk assessment.  
Ideally, these factors should be taken into consideration in the early stages of therapeutic protein 
product development.  Section III contains a detailed discussion of the nature of and the risk 
factors for immune responses to therapeutic protein products as well as possible mitigation 
strategies that may be employed. 
 
                                                 
3 See the draft guidance for industry Assay Development for Immunogenicity Testing of Therapeutic Proteins.  
When final, this guidance will reflect the Agencys current thinking on this topic.  
 
4 IgG and IgE antibody responses are those most often associated with clinical adverse events, and their generation 
generally requires collaboration between antigen-specific T-helper cells and B-cells. Murphy, K. (2011). The 
Humoral Immune Response. Janeway's Immunobiology. New York, Garland Science Publishing. 8th: 367-408.  
 
  2  Contains Nonbinding Recommendations 
 
III.  CLINICAL CONSEQUENCES 
 
Treatment of patients with therapeutic protein products may result in immune responses of 
varying clinical relevance, ranging from antibody responses with no apparent clinical 
manifestations to life-threatening and catastrophic reactions.  During therapeutic protein product 
development, elucidation of a specific underlying immunologic mechanism for immunologically 
related adverse events is encouraged, because this information can facilitate the development of 
strategies to help mitigate their risk (see section III.B.15).  The extent of information required to 
perform a risk-benefit assessment will vary among individual products, depending on product 
origin and features, the immune responses of concern, the target disease indication, and the 
proposed patient population. 
 
A.  Consequences for Efficacy 
 
Development of antibodies can limit product efficacy in patients treated with therapeutic protein 
products.  Neutralizing antibodies can block the efficacy of therapeutic protein products by 
specifically targeting domains critical for efficacy.  For example, antibodies binding to either the 
uptake or catalytic domain of a therapeutic enzyme may lead to loss of product efficacy.  Loss of 
efficacy is problematic for all products, but is of utmost concern if the product is a lifesaving 
therapeutic.  Neutralizing antibodies that cross-react with a nonredundant endogenous 
counterpart of the therapeutic protein product can also impact safety, as discussed in 
section III.B.  Both neutralizing and non-neutralizing antibodies may alter the pharmacokinetics 
of the product by enhancing clearance (and thereby shortening serum half-life) or, conversely, by 
prolonging serum half-life and product activity.  If present at high enough titer, a non-
neutralizing antibody may mistarget the therapeutic protein product into Fc receptor (FcR) 
bearing cells, thereby reducing or eliminating product efficacy (Brooks et al. 1998; Wang et al. 
2008).  Furthermore, although some antibody responses to therapeutic protein products may have 
no apparent effect on clinical safety or efficacy, they may promote the generation of neutralizing 
antibodies via the mechanism of epitope spreading of antibody responses (Disis et al. 2004; 
Hintermann et al. 2011).  Pharmacodynamic biomarkers may be useful in the assessment of 
antibody-mediated interference with product activity, although correlation with clinical response 
is usually necessary to determine clinical relevance. 
 
B.  Consequences for Safety 
 
The safety consequences of immunogenicity may vary widely and are often unpredictable in 
patients administered therapeutic protein products.  Therefore, a high index of suspicion should 
be maintained for clinical events that may originate from such responses, even if the initial risk 
assessment suggests a lower risk of immunogenicity.  The applicant should provide a rationale 
for the proposed immunogenicity testing paradigm, based on product- and patient-specific 
concerns.  The following sections describe a few of the major safety concerns associated with 
immunogenicity: 
 
  3  Contains Nonbinding Recommendations 
 
  1.  Anaphylaxis 
 
Anaphylaxis is a serious, acute allergic reaction characterized by certain clinical features.  The 
definition currently accepted by the Agency relies on clinical diagnostic criteria and does not 
specify a particular immunologic mechanism (Sampson et al. 2006) (also see Appendix A.1).  
Historically, the definition of anaphylaxis has invoked the involvement of specific IgE 
antibodies.  However, such a mechanistic definition may be problematic in the context of 
therapeutic protein product development and other clinical settings where it may not always be 
possible to identify a specific immunologic mechanism as the basis of an adverse event.  To 
capture all potential adverse events of interest, the Agency recommends identifying all cases 
meeting the clinical diagnostic criteria of anaphylaxis, regardless of the presumed 
pathophysiology.  Additional information, such as the assessment of serum histamine, serum 
tryptase, and complement components, following a reaction or the detection of product-specific 
IgE antibodies may help elucidate the pathophysiology of the anaphylactic response and thus 
guide control and mitigation strategies. 
 
Furthermore, the presence of anti-drug antibody (ADA) alone is not necessarily predictive of 
anaphylaxis or other hypersensitivity reactions.  Correlation with clinical response is typically 
required to determine the clinical relevance of these antibodies.  Determination of the underlying 
mechanism remains of interest, however, because anaphylaxis with confirmation of IgE 
involvement has certain prognostic implications for repeat exposure as well as for potential 
therapeutic options for mitigation. 
 
2.  Cytokine Release Syndrome 
 
Cytokine release syndrome is a symptom complex caused by the rapid release of 
proinflammatory cytokines from target immune cells (Stebbings et al. 2007; Stebbings et al. 
2013).  Although cytokine release syndrome is not directly related to immunogenicity, the 
clinical presentation of cytokine release syndrome overlaps with anaphylaxis and other 
immunologically related adverse reactions.  Distinguishing this symptom complex from these 
other types of adverse reactions is potentially useful for the purpose of risk mitigation.  Although 
the underlying mechanisms may not be fully understood, in some cases the mechanism appears 
to relate to the cross-linking of activating cell surface expressed receptors, which are the targets 
of the therapeutic protein product (e.g., CD28 expressed on T-cells).  A risk-based evaluation, 
focused on the mechanism of action of the therapeutic protein product as well as results of 
animal and in vitro evaluations should be performed to determine the need for collection of pre- 
and post-dose cytokine levels in the early phase of clinical development.  In case of a clinical 
adverse event, such an evaluation may provide evidence to support the clinical diagnosis of 
cytokine release syndrome and help distinguish this entity from other acute drug reactions (e.g., 
anaphylaxis, see Appendix A.2).  
 
3.  Infusion Reactions 
 
Therapeutic protein products may elicit a range of acute effects, from symptomatic discomfort to 
sudden, fatal reactions that have often been grouped as infusion reactions in the past.  
Although the term implies a certain temporal relationship, infusion reactions are otherwise not 
  4  Contains Nonbinding Recommendations 
 
well defined and may encompass a wide range of clinical events, including anaphylaxis and 
other events that may not be directly related to antibody responses, such as cytokine release 
syndrome.  In the absence of an agreed-upon definition for infusion reaction, the categorization 
of certain adverse events as infusion reactions without further detail is problematic and is not 
recommended.  Sponsors are encouraged to use more-descriptive terminology when possible, 
noting the timing, duration, and specific signs and symptoms observed upon administration of a 
therapeutic protein product and to provide data from mechanistic studies which may facilitate a 
mitigation strategy. 
 
4.  Non-Acute Reactions  
 
Anaphylaxis, cytokine release syndrome, and other acute reactions are temporally linked to 
administration of a therapeutic protein product.  Delayed hypersensitivity (i.e., serum sickness) 
and immune responses secondary to immune complex formation typically have a subacute 
presentation.  As a result, the association between administration of a therapeutic protein product 
and non-acute reactions may be more difficult to establish, and assessment of the underlying 
mechanism will likely require evaluation of circulating immune complexes and complement 
activation.  Clinical signs may include delayed onset of fever, rash, arthralgia, myalgia, 
hematuria, proteinuria, serositis, central nervous system complications, and hemolytic anemia in 
the face of an ongoing antibody response to the therapeutic protein product (Hunley et al. 2004; 
Goto et al. 2009).  When such a reaction is suspected, laboratory assessment for circulating 
immune complexes may help confirm the diagnosis.  The necessity and details of a laboratory 
assessment will depend on the individual situation and should be discussed with the respective 
review division for the therapeutic protein product.  
 
