Investigational Enzyme 
Replacement Therapy 
Products: Nonclinical 
Assessment 
Guidance for Industry 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
October 2019 
Pharmacology/Toxicology 
 
   
Investigational Enzyme 
Replacement Therapy 
Products: Nonclinical 
Assessment 
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) 
 
October 2019 
Pharmacology/Toxicology 
 
   
TABLE OF CONTENTS 
 
I.  INTRODUCTION............................................................................................................. 1 
II.  BACKGROUND ............................................................................................................... 2 
III.  NONCLINICAL STUDY CONSIDERATIONS ............................................................ 2 
A.  Nonclinical Program Objectives ................................................................................................... 2 
B.  Recommendations for Nonclinical Program Design ................................................................... 3 
1. ERT Products Used in Nonclinical Studies...................................................................................... 3 
2. Selecting Animal Species ................................................................................................................. 3 
3. Animal Models of Disease ............................................................................................................... 4 
4. Toxicology Studies ........................................................................................................................... 5 
5. Good Laboratory Practice ............................................................................................................... 7 
6. Product Development for Later-Phase Clinical Trials and Marketing Applications ...................... 8 
7. Nonclinical Study Reports ................................................................................................................ 8 
8. Communicating with CDER Pharmacology/Toxicology Staff ......................................................... 9 
  Contains Nonbinding Recommendations 
Investigational Enzyme Replacement Therapy  
Products:  Nonclinical Assessment 
Guidance for Industry1 
 
 
 
 
This guidance represents the current thinking of the Food and Drug Administration (FDA or Agency) on 
this topic.  It does not establish any rights for any person and is not binding on FDA or the public.  You 
can use an alternative approach if it satisfies the requirements of the applicable statutes and regulations.  
To discuss an alternative approach, contact the FDA office responsible for this guidance as listed on the 
title page. 
 
 
 
 
I.  INTRODUCTION  
 
The purpose of this guidance is to help sponsors design and conduct nonclinical studies during 
development of investigational enzyme replacement therapy (ERT) products.  Specifically, this 
guidance describes the Food and Drug Administrations (FDAs) current thinking about the 
substance and scope of nonclinical information needed to support initiation of clinical trials, 
ongoing clinical development, and marketing approval for investigational ERT products. 
 
This guidance is intended as an adjunct to the ICH guidances for industry M3(R2) Nonclinical 
Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for 
Pharmaceuticals (January 2010), M3(R2) Nonclinical Safety Studies for the Conduct of Human 
Clinical Trials and Marketing Authorization for Pharmaceuticals  Questions and Answers 
(February 2013), and S6(R1) Preclinical Safety Evaluation of Biotechnology-Derived 
Pharmaceuticals (May 2012).2  These ICH guidances provide general recommendations about 
the nonclinical safety studies of traditional small molecule and biotechnology-derived 
pharmaceuticals that support human clinical trials, as well as marketing authorization for 
pharmaceuticals. 
 
The recommendations in this guidance apply to ERT products indicated for lysosomal storage 
diseases and other diseases caused by inborn errors of metabolism.  This guidance does not apply 
to the development of pancreatic enzyme products. 
 
In general, FDAs guidance documents do not establish legally enforceable responsibilities.  
Instead, guidances describe the Agencys current thinking on a topic and should be viewed only 
                                                 
1 This guidance has been prepared by the Division of Gastroenterology and Inborn Errors Products in the Center for 
Drug Evaluation and Research at the Food and Drug Administration. 
 
2 We update guidances periodically.  To make sure you have the most recent version of a guidance, check the FDA 
guidance web page at https://www.fda.gov/drugs/guidance-compliance-regulatory-information/guidances-drugs. 
1  Contains Nonbinding Recommendations 
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 
 
ERT products are used to treat a wide array of rare inborn errors of metabolism disorders 
resulting from inheriting defective genes (e.g., Gaucher disease, Fabry disease, Pompe disease, 
and mucopolysaccharidoses).  These diseases generally manifest early in life.  The natural 
history varies within and across diseases.  For a given disease, there may be multiple phenotypic 
presentations, which can range from rapidly progressing changes that result in early death or 
devastating irreversible morbidity within a very short time frame to a slower progressive course 
that can extend into adulthood.  Treatments generally involve exogenously supplying the missing 
or defective protein. 
 