5.  Cross-Reactivity to Endogenous Proteins 
 
ADA can have severe consequences if it cross-reacts to and inhibits a nonredundant endogenous 
counterpart of the therapeutic protein product or related proteins (Macdougall et al. 2012; Seidl 
et al. 2012).  If the endogenous protein is redundant in biological function, inhibition of the 
therapeutic and endogenous proteins may not produce an obvious clinical syndrome until the 
system is stressed, because not all biological functions of an endogenous protein may be known 
or fully characterized (Stanley et al. 1994; Bukhari et al. 2011).  Moreover, the long-term 
consequences of such antibodies may not be known.  An additional potential consequence of 
cross-reactivity to an endogenous protein results from antibody responses to a therapeutic protein 
product that is a counterpart of an endogenous cell surface receptor or a counterpart of an 
endogenous cytokine that is membrane-expressed.  Such antibodies may cross-reactively bind to 
the respective cell surface receptors or proteins, causing cytokine release or other manifestations 
of cellular activation. 
 
For therapeutic protein product counterparts of endogenous proteins that are critical to normal 
fetal or neonatal development, neutralization of such endogenous proteins, resulting from 
antibodies to the therapeutic protein product that cross react to the endogenous counterpart, has 
the potential to negatively impact fetal or neonatal development when these immune responses 
are generated or boosted during pregnancy or breast feeding.  As part of the risk evaluation, 
sponsors should consider the potential transmission of antibodies to the fetus by the placenta or 
  5  Contains Nonbinding Recommendations 
 
to the developing neonate by human milk.  Therefore, the risk of neutralizing antibody 
development following administration of such therapeutic protein products to women of 
childbearing potential should be strongly considered in light of the potential benefit.  Moreover, 
the risk of neutralizing antibody development to endogenous proteins critical in growth and 
development beyond the neonatal period should be evaluated in studies in pediatric populations.  
 
Although animal studies may provide useful information regarding the possible consequences of 
inhibition of an endogenous protein, particularly for endogenous proteins that are highly 
evolutionarily conserved, such studies are not considered to be predictive of the likelihood of an 
immune response to a therapeutic protein product in humans.  Moreover, differences in the 
timing and extent of transplacental transfer of maternal antibodies may limit the utility of animal 
studies to assess in utero effects of cross-reactive antibodies to the endogenous counterpart of the 
therapeutic protein product. 
 
IV.  RECOMMENDATIONS FOR IMMUNOGENICITY RISK MITIGATION IN 
THE CLINICAL PHASE OF DEVELOPMENT OF THERAPEUTIC PROTEIN 
PRODUCTS 
 
Given the variety of factors that can affect immunogenicity, the risk assessment and the control 
and mitigation strategies will depend on the individual development program and should be 
considered at the earliest stage and at each subsequent stage of product development.  The extent 
of immunogenicity safety information required premarketing and postmarketing will vary, 
depending on the potential severity of the consequences of such immune responses and the 
likelihood of their occurrence. 
 
In terms of evaluating the clinical relevance of immune responses, the Agency has the following 
recommendations: 
 
Development of assays for anti-drug antibody (ADA) 
 
  Sponsors should develop and implement sensitive immunoassays commensurate with 
the overall product development program.5  Concomitant assessment of levels of 
therapeutic protein product in the sample is recommended to assess the potential for 
the presence of the product to interfere with detection of antibody in the assay. 
 
Product-specific antibody sampling considerations 
 
  Baseline samples for ADA testing should be collected, and the post-baseline sampling 
frequency and duration should reflect anticipated use of the product.  More frequent 
                                                 
5 See the draft guidance for industry Assay Development for Immunogenicity Testing of Therapeutic Proteins, where 
assay development is covered in detail.  When final, this guidance will reflect the Agencys current thinking on this 
topic.  Guidance on appropriate assay development for immunogenicity testing is also available in the ICH 
guidances for industry Q2A Text on Validation of Analytical Procedures and Q2B Validation of Analytical 
Procedures: Methodology. 
 
  6  Contains Nonbinding Recommendations 
 
sampling is appropriate during the initiation and early use of a new, chronically 
administered product; less frequent sampling may be appropriate after prolonged use.  
Repeat sampling should generally occur over periods of sufficient duration to 
determine whether these responses are persistent, neutralizing, and associated with 
clinical sequelae.  Samples for antibody assessment should be drawn prior to 
administration of the therapeutic protein product.  
 
  In addition to a prespecified sampling schedule, unscheduled sampling, triggered 
by suspected immunologically related adverse events, is necessary for 
establishing the clinical relevance of ADAs.  Future sampling considerations for 
patients whose samples test positive for antibody at the end of a study should be 
discussed with the respective review division for the therapeutic protein product.  
Informed consent should address the possibility for sampling beyond study 
termination. 
 
  Banking of serum samples from clinical trials under appropriate storage conditions 
for future testing is always advisable. 
 
Dosing 
 
  For first-in-human trials, a conservative approach in an appropriate medical setting 
with access to immediate supportive care in the event of a serious adverse event, such 
as anaphylaxis, should be taken.  Staggered dosing among individual patients and 
dosing cohorts is appropriate.  The trial design should include prespecified dose 
escalation criteria and adequate time intervals between dosing cohorts and, as 
appropriate for the pharmacokinetics and pharmacodynamics of the product, between 
individuals within a dosing cohort to assess toxicities prior to administration of 
subsequent doses or treatment of additional individuals.  The need for such an 
approach will depend on individual circumstances.6  Aside from first-in-human trials, 
there may be other situations where a similarly conservative approach is indicated, 
e.g., change in the route of administration, change in formulation, change in container 
closure system.  As development progresses, dosing strategies and safety parameters 
can be modified based on clinical experience with the product and other products of 
the same class. 
 
  Because it may be difficult to predict the incidence of product-specific antibodies in 
different clinical trial scenarios, dosing regimens in subsequent studies should be risk 
based and take into account the following: data from initial trials; the potential for and 
predicted effects of cross-reactivity to endogenous proteins; the severity of effects of 
neutralization of the therapeutic protein product (e.g., a lifesaving versus adjunctive 
treatment product); clinical parameters that impact immunogenicity in different 
                                                 
6 See the guidance for industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for 
Therapeutics in Adult Healthy Volunteers. 
  7  Contains Nonbinding Recommendations 
 
patient populations; and the adequacy of proposed safety monitoring (Koren et al. 
2008). 
 
  Higher doses of therapeutic protein products do not uniformly overcome high titer 
and/or sustained or neutralizing antibody responses and may impact safety, e.g., may 
precipitate immune complex mediated disease or cause other toxicities.  The 
appropriateness of such a dose escalation strategy will depend on the specific product, 
the magnitude of the antibody response, and the disease indication.  A protocol 
defining specific safety monitoring evaluations and stopping rules should be 
developed prior to implementation of dose escalation to overcome an antibody 
response. 
 
Adverse events 
 
  The development of neutralizing antibody activity or the presence of sustained 
antibody titers may lead to loss of efficacy or an increased risk of an adverse reaction. 
In certain high-risk situations (e.g., assessment of a product with a nonredundant 
endogenous counterpart), real-time assessments for antibodies during a clinical trial 
may be recommended for safety reasons.  Real-time assessments entail analyses of 
the samples as soon as possible after sampling, before banking of the samples, and 
prior to additional dosing.  The need for such intensive monitoring will depend on 
individual circumstances. 
 
  If clinically relevant immune responses are observed, sponsors are encouraged to 
study the underlying mechanism and identify any critical contributing factors.  These 
investigations can facilitate development and adoption of potential control and 
mitigation strategies, including modification of product formulation and screening of 
higher-risk patients (see section V). 
 
  In some cases, sponsors may choose to explore premedication, desensitization, or 
immune tolerance induction procedures as potential mitigation strategies.  Given the 
risks associated with desensitization/immune tolerance induction procedures and the 
potential for premedication to mask early signs and symptoms of adverse events, the 
appropriateness of such procedures will depend on the nature of the specific 
indication, the target patient population, and the stage of development.  
 
Comparative immunogenicity studies 
 
  For all comparative immunogenicity studies (e.g., those comparing immunologically 
related adverse events, antibody incidence, titer, or neutralizing activity to product 
pre- and post-manufacturing changes), a strong rationale and, when possible, 
prespecified criteria should be provided to justify what differences in incidence or 
severity of immune responses would constitute an unacceptable difference in product 
  8  Contains Nonbinding Recommendations 
 
safety.7  The same antibody assay should be used to enable valid comparisons (see 
Appendix A.6). 
 
Postmarketing safety monitoring  
 
 Robust postmarketing safety monitoring is an important component in ensuring the 
safety and effectiveness of therapeutic protein products.  Because some aspects of 
postmarketing safety monitoring are product-specific, FDA encourages sponsors to 
consult with the appropriate FDA review division to discuss the sponsors proposed 
approach to postmarketing safety monitoring.  Rare, but potentially serious, safety risks 
(e.g., immunologically related adverse events) may not be detected during preapproval 
clinical testing, because the size of the population exposed may not be large enough to 
assess rare events.  In some cases, such risks may need to be evaluated through 
postmarketing surveillance or required studies or clinical trials. 
 
V.  PATIENT- AND PRODUCT-SPECIFIC FACTORS THAT AFFECT 
IMMUNOGENICITY 
 
A.  Patient-Specific Factors That Affect Immunogenicity 
 
Factors related to the target patient population may increase or decrease the potential for and the 
risk associated with an immune response.  Therefore, caution is recommended when moving 
from one patient population to another, and a new risk assessment should be performed for each 
new patient population considered for treatment. 
 
1.   Immunologic Status and Competence of the Patient 
 
Patients who are immune suppressed may be at lower risk of mounting immune responses to 
therapeutic protein products compared to healthy volunteers with intact immune responses.  For 
example, 95 percent of immune-competent cancer patients generated neutralizing antibody to a 
granulocyte-macrophage colony-stimulating factor (GM-CSF) product; but only 10 percent of 
immune-compromised cancer patients did so in response to a GM-CSF product (Ragnhammar et 
al. 1994).  Immune suppressive agents may diminish the immune response to therapeutic protein 
products.  Thus, agents that kill antigen-activated lymphocytes and/or elicit activity of regulatory 
T-cells, such as methotrexate, have been shown to have a substantial effect on immunogenicity 
of co-administered monoclonal or other antibody products (Baert et al. 2003).  In contrast to 
immune-deficient patients, patients with an activated immune system (e.g., patients with certain 
infections or autoimmune disease) may have augmented responses.  Immune response generation 
may also be affected by patient age, particularly at the extremes of the age range (LeMaoult et al. 
1997; PrabhuDas et al. 2011; Cuenca et al. 2013; Goronzy and Weyand 2013).  Particular 
caution with regard to immunogenicity and immune responses should be used in studies 
                                                 
7 For information on proposed biosimilar products, see the draft guidance for industry Scientific Considerations in 
Demonstrating Biosimilarity to a Reference Product.  When final, this guidance will represent the FDAs current 
thinking on this topic. 
  9  Contains Nonbinding Recommendations 
 
evaluating novel therapeutics in healthy volunteers (Li et al. 2001; Stebbings et al. 2007; 
Colombel et al. 2010; Garces et al. 2013). 
 
Recommendation 
 
In the development of therapeutic protein products, a rationale should be provided to support the 
selection of an appropriate study population, especially for first-in-human studies.  The potential 
influence of concomitant medications on ADAs should be taken into consideration during all 
stages of clinical development. 
 
2.  Prior Sensitization/History of Allergy  
 
Prior exposure to a therapeutic protein product or to a structurally similar protein may lead to 
pre-existing antibodies at baseline.  This is a particular concern for patients receiving a 
replacement product, such as clotting factors or an enzyme replacement therapy, who may have 
antibodies to a previous product that could cross-react to an analogous product.  
 
Sensitization to the excipients or process/product-related impurities of a therapeutic protein 
product may also predispose a patient to an adverse clinical consequence.  For example, products 
produced from transgenic sources may contain allergenic foreign proteins, such as milk protein 
or protein from chicken eggs. 
 
Because patient history may not capture all prior exposures that could generate a pre-existing 
antibody response or predict anaphylaxis, screening for pre-existing antibodies, e.g., inhibitors or 
neutralizing antibodies in factor replacement therapy, should be considered when appropriate.  
 
Recommendation 
 
Screening for a history of relevant allergies pertaining to the source material of the therapeutic 
protein product (e.g., produced in transgenic hen eggs versus mammalian cells) is recommended, 
and the appropriateness of additional clinical or laboratory tests prior to administration should be 
considered in light of the overall risk-benefit assessment. 
 
3.  Route of Administration, Dose, and Frequency of Administration 
 
Route of administration can affect the risk of sensitization.  In general, intradermal, 
subcutaneous, and inhalational routes of administration are associated with increased 
immunogenicity compared to intramuscular and intravenous (IV) routes.  The IV route is 
generally considered to be the least likely to elicit an immune response.  In conjunction with the 
route of administration, dose and frequency can also affect immunogenicity (Rosenberg and 
Worobec 2004).  For example, in certain circumstances, a lower dose administered intermittently 
may be more immunogenic than a larger dose administered without interruption.  It should be 
noted that the effects of dose and frequency on ADA development may be affected by other 
factors, such as route of administration, product origin, and product-related factors that influence 
immunogenicity.  
 
  10  Contains Nonbinding Recommendations 
 
Recommendations 
 
Immunogenicity should be considered when selecting an appropriate route of administration, 
especially for high-risk therapeutic protein products (e.g., therapeutic counterparts of 
nonredundant endogenous proteins) in first-in-human dosing.  
 
Changes in the route of administration or dosing during product development may be associated 
with changes in the immunogenicity profile, and clinical safety data should be obtained to 
support such changes. 
 
4.  Genetic Status  
 
Genetic factors may modulate the immune response to a therapeutic protein product.  In 
particular, some human leukocyte antigen (HLA) haplotypes may predispose patients to 
development of undesirable antibody responses to specific products (Hoffmann et al. 2008).  If 
both appropriate and feasible, HLA mapping studies may help define a subset of the patient 
population at increased risk.  Moreover, genetic polymorphisms in cytokine genes may 
upregulate or downregulate immune responses (Donnelly et al. 2011). 
 
Recommendation 
 
Evaluation of genetic factors that may modulate the immune response to a therapeutic protein 
product is recommended in circumstances in which a subset of treated patients lose the clinical 
benefit of treatment or experience severe adverse events.  For example, the subset of patients that 
generate neutralizing antibodies to IFN-beta products are more likely to possess distinct HLA 
haplotypes (Hoffmann et al. 2008).  Thus, knowledge of the heightened susceptibility of patients 
with such HLA haplotypes may allow for measures to prevent such responses or for pursuit of 
other treatment options. 
 
5.  Status of Immune Tolerance to Endogenous Protein  
 
Humans are not equally immunologically tolerant to all endogenous proteins.  Thus, the 
robustness of immune tolerance to an endogenous protein affects the ease with which a 
therapeutic protein product counterpart of that endogenous protein can break such tolerance.  
Immunological tolerance in both protein-specific T- and B-cells depends on many factors, 
prominent among which is the abundance of the endogenous protein: immune tolerance is 
weaker for low-abundance and stronger for high-abundance proteins (Weigle 1980; Goodnow 
1992; Haribhai et al. 2003). 
 
The human immune system is not fully tolerant to low-abundance endogenous proteins, such as 
cytokines and growth factors, for which serum levels may be in the nanogram (ng)/milliter (mL) 
to picogram (pg)/mL range.  This point is underscored by the presence of autoantibodies to 
cytokines and growth factors in healthy individuals, the development of antibodies to 
inflammatory cytokines, and the breaking of tolerance to endogenous proteins by administration 
of exogenous recombinant therapeutic protein products (Hermeling et al. 2004; Rosenberg and 
Worobec 2004; Rosenberg and Worobec 2005; Koren et al. 2008). 
  11  Contains Nonbinding Recommendations 
 
 
When a human therapeutic protein product is intended as a replacement for an absent or deficient 
endogenous protein, patients with genetic mutations conferring a protein knock out phenotype 
may respond to the therapeutic protein product as to a foreign protein or neoantigen or may 
already be sensitized as a result of previous exposure to a similar therapeutic protein or related 
proteins from other sources.  Such responses may abrogate the efficacy of the replacement 
therapy.  
 