Although an ERT product is intended to resemble the natural product as much as possible, the 
commercial protein is not produced endogenously and may not interact with the same target in 
the same way as the natural product.  ERT products may be chemically modified for reasons 
such as extending half-lives or targeting delivery to specific sites such as the brain.  Therefore, 
there is a potential for toxicities beyond hypersensitivity reactions (e.g., toxicity resulting from 
direct or indirect effects of excess enzyme levels or a possible toxic effect of the ERT to 
nontarget tissues).  Given the wide array of clinical indications, variations in the natural history 
of disease, and enzyme product types, no single nonclinical program can be designed to fit all 
ERT products. 
 
 
III.  NONCLINICAL STUDY CONSIDERATIONS 
 
A.  Nonclinical Program Objectives 
 
Nonclinical studies conducted to support clinical investigations for ERT products should address 
the following objectives: 
 
  Pharmacodynamic characterizations, including proof-of-concept (POC) studies, to 
demonstrate functional enzyme replacement and identify biologically active dose levels 
 
  Safety assessments, including toxicology studies, to inform selection of a safe starting 
dose, dose-escalation schedule, and dosing frequency; demonstrate the feasibility and 
safety of the investigational products proposed clinical route of administration (ROA); 
and identify safety parameters that can guide clinical monitoring of safety in humans 
 
2  Contains Nonbinding Recommendations 
B.  Recommendations for Nonclinical Program Design 
 
When planning the nonclinical development program, sponsors should consider the following 
issues that can affect the timing, duration, and type of supportive nonclinical studies needed to 
initiate clinical trials: 
 
  The proposed clinical indication and population, such as whether children or adults will 
be studied, and the rate of disease progression to death or irreversible morbidity in that 
population 
 
  Pharmacodynamic data, including changes in known disease-specific biomarkers that 
suggest the prospect of direct benefit to support first-in-human clinical trials in pediatric 
patients 
 
  The availability of existing relevant nonclinical or clinical safety and pharmacology 
information for the specific ERT product under investigation 
 
  The availability of existing relevant safety information about the proposed clinical 
delivery device or delivery procedure for the product, or with any related device or 
procedure, as appropriate 
 
  The availability of appropriate species and models, either normal or enzyme deficient, for 
testing the investigational ERT product for the expected biological response with 
pathophysiology of the disease relevant to the target patient population 
 
1.  ERT Products Used in Nonclinical Studies 
 
The investigational ERT product that will be administered in clinical trials should be used in the 
nonclinical studies that are used to determine a safe dose in humans.  Sponsors should 
characterize each lot of an investigational ERT product used in the nonclinical studies according 
to prospectively established criteria, consistent with the stage of product development.  Sponsors 
should also highlight and discuss in the investigational new drug application (IND) similarities 
and differences between the drug substance and the drug product intended for use in nonclinical 
studies and for clinical trials, including differences in excipients. 
 
2.  Selecting Animal Species 
 
Sponsors should conduct nonclinical evaluations in relevant species.  Assessing factors for 
determining a relevant species necessitates considering the specific ERT product and clinical 
indication.  Some additional factors sponsors should consider when determining the most 
relevant species for testing ERT products include (1) comparability of molecular attributes, 
including the interspecies homology of the enzyme and the cell-surface receptors mediating 
uptake of the circulating ERT product; (2) distribution of the native enzyme and/or ERT product 
compared with that of humans; and (3) feasibility of using the planned clinical delivery system 
3  Contains Nonbinding Recommendations 
or procedure, as appropriate.  Sponsors should also justify the appropriateness of each animal 
species.3 
 
3.  Animal Models of Disease 
 
Although studies in animal models of human diseases caused by single enzyme defects are not 
expected to replace clinical studies in humans, they can provide, in selected situations, 
complementary and supportive information that can facilitate the progression of a human ERT 
development program.  The extent to which the information obtained from such animal models 
may be relevant to the human disease of interest depends on many factors.  To this end, it is 
desirable that the selected animal disease model displays a phenotype that mimics to a large 
extent the clinical manifestations and the overall course of the human disease.  Greater 
credibility and relevance can be given to animal data if the metabolic pathways and essential 
intermediary components are conserved between the selected animal species and humans.  It is 
essential not only that the inactivated animal gene is the same as the human gene but also that the 
animal and human genes serve the same biological functions and that the metabolic pathways are 
preserved across species.  If reliance on animal data is proposed, the IND should include 
information supporting the usefulness and/or ability of the models to mimic the target disease 
population. 
 
Animal models of disease may provide proof of concept that the ERT of interest has the desired 
biological effect.  They may also help identify a biologically effective dose or doses and dosing 
regimen or regimens and confirm the effectiveness of the animal dose through studies that 
measure reduction in tissue substrate levels or other related histopathological changes that can be 
linked mechanistically to the enzymatic defect.  They can further provide evidence of biological 
activity by demonstrating improvement of the disease manifestations (including effects on 
survival). 
 