Recommendations 
 
For a therapeutic protein product that is a counterpart of an endogenous protein, the robustness of 
immune tolerance to the endogenous protein should be investigated before initiating a clinical 
trial and such evaluation should consider the following as preeminent risks:  if the clinical study 
is a first-in-human use, if the endogenous protein has a nonredundant physiological function, and 
if immune responses to the endogenous protein have been detected in the context of autoimmune 
diseases.  Suggested evaluations include: 
 
  Quantitating or gathering information on the level of the endogenous protein in 
serum in the steady state, as well as in conditions that may specifically elicit its 
production (Weigle 1980). 
  Assessing for or gathering information on the presence of pre-existing antibodies 
in healthy individuals and patient populations and on the frequency and role of 
such antibodies in autoimmune diseases (Bonfield et al. 2002; Hellmich et al. 
2002). 
  Evaluating immunogenicity, immune cell activation, inflammatory responses, and 
cytokine release in relevant animal studies to obtain insight and provide guidance 
for clinical safety assessments (Koren 2002) (also see Appendix A.5). 
  In patients requiring factor/enzyme replacement therapies, evaluation of patient 
tissue samples for detection of endogenous protein or peptides (e.g., cross-
reactive immunologic material (CRIM)), as well as for genetic mutations and 
HLA alleles (as appropriate), should be strongly considered to better predict the 
development of immune responses to the replacement therapy and to evaluate the 
need for tolerance induction mitigation strategies (Pandey et al. 2013). 
  Evaluating the extent of polymorphisms, including single nucleotide 
polymorphisms, when appropriate, in relevant patient populations to identify 
potential mismatches with the therapeutic protein product (Jefferis and Lefranc 
2009; Viel et al. 2009; Pandey et al. 2013). 
 
 
B.  Product-Specific Factors That Affect Immunogenicity 
 
Product-specific factors may increase or decrease the potential for and the risk associated with an 
immune response.  Immunogenicity testing should be considered when changes are made to 
product-specific factors.    
 
 
  12  Contains Nonbinding Recommendations 
 
1.  Product Origin (foreign or human) 
 
Immune responses to nonhuman (i.e., foreign) proteins are expected and, as previously 
explained, may be anticipated for endogenous human proteins.  Moreover, mismatches between 
the sequence of the endogenous protein of the patient and that of the therapeutic protein product 
caused by naturally occurring polymorphisms are one risk factor for the development of immune 
responses to the therapeutic protein product (Viel et al. 2009).  However, the rapidity of 
development, the strength (titer), and the persistence of the response may depend on a number of 
factors, including the following: previous and ongoing environmental exposure and the mode of 
such exposure; the presence in the product of immunity-provoking factors, such as product 
aggregates and materials with adjuvant activity; and the products inherent immunomodulatory 
activity (see section V.B.6).  For example, environmental exposure to bacterial proteins from 
either commensal or pathogenic bacteria on skin or in the gut may predispose to generation of 
immune responses when such bacterial proteins (either recombinantly or naturally derived) are 
used as therapeutics. 
 
For proteins derived from natural sources, antibodies can develop not only to the desired 
therapeutic protein product, but also to other foreign protein components potentially present in 
the product.  Furthermore, such foreign proteins may contain regions of homology to 
endogenous human proteins.  The capacity of the foreign protein to break tolerance and induce 
antibody responses to the homologous human factor should be evaluated in the clinical trial.  For 
example, during treatment with a bovine thrombin product, immune responses to bovine 
coagulation factor V, incidentally present in the product, led to development of antibodies that 
cross-reacted to human factor V and resulted in life-threatening bleeding in some patients 
(Kessler and Ortel 2009). 
 
For monoclonal antibodies, product origin is an important factor that can influence 
immunogenicity.  Although mouse antibodies have been shown to robustly elicit immune 
responses in humans as compared to chimeric, humanized and human monoclonal antibodies, it 
should be noted that chimeric, humanized and human monoclonal antibodies can also elicit a 
high rate of immunogenicity depending on the dosing regimen and patient population (Singh 
2011).  In fact, some human antibodies developed using phage display may have significant 
ADA responses. 
 
Moreover,  novel structural formats, including fusion proteins, bispecific or multispecific 
antibodies (bivalent or tetravalent), single chain fragments, single domain antibodies, and 
specifically engineered antibodies with mutations in the constant or variable regions, may elicit 
immune responses, as such novel structures may create neoantigens or expose cryptic epitopes.  
In addition, site-specific mutations in constant regions may create novel allotypes, and the use of 
an in vitro affinity maturation process may result in novel idiotypes.  An understanding of the 
increased immunogenicity associated with certain antibody products will require more complete 
characterization of the ADA response, such as identification of the target epitope(s) (Singh 
2011). 
 
  13  Contains Nonbinding Recommendations 
 
Recommendations 
 
All therapeutic protein products should be evaluated for their content of and immune responses 
directed to incidental product components, including proteins and nonprotein components.  A 
risk-based evaluation of potential immune responses to such process- and product-related 
impurities should be performed, and a testing program should be designed based on this 
evaluation.  Foreign proteins intended for therapeutic use should be evaluated for molecular 
regions that bear strong homology to endogenous human proteins.  When such homologies exist, 
assessment of antibodies to the homologous human protein should be made in addition to 
assessment of antibodies to the foreign therapeutic protein.   
 
When developing assays to assess the immunogenicity of novel antibody-related products, 
appropriate controls should be incorporated into the assays to determine if the ADA response is 
directed against novel epitopes. 
 
2.  Primary Molecular Structure/Posttranslational Modifications  
 
Primary sequence, higher-order structure, species origin, and molecular weight of therapeutic 
protein products are all important factors that may contribute to immunogenicity.  Primary 
sequence analysis can reveal potentially immunogenic sequence differences in proteins that are 
otherwise relatively conserved between humans and animals.  In some cases, nonhuman epitopes 
may elicit T-cell help or facilitate epitope spreading to generate an antibody response to the 
conserved human sequences (Dalum et al. 1997).  Per section V.A.4, it is important to note that 
therapeutic protein products of human origin may elicit immune responses in subsets of patients 
with distinct HLA haplotypes as well as in patients whose endogenous protein amino acid 
sequence differs from that of the therapeutic protein product, even by single nucleotide 
polymorphisms. 
 
Additional advanced analyses of primary sequence are also likely to detect HLA class II binding 
epitopes in nonpolymorphic human proteins.  Such epitopes may elicit and activate regulatory T-
cells, which enforce self-tolerance, or, opposingly, could activate T-helper (Th) cells when 
immune tolerance to the endogenous protein is not robust (Barbosa and Celis 2007; Tatarewicz 
et al. 2007; De Groot et al. 2008; Weber et al. 2009).  However, if considered appropriate, 
engineering of changes to the primary sequence to eliminate immunogenic Th cell epitopes or 
addition of tolerogenic T-cell epitopes should be done cautiously, because these modifications 
may alter critical product quality attributes such as aggregation, deamidation, and oxidation and 
thus alter product stability and immunogenicity.  Therefore, extensive evaluation and testing of 
critical product attributes should be performed following such changes.  Primary sequence 
considerations are especially important in evaluation of the immunogenicity of fusion proteins, 
because immune responses to neoantigens formed in the joining region may be elicited (Miller et 
al. 1999) and may then spread to conserved segments of the molecule.  Fusion proteins 
consisting of a foreign protein and an endogenous protein are of particular concern because of 
the capacity of the foreign protein to elicit T-cell help for generation of an antibody response to 
the endogenous protein partner.  Similarly, bioengineered proteins involve the introduction of 
sequences not normally found in nature and may thus contain neo-epitopes.  These epitopes have 
  14  Contains Nonbinding Recommendations 
 
the potential to broadly elicit immune responses or may instead interact with HLA alleles found 
only in a subset of patients to induce immune responses (Kimchi-Sarfaty et al. 2013).  
 
Chemical modifications of therapeutic protein products, such as oxidation, deamidation, 
aldehyde modification, and deimination, may elicit immune responses by, for example, 
modifying primary sequence, causing aggregate formation, or altering antigen processing and 
presentation.  Importantly, such changes may be well controlled during manufacture and storage, 
but may occur in vivo in the context of the relatively high pH of the in vivo environment or in 
inflammatory environments and cause loss of activity as well as elicitation of immune responses.  
Evaluation of therapeutic protein products in the context of the in vivo environments to which 
they are targeted can reveal susceptibility to such chemical modifications. (Huang et al. 2005; 
Demeule et al. 2006; Makrygiannakis et al. 2006).  Susceptibility to chemical modifications of 
therapeutic protein products, and thus the possibility of loss of activity or induction of immune 
responses in vivo, should prompt consideration of careful protein engineering. 
 
Recommendations  
 
Careful consideration should be given to the primary sequences chosen for development of 
therapeutic protein products in general, and especially of therapeutic protein product counterparts 
of endogenous proteins in view of potential polymorphisms in endogenous proteins across 
human populations.  
 
The ADA response to fusion molecules or engineered versions of therapeutic protein products 
should utilize assays that are able to assess reactivity to the whole molecule as well as to its 
distinct components.  Immune responses directed to the intact protein product, but not reactive 
with either of the separate partner proteins, may be targeting novel epitopes in the fusion region.  
 
Evaluation of therapeutic protein products in the in vivo milieu in which they function (e.g., in 
inflammatory environments or at physiologic pH) may reveal susceptibilities to modifications 
(e.g., aggregation and deamidation) that result in loss of efficacy or induction of immune 
responses.  Such information may facilitate product engineering to enhance the stability of the 
product under such stress conditions.  Sponsors should consider obtaining this information early 
in product design and development.  
 
3.  Quaternary Structure: Product Aggregates and Measurement of Aggregates  
 
Protein aggregates are defined as any self-associated protein species, with monomer defined as 
the smallest naturally occurring and/or functional subunit.  Aggregates are further classified 
based on five characteristics: size, reversibility/dissociation, conformation, chemical 
modification, and morphology (Narhi et al. 2012).  Aggregates ranging from dimer to visible 
particles that are hundreds of micrometers in size (Narhi et al. 2012) have been recognized for 
their potential to elicit immune responses to therapeutic protein products for over a half-century 
(Gamble 1966).  Mechanisms by which protein aggregates may elicit or enhance immune 
responses include the following: extensive cross-linking of B-cell receptors, causing efficient B-
cell activation (Dintzis et al. 1989; Bachmann et al. 1993); and enhancing antigen uptake, 
processing, and presentation; and triggering immunostimulatory danger signals (Seong and 
  15  Contains Nonbinding Recommendations 
 
Matzinger 2004).  Such mechanisms may enhance recruitment of the T-cell help needed for 
generation of high-affinity, isotype-switched IgG antibody, the antibody response most often 
associated with neutralization of product efficacy (Bachmann and Zinkernagel 1997). 
 
The potential clinical consequences of immune responses induced by protein aggregates may in 
large measure depend on the loss or preservation of native epitopes in the aggregate.  Some 
antibodies generated by aggregates containing native protein can bind to monomeric protein as 
well as to the aggregate, with the potential to inhibit or neutralize product activity.  In contrast, 
some antibodies to denatured/degraded protein bind uniquely to the aggregated material, but not 
to native protein monomers, such as was the case with early preparations of human intravenous 
immune globulin (IVIG) (Barandun et al. 1962; Ellis and Henney 1969).  Responses to 
aggregates containing degraded epitopes have been shown to cause anaphylaxis, but do not 
inhibit or neutralize activity of the native protein (Ellis and Henney 1969). 
 
Critical information is lacking regarding the types and quantities of aggregates needed to 
generate immune responses for any given therapeutic protein product (Marszal and Fowler 
2012), although there is evidence that higher-molecular-weight aggregates and particles are more 
potent in eliciting such responses than lower-molecular-weight aggregates (Dintzis et al. 1989; 
Bachmann et al. 1993; Joubert et al. 2012).  The aggregates formed and the quantities that 
efficiently elicit immune responses also may differ for different products and in different clinical 
scenarios.  Furthermore, the use of any single method for assessment of aggregates is not 
sufficient to provide a robust measure of protein aggregation.  For example, sole use of size 
exclusion chromatography may preclude detection of higher-molecular-weight aggregates that 
fail to traverse the column prefilter, yet may be the most crucial species in generating immune 
responses.  Moreover, it has been recognized that subvisible particulates in the size range of 0.1
10 microns have a strong potential to be immunogenic, but are not precisely monitored by 
currently employed technologies (Berkowitz 2006; Roda et al. 2009; Gross and Zeppezauer 
2010; Mahler and Jiskoot 2012).  These very large aggregates may contain thousands to millions 
of protein molecules and may be homogeneous or heterogeneous (e.g., protein molecules 
adhered to glass or metal particles). 
 
Recommendations  
 
It is critical for manufacturers of therapeutic protein products to minimize protein aggregation to 
the extent possible.  Strategies to minimize aggregate formation should be developed as early as 
feasible in product development.  This can be done by using an appropriate cell substrate, 
selecting manufacturing conditions that minimize aggregate formation, employing a robust 
purification scheme that removes aggregates to the greatest extent possible, and choosing a 
formulation (see section V.B.7) and container closure system (see section V.B.8) that minimize 
aggregation during storage.  It is particularly important that product expiration dating take into 
account any increase in protein aggregates associated with protein denaturation or degradation 
during storage.  
 
Methods that individually or in combination enhance detection of protein aggregates should be 
employed to characterize distinct species of aggregates in a product.  Methods for measuring 
aggregation are constantly evolving and improving.  Constant improvement and development of 
  16  Contains Nonbinding Recommendations 
 
these methods should be considered in choosing one or more appropriate assays.  Assays should 
be validated for use in routine lot release and stability evaluations, and several of them should be 
employed for comparability assessments.  Animal studies may be useful in identifying aggregate 
species that have the potential to be immunogenic, although additional considerations (amount 
and types of aggregates, route of administration, etc.) may determine the extent to which such 
aggregate species pose clinical risk. 
 
Assessment should be made of the range and levels of subvisible particles (210 microns) 
present in therapeutic protein products initially and over the course of the shelf life.  Several 
methods are currently qualified to evaluate the content of subvisible particulates in this size 
range (Mahler and Jiskoot 2012). As more methods become available, sponsors should strive to 
characterize particles in smaller (0.12 microns) size ranges.  Sponsors should conduct a risk 
assessment of the impact of these particles on the clinical performance of the therapeutic protein 
product and develop control and mitigation strategies based on that assessment, when 
appropriate.  
 
4.  Glycosylation/Pegylation  
 
Glycosylation may strongly modulate immunogenicity of therapeutic protein products.  Although 
foreign glycoforms such as mammalian xenogeneic sugars (Chung et al. 2008; Ghaderi et al. 
2010), yeast mannans (Bretthauer and Castellino 1999), or plant sugars (Gomord and Faye 2004) 
may trigger vigorous innate and acquired immune responses, glycosylation of proteins with 
conserved mammalian sugars generally enhances product solubility and diminishes product 
aggregation and immunogenicity.  Glycosylation indirectly alters protein immunogenicity by 
minimizing protein aggregation, as well as by shielding immunogenic protein epitopes from the 
immune system (Wei et al. 2003; Cole et al. 2004).  Pegylation of therapeutic protein products 
has been found to diminish their immunogenicity via similar mechanisms (Inada et al. 1995; 
Harris et al. 2001), although immune responses to the polyethylene glycol (PEG) itself have been 
recognized and have caused loss of product efficacy and adverse safety consequences (Liu et al. 
2011).  Anti-PEG antibodies have also been found to be cross-reactive between pegylated 
products (Garay et al. 2012; Schellekens et al. 2013). 
 
Recommendations  
 
For proteins that are normally glycosylated, use of a cell substrate production system and 
appropriate manufacturing methods that glycosylate the therapeutic protein product in a 
nonimmunogenic manner is recommended.   
 
For pegylated therapeutic protein products, the ADA assay should be able to detect both the anti-
protein antibodies and antibodies against the PEG moiety.  The same principle may apply to 
modifications where the therapeutic protein products that are not pegylated but are modified with 
other high molecular weight entities, e.g., hydroxyethyl starch. 
 