In addition, studies in animal models can support future reliance on noninvasive biomarkers 
(e.g., disease-specific substrate levels and/or enzyme-reaction products in the circulation) as long 
as the rate and extent of improvement of the biomarker levels correlate with a reduction in tissue 
substrate deposition/tissue damage and/or improvements in organ function.  Such biomarkers 
may later be used as pharmacodynamic markers of disease activity in humans in clinical trials 
and may even be qualified as surrogate markers. 
 
If studies in animal models of disease are used to support the safety of an ERT product, sponsors 
should incorporate safety endpoints in the POC studies.  FDA encourages sponsors to discuss 
the adequacy of study designs (e.g., number of animals used and plans for tissue collection and 
evaluation) with the review division before initiating the study. 
 
                                                 
3 We support the principles of the 3Rs (reduce/refine/replace) for animal use in testing when feasible.  FDA 
encourages sponsors to consult with review divisions when considering a nonanimal testing method believed to be 
suitable, adequate, validated, and feasible.  FDA will consider if the alternative method could be assessed for 
equivalency to an animal test method. 
4  Contains Nonbinding Recommendations 
4.  Toxicology Studies 
 
Sponsors should perform an appropriate nonclinical safety assessment to support the proposed 
clinical development program.  Healthy animals represent the standard test system employed to 
conduct traditional toxicological studies.  For studies to support ERT clinical trials, sponsors can 
consider study designs that use animal models of disease that incorporate important safety 
parameters that allow for assessing the potential toxicity of an investigational ERT product.  
Sponsors should consider POC studies in relevant animal disease models modified to assess 
toxicology endpoints prospectively, including microscopic examinations of tissues, as support 
for initiating human clinical trials.  Using animal disease models for toxicity testing may also 
allow sponsors to detect toxicity caused by the interaction of the drug and the disease in ways 
that would not be observed in healthy animals.  To obtain agreement on study design, sponsors 
should discuss such study designs with the review division before initiating a study. 
 
The nonclinical safety assessment, whether conducted in healthy animals or in animal disease 
models, should be sufficiently comprehensive to permit identifying, characterizing, and 
quantifying potential local and systemic toxicities, their onset (i.e., acute or delayed), the effect 
of the product dose level on toxicity findings, and the possibility for reversing any toxicities (if 
applicable). 
 
The overall design of the nonclinical studies should support the safety of the proposed clinical 
trial.  Nonclinical toxicology study designs should include the following, as applicable: 
 
  An adequate number of animals per sex that are appropriately randomized to each group.  
The number of animals needed will depend on existing safety concerns for the 
investigational ERT product, the species, the model, and the delivery system.  If safety 
data are generated from POC studies to support clinical trials, sponsors should use an 
adequate number of animals to appropriately assess the safety endpoints.  FDA 
recommends sponsors consult with the review division for design of these studies before 
initiating a study. 
 
  Animals with the appropriate age and developmental status as related to the proposed 
clinical trial population. When a first-in-human clinical trial for an ERT product will 
enroll pediatric patients, sponsors should conduct toxicity studies that use juvenile 
animals before initiating a clinical trial.  The major issue is the potential for adverse 
effects on the developing organ systems in young pediatric patients (e.g., central nervous 
system, reproductive tract, immune system, and skeletal system).  ICH M3(R2) and the 
guidance for industry Nonclinical Safety Evaluation of Pediatric Drug Products 
(February 2006) provide recommendations for determining the need for juvenile animal 
studies.  Sponsors can submit the protocol for the juvenile animal toxicology studies to 
the review division for the divisions concurrence on appropriate toxicology endpoints 
before conducting the study.  The juvenile animal toxicity studies potentially may be 
waived when (1) clinical development is initiated in adult patients, (2) there are no 
specific safety concerns from studies in adult animals or adult patients, and (3) target 
organs with identified toxicity concerns are not undergoing development at the time of 
treatment. 
5  Contains Nonbinding Recommendations 
 
  Appropriate control groups.  A control group should be included in all toxicology studies 
with ERT products.  An appropriate control group should be age-matched animals that 
have been administered the formulation vehicle only.  When it is necessary to co-
administer an antihistamine (e.g., diphenhydramine) to control hypersensitivity reactions 
to the ERT product, sponsors should include in the study a vehicle control group and a 
vehicle-plus-antihistamine control group.  Sponsors should also provide justification for 
the specific control group or groups selected. 
 