  17  Contains Nonbinding Recommendations 
 
5.  Impurities with Adjuvant Activity  
 
Adjuvant activity can arise through multiple mechanisms, including the presence of microbial or 
host-cell-related impurities in therapeutic protein products (Verthelyi and Wang 2010; Rhee et 
al. 2011; Eon-Duval et al. 2012; Kwissa et al. 2012).  These innate immune response modulating 
impurities (IIRMIs), including lipopolysaccharide, glucan and flagellin, high-mobility group 
protein B1 (HMGB1), and nucleic acids, exert immune-enhancing activity by binding to and 
signaling through toll-like receptors or other pattern-recognition receptors present on B-cells, 
dendritic cells, and other antigen-presenting cell populations (Iwasaki and Medzhitov 2010; 
Verthelyi and Wang 2010).  This signaling prompts maturation of antigen-presenting cells and/or 
serves to directly stimulate B-cell antibody production. 
 
Recommendations  
 
It is very important for manufacturers to minimize the types and amounts of such microbial or 
host-cell-related impurities in therapeutic protein products. 
  
Assays to evaluate the types of IIRMIs present should be tailored to the relevant cell substrate.  
Because even trace levels of IIRMIs can modify the immunogenicity of a therapeutic protein 
product, the assays used to detect them should have sensitivities to assess levels that may lead to 
clinically relevant immune responses.  
 
If biomarkers are used to detect and compare the presence of IIRMIs, they should be tailored to 
the IIRMIs that could be present in the product.  Examples of biomarkers could include cytokine 
release and transcription factor activation from defined cell populations.  
 
6.  Immunomodulatory Properties of the Therapeutic Protein Product  
 
The immunomodulatory activity of any given therapeutic protein product critically influences 
not only the immune response directed to itself but also immune responses directed to other co-
administered therapeutic protein products, endogenous proteins, or even small drug molecules 
and may not be predictable.  For example, interferon-alpha (Gogas et al. 2006; Tovey and 
Lallemand), interleukin-2 (Franzke et al. 1999), and GM-CSF (Hamilton 2008) are not only 
relatively immunogenic of themselves but also are known to upregulate immune responses to 
endogenous proteins and to induce clinical autoimmunity.  Immunosuppressive therapeutic 
proteins may globally downregulate immune responses, raising the possibility of serious 
infections.  However, not all immunosuppressive therapeutic protein products suppress responses 
to themselves.  For example, integrin and TNF monoclonal antibodies tend to be immunogenic.  
Thus, the immunogenicity of such therapeutic protein products should be evaluated empirically. 
 
Recommendations 
 
The immunomodulatory properties of therapeutic protein products, their effects on immune 
responses to themselves, and their capacity to induce autoimmunity should be monitored from 
the earliest stages of product development (Franzke et al. 1999; Gogas et al. 2006; Hamilton 
2008). 
  18  Contains Nonbinding Recommendations 
 
 
Vaccination using live attenuated organisms should be avoided when the therapeutic protein 
product is immunosuppressive.  Updated vaccination status, compliant with local health care 
standards, is recommended for patients before administration of the therapeutic protein product.  
 
7.  Formulation  
 
Formulation components are principally chosen for their ability to preserve the native 
conformation of the therapeutic protein in storage by preventing denaturation due to hydrophobic 
interactions, as well as by preventing chemical degradation, including truncation, oxidation, and 
deamidation (Cleland et al. 1993; Shire et al. 2004; Wakankar and Borchardt 2006).  Large 
protein excipients in the formulation, such as human serum albumin (HSA), may affect 
immunogenicity positively or negatively.  Excipients such as HSA, although added for their 
ability to inhibit hydrophobic interactions, may coaggregate with the therapeutic protein or form 
protein adducts under suboptimal storage conditions (Braun and Alsenz 1997).  Polysorbate, a 
nonionic detergent, is the most commonly used alternative to HSA.  The stability of both types of 
excipients (i.e., HSA and polysorbate) should be kept in mind for formulation purposes because 
they too are subject to modifications (e.g., oxidation), which may then pose a threat to the 
integrity of the therapeutic protein product.  
 
Formulation may also affect immunogenicity of the product by altering the spectrum of 
leachables from the container closure system.  Leachables from rubber stoppers have been shown 
to possess immune adjuvant activity, as shown in an animal experiment (Mueller et al. 2009).  
Organic compounds with immunologic activity as well as metals have been eluted from 
container closure materials by polysorbate-containing formulations, leading to increased 
oxidation and aggregation (Seidl et al. 2012).  
 
Recommendations  
 
Excipients should be evaluated for their potential to prevent denaturation and degradation of 
therapeutic protein products during storage.  Interactions between excipients and therapeutic 
proteins should be carefully evaluated, especially in terms of co-aggregation or formation of 
protein-excipient adducts.  
 
Excipient stability should be carefully considered when establishing product shelf life.  
Thorough analyses of leachables and extractables should be performed to evaluate the capacity 
of container closure materials to interact with and modify the therapeutic protein product.  A risk 
assessment should be conducted, and control and risk mitigation strategies should be developed 
as appropriate.  
 
8.  Container Closure Considerations  
 
Interactions between therapeutic protein products and the container closure may negatively affect 
product quality and immunogenicity.  These interactions are more likely with prefilled syringes 
of therapeutic protein products.  These syringes are composed of multiple surfaces and materials 
that interact with the therapeutic protein product over a prolonged time period and thus have the 
  19  Contains Nonbinding Recommendations 
 
potential to alter product quality and immunogenicity.  The following are other container closure 
considerations pertinent to immunogenicity:  
 
  Glass and air interfaces can denature proteins and cause aggregation in glass 
syringes and vials.  
 
  Glass vials have been known to delaminate at higher pH and with citrate 
formulations, potentially creating protein-coated glass particles, which may 
enhance immunogenicity of the therapeutic protein product (Fradkin et al. 2011).  
 
  Silicone oil-coated syringe components provide a chemical and structural 
environment on which proteins can denature and aggregate. 
 
  Appropriate in-use stability studies should be performed to confirm that 
conditions needed to maintain product quality and prevent degradation are 
adequately defined. 
 
  Leached materials from the container closure system may be a source of materials 
that enhance immunogenicity, either by chemically modifying the therapeutic 
protein product or by having direct immune adjuvant activity, including the 
following:  
 
  Organic compounds with immunomodulatory activity may be eluted from 
o
container closure materials by polysorbate-containing formulations: a 
leachable organic compound involved in vulcanization was found in a 
polysorbate formulated product when the stopper surfaces were not Teflon 
coated (Boven et al. 2005).  
 
  Metals that oxidize and aggregate therapeutic protein products or activate 
o
metalloproteinases have been found in various products contained in 
prefilled syringes or in vials.  For example, tungsten oxide that leached 
from the syringe barrel was reported to cause protein aggregation (Bee et 
al. 2009); and leached metals from vial stoppers caused increased 
proteolysis of a therapeutic protein because of activation of a 
metalloproteinase that co-purified with the product.  
 
Recommendations  
 
Whenever possible, sponsors should obtain detailed information regarding a description of all 
raw materials used in the manufacture of the container closure systems for their products.  
Sponsors should conduct a comprehensive extractables and leachables laboratory assessment 
using multiple analytical techniques to assess the attributes of the container-closure system that 
could interact with and degrade protein therapeutic products. 
 
Because the United States Pharmacopeia elastomeric closures for injections tests do not 
adequately characterize the impact of leachables in storage containers on therapeutic protein 
  20  Contains Nonbinding Recommendations 
 
products under real-time storage conditions, leachables must be evaluated for each therapeutic 
protein product in the context of its storage container under real-time storage conditions8.  
 
Testing for leachables should be performed on the product under stress conditions,9 as well as 
under real-time storage conditions, because in some cases the amount of leachables increases 
dramatically over time and at elevated temperatures.  Product compatibility testing should be 
performed to assess the effects of container closure system materials and all leachables on 
product quality.  
 
9.  Product Custody  
 
Products in their intended primary packaging container closure system should be tested for 
stability in protocols that include appropriate in-use conditions (e.g., light, temperature, and 
agitation) to identify conditions and practices that may cause product denaturation and 
degradation.  
 
Given that most therapeutic protein products degrade on exposure to heat and light or with 
mechanical agitation, to ensure product quality, health care practitioners and patients should be 
educated regarding product storage, handling, and administration.  
 
A secure supply chain is critical.  Appropriate temperature-controlled transport and storage is of 
utmost importance in preserving product quality.  For example, the storage of epoetin- under 
inappropriate conditions by unauthorized vendors was associated with high levels of aggregates 
and antibody-mediated pure red cell aplasia (Fotiou et al. 2009).  
 
Recommendations  
 
Patient educational materials (e.g., FDA-approved patient labeling providing instructions for use 
as required under 21 CFR 201.57 and 201.80) should explicitly identify appropriate storage and 
handling conditions of the product.  Appropriate patient instruction by caregivers is vital to 
ensuring product quality and helping to minimize adverse impacts on product quality during 
product storage and handling.  Appropriate temperature-controlled transport and storage should 
be ensured.  
 
 
VI.  CONCLUSION  
 
The consequences of immune responses to therapeutic protein products can range from no 
apparent effect to serious adverse events, including life-threatening complications such as 
anaphylaxis, neutralization of the effectiveness of lifesaving or highly effective therapies, or 
neutralization of endogenous proteins with nonredundant functions.  Although immunogenicity 
risk factors pertaining to product quality attributes and patient/protocol factors are understood, 
immune responses to therapeutic protein products cannot be predicted based solely on 
                                                 
8 21 CFR 600.11(b) and (h). 
9 See the ICH guidance for industry Q1A(R2) Stability Testing Of New Drug Substances And Products. 
  21  Contains Nonbinding Recommendations 
 
characterization of these factors, but should be evaluated in the clinic.  A risk-based approach, as 
delineated in this guidance, provides investigators with the tools to develop novel protein 
therapeutics, evaluate the effect of manufacturing changes, and evaluate the potential need for 
tolerance-inducing protocols when severe consequences result from immunogenicity.  
  22  Contains Nonbinding Recommendations 
 
 
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APPENDIX A 
 
1.  Diagnosis of Anaphylaxis 
 
The diagnosis of anaphylaxis is based on the following three clinical criteria, with anaphylaxis 
considered as highly likely when one of these criteria is fulfilled (Sampson et al. 2006):  
 
a.  Acute onset of an illness (minutes to several hours) with involvement of the skin, 
mucosal tissue, or both (e.g., generalized hives, pruritus or flushing, swollen lips-
tongue-uvula), and at least one of the following: 
  Respiratory compromise (e.g., dyspnea, wheeze-bronchospasm, stridor, reduced 
peak expiratory flow, hypoxemia) 
  Reduced blood pressure or associated symptoms of end-organ dysfunction (e.g., 
hypotonia [collapse], syncope, incontinence)  
 
b.  Two or more of the following that occur rapidly after exposure to a likely allergen for 
that patient (minutes to several hours):  
  Involvement of the skin-mucosal tissue (e.g., generalized hives, itch-flush, 
swollen lips-tongue-uvula) 
  Respiratory compromise (e.g., dyspnea, wheeze-bronchospasm, stridor, reduced 
peak expiratory flow, hypoxemia) 
  Reduced blood pressure or associated symptoms (e.g., hypotonia [collapse], 
syncope, incontinence) 
  Persistent gastrointestinal symptoms (e.g., crampy abdominal pain, vomiting)  
 
c.  Reduced blood pressure after exposure to known allergen for that patient (minutes to 
several hours):  
  Infants and children: low systolic blood pressure (age specific) or greater than 30-
percent decrease in systolic blood pressure  
  Adults: systolic blood pressure of less than 90 mm Hg or greater than 30-percent 
decrease from that persons baseline 
 
Although none of the clinical criteria provide 100-percent sensitivity and specificity, it is 
believed that these criteria are likely to capture more than 95 percent of cases of anaphylaxis 
(Sampson et al. 2006). 
 
Laboratory tests for evaluating anaphylaxis:  
 
At present, there are no sensitive and specific laboratory tests to confirm the clinical diagnosis of 
anaphylaxis.  Skin testing and in vitro diagnostic tests to determine the level of specific IgE 
antibodies directed against the therapeutic protein product, mediator release, or basophil 
activation may be useful for characterizing the underlying pathophysiology and may provide 
insight into potential mitigation strategies (Simons 2010; Lee and Vadas 2011).  However, the 
results of unvalidated tests should be interpreted with caution; and the clinical relevance of 
positive results from unvalidated tests may be uncertain during product development. 
  31  Contains Nonbinding Recommendations 
 
 
2.  Cytokine Release Syndrome  
 
Monoclonal antibodies specific for cell surface receptors or for cell membrane expressed 
cytokines, as well as antibodies that develop in patients to therapeutic protein products that bind 
to cell surface receptors, have the potential to augment a products intrinsic agonist activity and 
exacerbate infusion-related toxicities.  In vitro assessments of the capacity of such therapeutic 
protein products to mediate cellular activation, including proliferation and cytokine release in 
human whole blood or peripheral blood mononuclear cells, are recommended.  For products with 
the potential to incur a cytokine release syndrome, an initial starting dose below that obtained by 
traditional calculations and slower infusion rates, where applicable, may also be recommended 
(Duff 2006).  Pre- and post-administration levels of C-reactive protein and cytokines, such as 
TNF-; IL-2; IL-6; IL-10; and IFN-, and certain clinical signs and symptoms, such as an acute 
elevation of body temperature, erythema, and hypotension, may serve as markers of a 
proinflammatory response pertaining to cytokine release.  
 
Data from both animal studies and in vitro assessments may provide information to guide 
development of therapeutic protein products with the potential to induce cytokine release.  
Although data from both animal studies and in vitro assessments may supplement each other, 
they generally are not fully predictive of the clinical occurrence or outcome.  Therefore it is 
imperative that great caution is always exercised in the clinical development of products with the 
potential to mediate receptor cross-linking (see sections III.B.1 and III.B.2).  Although the 
traditional animal models used for toxicology testing (i.e., rat, mouse, dog, and cynomolgus 
monkey) rarely demonstrate overt toxicities related to lymphocyte activation and cytokine 
release, specific markers related to T-cell activation and cytokine release can be measured in 
routine toxicology studies, provided that the drug is pharmacologically active in the test species.  
These data may then be useful for predicting the potential for these agents to induce a cytokine 
release syndrome in the clinic or for evaluating the activity of second-generation agents that have 
been modified to reduce their level of T-cell activation.  For example, cytokine production can 
be measured in blood samples obtained from treated animals during pharmacokinetic or general 
toxicology studies, provided that the amount of samples obtained does not compromise the 
health of the animals or the ability to evaluate the toxicology endpoints at study termination.  
When evaluation of cytokine release is included in animal testing, measurement of a cytokine 
panel that is as broad as possible and includes IL-2, IL-6, IFN-, and TNF-, as well as other 
relevant cytokines indicative of cytokine release syndrome, is recommended.  Such proposed 
animal studies should be discussed with FDA prior to initiation (Hsu et al. 1999; Norman et al. 
2000).  In vitro assessments of cellular activation, including proliferation and cytokine release in 
human whole blood or peripheral blood mononuclear cells, are important assessment tools that 
can help in overcoming the known limitations of animals in modeling activating stimuli in some 
T-cell subsets (Stebbings et al. 2007; Hellwig et al. 2008; Findlay et al. 2011; Romer et al. 2011; 
Stebbings et al. 2013).  The impact of product cross-linking of cellular receptors should be 
considered in such studies.  Signs of cellular activation in vitro should also be taken as an 
indication that the product has the potential to induce toxicities in the clinic, regardless of 
negative findings from animal studies.  
 
 
  32  Contains Nonbinding Recommendations 
 
3.  Non-Acute Immune Responses  
 
Type III hypersensitivity responses, including those mediated by immune complexes and T-cells 
(delayed hypersensitivity responses in the older literature), are relatively rare with respect to 
therapeutic protein products; and a high degree of clinical suspicion is necessary for the 
diagnosis (Dharnidharka et al. 1998; Hunley et al. 2004; Gamarra et al. 2006; Goto et al. 2009).  
Signs and symptoms of immune complex deposition may include fever, rash, arthralgia, myalgia, 
hematuria, proteinuria, serositis, central nervous system complications, and hemolytic anemia.  
Immune complexes, composed of antibody and a therapeutic protein product, have been 
responsible for the development of glomerulonephritis and nephrotic syndrome in patients 
undergoing tolerance induction treatment (with factor IX and -glucosidase) in the face of a high 
titer and sustained antibody response (Dharnidharka et al. 1998; Hunley et al. 2004).  There have 
been case reports of immune complex disease with immune responses to monoclonal antibodies 
(Gamarra et al. 2006; Goto et al. 2009) and situations in which large doses of a monoclonal 
antibody targeting high levels of a circulating multivalent antigen may increase the likelihood of 
immune complex deposition (Gonzalez and Waxman 2000). 
 