  Appropriate dose levels.  Results obtained from POC studies should guide selection of 
the target dose levels both for nonclinical safety assessment and for clinical development. 
ICH M3(R2) and its subsequent questions-and-answers document provide considerations 
for selecting high doses for general toxicity studies.  In general, in the absence of frank 
toxicity, the exposure at the highest doses tested for ERT products should be severalfold 
greater than the exposure expected at the highest proposed clinical dose regimen.  The 
highest dose level used in nonclinical studies may be restricted because of animal size, 
tissue volume or size, ROA, or product-manufacturing capacity.  Sponsors should 
provide justification, with supporting data, for the specific dose levels selected. 
 
  The dosing schedule should reflect the expected clinical exposure, to the extent possible. 
 
  An adequate duration of dosing.  Decisions regarding the dosing duration in the 
nonclinical studies conducted to support first-in-human dosing of ERT products should 
consider two key issues:  (1) the treatment of diseases caused by inborn errors of 
metabolism is expected to be chronic, and the first-in-human clinical trials are generally 
expected to be chronic dosing studies; and (2) greater uncertainty about risk may be 
acceptable in the setting of a disease with a rapid course to death or irreversible 
morbidity.  For these reasons, sponsors should not only design the nonclinical study plan 
to support chronic dosing in patients who enter the first-in-human clinical trial but also 
consider the disease phenotype of the patients who will be enrolled in the trial. 
 
If the clinical trial entry criteria define a phenotype that is anticipated to progress rapidly 
to irreversible morbidity or mortality over the course of approximately 1 year, then 
repeat-dose toxicology studies in a rodent and a nonrodent species of 1-month dosing 
duration may be sufficient to initiate clinical trials.  Initial dosing in these patients can 
also be supported by POC studies of appropriate duration in animal disease models, with 
adequate toxicological assessments.  A 3-month toxicity study in one species may be 
needed before submission of a marketing application.  Three-month studies in two 
species may be needed if the toxicological findings of the 1-month studies in the rodent 
and the nonrodent are not similar.  Sponsors could conduct the 3-month toxicity study or 
studies in parallel with the first-in-human clinical trial.  In the event of severe toxicity, 
including recovery animals can provide useful information. 
 
If the clinical trial entry criteria define a phenotype that would be expected to have slower 
disease progression, then toxicology studies of at least 3 months duration in a rodent and 
a nonrodent species will be needed to initiate first-in-human clinical trials; this is 
6  Contains Nonbinding Recommendations 
because, given the chronic nature of these rare diseases and unmet medical need, first-in-
human clinical trials may be anticipated to initiate chronic dosing. 
 
In cases for which short-term clinical dosing (e.g., less than 1 month) is proposed and 
considered appropriate, shorter-duration toxicology studies may be acceptable as 
discussed in ICH M3(R2).  Sponsors should complete longer-duration toxicology studies 
to support chronic clinical dosing as discussed above. 
 
  An ROA that mimics the intended clinical route as closely as possible.  If the delivery 
device could impact toxicology, then we recommend sponsors use the delivery device 
intended for use in the clinical trials to administer the investigational ERT product in the 
definitive toxicology studies if possible.  If it is not possible to replicate the clinical ROA 
in the animal model, then sponsors should propose and scientifically justify alternative 
routes or methods as a part of the nonclinical development plan. 
 
  Safety endpoints that capture potential toxicities.  Standard parameters evaluated should 
include mortality (with cause of death determined, if possible), clinical observations, 
body weights, physical examinations, food consumption, clinical pathology (serum 
chemistry, hematology, coagulation, urinalysis), organ weights, gross pathology, and 
histopathology.  Additional developmental endpoints may be appropriate when 
conducting juvenile animal studies. 
 
  Assessment of the effect of antidrug antibodies (ADA) on drug exposure and in response 
to the administration of the ERT product.  This information is needed to assess the effect 
of ADA formation on the interpretation of the toxicology study findings. 
 
These nonclinical data can help guide first-in-human dose selection.  For example, data 
generated from the toxicology studies may establish a no observed adverse effect level, which 
can help sponsors select the starting dose level for the clinical trial. Alternatively, sponsors can 
consider a minimum efficacious dose (MED), as predicted by a relevant animal disease model, 
when selecting a starting dose.  In addition, this information may allow sponsors to avoid or 
mitigate significant toxicities in patients. 
 