If patients develop signs or symptoms suggestive of immune complex disease, appropriate 
laboratory assessments for circulating immune complexes and complement activation should be 
undertaken; and the administration of the therapeutic protein product should be suspended.  In 
certain situations, development of tolerance induction therapies that eliminate the antibody 
response may be appropriate prior to further attempts at treatment.  
 
 
4.  Antibody Responses to Therapeutic Protein Products  
 
Antibodies to therapeutic protein products are classified as either neutralizing or non-
neutralizing.  Neutralizing antibodies bind to distinct functional domains of the therapeutic 
protein product and preclude their activity.  For example, antibodies to therapeutic enzymes may 
bind to either the catalytic site, blocking catalysis of substrate, or to the uptake domain, 
preventing uptake of the enzyme into the cell.  In rare circumstances, neutralizing antibody may 
act as a carrier and enhance the half-life of the product and prolong its therapeutic effect.  As 
discussed in section III of this guidance, non-neutralizing antibodies bind to areas of the 
therapeutic protein product other than specific functional domains and may exhibit a range of 
effects on safety and efficacyenhanced or delayed clearance of the therapeutic protein product, 
which may prompt consideration of dosing changes, induction of anaphylaxis, diminished 
efficacy of the product by causing uptake of the therapeutic protein product into FcR-expressing 
cells rather than the target cells, and facilitation of epitope spreading, allowing the emergence of 
neutralizing antibodies.  However, they may have no apparent effect on either safety or efficacy.  
 
The development of neutralizing antibody is expected with administration of nonhuman proteins 
and in patients receiving factor/enzyme replacement therapies to whom such therapeutic protein 
products appear as foreign.  However, neutralizing antibody to a therapeutic protein product that 
cross reacts to an endogenous protein does not always arise in situations in which the 
endogenous factor is defective or absent by genetic mutation, as in the case of hemophilia A or 
lysosomal storage diseases.  Neutralizing antibodies can develop in healthy individuals to some 
  33  Contains Nonbinding Recommendations 
 
normal endogenous proteins because immune tolerance to some endogenous proteins is not 
robust and can be broken by a therapeutic protein homolog with sufficient provocation.  For 
example, healthy volunteers treated with a thrombopoietin (TPO)-type protein mounted a 
neutralizing antibody response to the therapeutic, which cross-reactively neutralized endogenous 
TPO, inducing a prolonged state of thrombocytopenia in those formerly healthy individuals (Li 
et al. 2001).  Thus, treatment with therapeutic counterparts of endogenous proteins serving a 
unique function or endogenous proteins present at low abundance should be undertaken with 
utmost caution.  Neutralizing antibody to a therapeutic protein product can also be catastrophic 
when it neutralizes the efficacy of a lifesaving therapeutic such as therapeutic enzymes for 
lysosomal storage disorders, and immune tolerance induction should be considered in such 
circumstances (Wang et al. 2008).  
 
Loss of efficacy of mAbs in patients caused by immune responses to the mAb can be highly 
problematic, and the clinical consequences should not be minimized.  Sponsors may consider 
development of immune tolerance induction regimens in such patients.  
 
As discussed in section III.B.5 of the guidance, if the endogenous protein is redundant in 
biological function (e.g., Type I interferons), neutralization of the therapeutic and endogenous 
protein may not appear to produce an obvious clinical syndrome.  However, the more subtle 
effects of blocking endogenous factors, even though redundant in some functions, may not be 
apparent until the system is stressed, as not all biological functions of a factor may be known or 
fully characterized.  Moreover, the effects of long-term persistence of neutralizing antibody, as 
have been observed, for example, in a small percentage of patients with antibodies to IFN- 
(Bellomi et al. 2003), would not be known from short-term follow-up and should be studied 
longer term.  Generally, for products given chronically, one year or more of immunogenicity 
data should be collected and evaluated unless a shorter duration can be scientifically justified.  
However, longer-term evaluation may be warranted depending on the frequency and severity of 
the consequences.  In some cases, these studies may be done in the postmarket setting.  
Agreement with the Agency should be sought regarding the extent of data required before and 
after marketing. 
 
In some circumstances, antibody responses, regardless of apparent clinical effect, should be 
serially followed until the levels return to baseline or an alternative approach is discussed with 
the Agency.  Moreover, for patients in whom a therapeutic protein product appears to lose 
efficacy, regardless of the duration of the treatment course, it is important that an assessment be 
undertaken to determine whether the loss of efficacy is antibody mediated. 
 
For patients who develop neutralizing antibodies or are considered at very high risk of 
developing neutralizing antibodies to a lifesaving therapeutic protein product (e.g., CRIM-
negative patients with a deletion mutation for a critical enzyme who are given enzyme 
replacement therapy), consideration should be given to tolerance induction regimens in a 
prophylactic setting, before or concomitant with the onset of treatment (Wang et al. 2008; 
Mendelsohn et al. 2009; Messinger et al. 2012).  Given the degree of immune suppression of 
such regimens, although far less than that of a therapeutic regimen to reverse an ongoing 
antibody response, careful safety monitoring should be undertaken throughout the duration of the 
protocol.  
  34  Contains Nonbinding Recommendations 
 
 
 
5.  Utility of Animal Studies 
 
Immunogenicity assessments in animals are conducted to assist in the interpretation of animal 
study results (e.g., toxicology studies) and in the design of subsequent clinical and nonclinical 
studies (for additional information, see the ICH guidance for industry S6(R1) Preclinical Safety 
Evaluation of Biotechnology-Derived Pharmaceuticals).10  They are generally limited in their 
ability to predict the incidence of human immune responses to a therapeutic protein product, but 
they may be useful in describing the consequences of antibody responses, particularly when an 
evolutionarily conserved, nonredundant endogenous protein is inhibited by cross-reactive 
antibodies generated to its therapeutic protein product counterpart.  When available, animal 
models including hyperimmunized mice or gene knock out mice can be used to address potential 
consequences of inhibition of endogenous proteins.  A special case is that of endogenous 
proteins that are vital to embryonic or fetal development whose elimination is embryonically 
lethal.  In such situations, the use of conditional knock out mice may be useful for assessing 
potential consequences of neutralizing antibodies.  As in human studies, consideration should be 
given to the potential transmission of antibodies to developing neonates by breast milk. 
 
In contrast to proteins that mediate biologically unique functions, animal models are generally 
not useful for predicting consequences of immune responses to therapeutic protein products that 
are counterparts to endogenous proteins with redundant biological functions.  Mice that are 
transgenic for genes encoding human proteins, humanized mice (i.e., immune-deficient mice 
with human immune systems), and mouse models of human diseases are increasingly being 
developed and may be considered for use in addressing multiple clinical issues, including 
immunogenicity. 
 
In addition to appropriate animal studies, consideration should be given to in vitro and in silico 
analyses that may supplement animal studies to better or further elucidate risk for 
immunogenicity. 
 
 
6.  Comparative Immunogenicity Studies 
 
The need for and the extent and timing of clinical immunogenicity studies in the context of 
evaluating the effects of a manufacturing change will depend on such factors as the degree of 
analytical comparability between the product before and after the manufacturing change, 
findings from informative comparative animal studies, and the incidence and clinical 
consequences of immune responses to the product prior to the manufacturing change.  For 
example, if the clinical consequence of an immune response is severe (e.g., when the product is a 
therapeutic counterpart of an endogenous protein with a critical, nonredundant biological 
                                                 
10 ICH guidance for industry S6(R1) Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals is 
available at http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm)) 
  35  Contains Nonbinding Recommendations 
 
function or is known to provoke anaphylaxis), more extensive immunogenicity assessments will 
likely be needed.11 
                                                 
11 Guidance on development programs for biosimilar products is available in the draft guidance for industry 
Scientific Considerations in Demonstrating Biosimilarity to a Reference Product.  When final, this guidance will 
reflect the Agencys current thinking on this topic. 
  36