5.  Good Laboratory Practice 
 
According to 21 CFR 312.23, each toxicology study intended primarily to support the safety of a 
proposed clinical investigation is subject to good laboratory practice (GLP) regulations under 
21 CFR part 58.  However, some toxicology assessments, although of sufficient quality and 
integrity, may not fully comply with the GLP regulations.  For example, toxicology data for 
investigational ERT products are sometimes collected in POC studies that may use an animal 
model of disease requiring unique animal-care issues and technical expertise unavailable at a 
GLP testing facility.  If the study is not conducted in compliance with GLP regulations, sponsors 
must include in the final study report a brief statement about the reason for the noncompliance 
(21 CFR 312.23(a)(8)(iii)).  In addition, sponsors need to demonstrate that non-GLP studies 
submitted to support safety of an investigational ERT product are rigorous and adequately 
controlled to maintain reliability, quality, and integrity. 
7  Contains Nonbinding Recommendations 
 
All nonclinical studies that incorporate safety parameters in the study design should be 
conducted using a prospectively designed study protocol.  Results derived from these studies 
should be of sufficient quality and integrity to support the proposed clinical trial.  Sponsors 
should include in the nonclinical study report a summary of all deviations from the prospectively 
designed study protocol and their potential effect on study integrity and outcome. 
 
6.  Product Development for Later-Phase Clinical Trials and Marketing Applications 
 
As development of an investigational ERT product progresses to later-phase clinical trials, 
sponsors should consider conducting additional nonclinical studies to address any outstanding 
issues.  For example, if manufacturing or formulation changes occur such that comparison of the 
later-phase ERT product with the product used in early-phase clinical trials is uncertain, sponsors 
may need additional in vitro and/or in vivo nonclinical studies to bridge the two products.  Such 
bridging studies allow data collected with the early-phase product to support later-phase 
development or licensure.  Additional nonclinical studies might be warranted if the ROA or 
patient population changes significantly from the early-phase clinical trials. 
 
Toxicity studies of 3 months duration generally should be considered sufficient to support a 
marketing application for an ERT product.  However, if the 3-month toxicity studies reveal 
concerning findings, then FDA may recommend toxicity studies up to 6 months duration to 
address outstanding concerns.  In general, we recommend conducting a battery of reproductive 
toxicity studies, as described in the ICH guidance for industry S5(R2) Detection of Toxicity to 
Reproduction for Medicinal Products: Addendum on Toxicity to Male Fertility (April 1996) 
(refer to ICH M3(R2) regarding the timing of these studies).  However, flexibility in timing or 
requirements for specific studies may be warranted in certain cases with adequate justification.  
Certain studies can be waived or delayed until after approval of the marketing application 
depending on the indicated patient population. 
 
Genotoxicity studies are not considered applicable to ERT products, so FDA does not 
recommend these studies.  Evaluating carcinogenic potential generally is not needed to support a 
marketing application.  However, chemically modified ERT products (e.g., a recombinant human 
enzyme conjugated with a chemical linker) may need an assessment to address the potential for 
genotoxicity and/or carcinogenicity. 
 
7.  Nonclinical Study Reports 
 
Sponsors should submit a report for each in vitro and in vivo nonclinical study intended to 
demonstrate the safety of an investigational ERT product.  Complete reports of pharmacology 
and POC studies generally are not required for an IND; however, sponsors should submit 
complete study reports if the POC studies with safety information are used to support clinical 
trials.  Each complete study report should include, but not be limited to, the following:  (1) a 
prospectively designed protocol and listing of all protocol amendments; (2) a detailed description 
of the study design (e.g., the test system used, animal species or model used, control and 
investigational products administered, dose levels, detailed procedures for product 
administration, and collection of all study protocol parameters); (3) complete data sets for all 
8  Contains Nonbinding Recommendations 
parameters evaluated, including individual animal data and tabulated/summary data; and (4) 
analysis and interpretation of the results obtained. 
 
8.  Communicating with CDER Pharmacology/Toxicology Staff 
 
We recommend sponsors communicate with the Center for Drug Evaluation and Research 
(CDER) pharmacology/toxicology staff of the relevant review division, through the division 
project management staff, early in the investigational ERT product development program.  
Nonclinical testing programs for ERT products often need to be highly individualized; therefore, 
discussions with the review division may be needed regarding CDER expectations for the 
specific product and indication.  If sponsors plan to leverage toxicology information obtained 
from the POC study to support initiating the first-in-human clinical trial, a pre-IND meeting with 
the review division to discuss design of the POC study before its initiation improves the chances 
that the study data will be adequate to support first-in-human clinical trials.  This interaction can 
serve to facilitate more rapid access to treatment for patients. 
9