M9 Biopharmaceutics 
Classification System-
Based Biowaivers 
Guidance for Industry 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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) 
 
May 2021 
ICH 
 
   
 
M9 Biopharmaceutics 
Classification System-
Based Biowaivers 
Guidance for Industry 
 
 
 
 
 
 
Additional copies are available from: 
 
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Center for Drug Evaluation and Research 
Food and Drug Administration 
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and/or 
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Email: ocod@fda.hhs.gov 
https://www.fda.gov/vaccines-blood-biologics/guidance-compliance-regulatory-information-biologics/biologics-guidances 
 
 
 
 
 
 
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) 
 
May 2021 
ICH 
  Contains Nonbinding Recommendations 
 
FOREWORD 
 
The International Council for Harmonisation of Technical Requirements for Pharmaceuticals 
for Human Use (ICH) has the mission of achieving greater regulatory harmonization 
worldwide to ensure that safe, effective, and high-quality medicines are developed, registered, 
and maintained in the most resource-efficient manner.   By harmonizing the regulatory 
expectations in regions around the world, ICH guidelines have substantially reduced 
duplicative clinical studies, prevented unnecessary animal studies, standardized safety 
reporting and marketing application submissions, and contributed to many other 
improvements in the quality of global drug development and manufacturing and the products 
available to patients.  
 
ICH is a consensus-driven process that involves technical experts from regulatory authorities 
and industry parties in detailed technical and science-based harmonization work that results in 
the development of ICH guidelines.   The commitment to consistent adoption of these 
consensus-based guidelines by regulators around the globe is critical to realizing the benefits 
of safe, effective, and high-quality medicines for patients as well as for industry.   As a 
Founding Regulatory Member of ICH, the Food and Drug Administration (FDA) plays a 
major role in the development of each of the ICH guidelines, which FDA then adopts and 
issues as guidance to industry.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
    Contains Nonbinding Recommendations 
 
TABLE OF CONTENTS 
 
 
I.  INTRODUCTION (1) ............................................................................................ 1 
A.  Background and Objective (1.1) ................................................................................ 1 
B.  Scope (1.2) ............................................................................................................... 2 
II.  BIOPHARMACEUTICS CLASSIFICATION OF THE DRUG SUBSTANCE (2)
   ............................................................................................................................... 2 
A.  Solubility (2.1).......................................................................................................... 2 
B.  Permeability (2.2) ..................................................................................................... 3 
III.  ELIGIBILITY OF A DRUG PRODUCT FOR A BCS-BASED BIOWAIVER (3)
   ............................................................................................................................... 4 
A.  Excipients (3.1) ........................................................................................................ 5 
B.  In Vitro Dissolution (3.2) .......................................................................................... 7 
IV.  DOCUMENTATION (4) ....................................................................................... 8 
V.  GLOSSARY (5) ..................................................................................................... 9 
ANNEX I: CACO-2 CELL PERMEABILITY ASSAY METHOD CONSIDERATIONS
   ............................................................................................................................. 10 
ANNEX II: FURTHER INFORMATION ON THE ASSESSMENT OF EXCIPIENT 
DIFFERENCES .............................................................................................................. 13 
 
i 
  Contains Nonbinding Recommendations 
 
M9 Biopharmaceutics Classification System-Based Biowaivers
 
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 (1)2 
 
A.  Background and Objective (1.1) 
 
Two drug products containing the same drug substance or substances are considered 
bioequivalent if their bioavailabilities (rate and extent of drug absorption) after administration 
in the same molar dose lie within acceptable predefined limits.   These limits are set to 
ensure comparable in vivo performance, i.e., similarity in terms of safety and efficacy.   In in 
vivo bioequivalence studies, the pivotal pharmacokinetic parameters area under the 
concentration time curve (AUC)3 and maximum concentration (C ), are generally used to 
max
assess the rate and extent of drug absorption. 
 
The biopharmaceutics classification system (BCS)-based biowaiver approach is intended to 
reduce the need for in vivo bioequivalence studies, i.e., it can provide a surrogate for in vivo 
bioequivalence.   In vivo bioequivalence studies may be exempted if an assumption of 
equivalence in in vivo performance can be justified by satisfactory in vitro data.   The BCS is 
a scientific approach based on the aqueous solubility and intestinal permeability 
characteristics of the drug substance or substances.   The BCS categorizes drug substances 
into one of four BCS classes as follows: 
 
  Class I: high solubility, high permeability 
  Class II: low solubility, high permeability 
  Class III: high solubility, low permeability 
  Class IV: low solubility, low permeability 
 
 
1  This guidance was developed within the Expert Working Group (Multidisciplinary) of the International 
Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) and has been 
subject to consultation by the regulatory parties, in accordance with the ICH process.   This document has been 
endorsed by the ICH Assembly at Step 4 of the ICH process, November 2019.  At Step 4 of the process, the 
final draft is recommended for adoption to the regulatory members of the ICH regions. 
2 Arabic numbers reflect the organizational breakdown of the document endorsed by the ICH Steering 
Committee at Step 4 of the ICH process, November 2019. 
3 Words found in the GLOSSARY (p. 9) are bolded at first use in this guidance. 
1  Contains Nonbinding Recommendations 
 
This guidance provides recommendations to support the biopharmaceutics classification of 
drug substances and the BCS-based biowaiver of bioequivalence studies for drug products.  
The BCS-based biowaiver principles may be applied to bioequivalence purposes not 
explicitly noted in the guidance, provided they can be supported by a thorough scientific 
rationale. 
 
The contents of this document do not have the force and effect of law and are not meant to 
bind the public in any way, unless specifically incorporated into a contract.   This document 
is intended only to provide clarity to the public regarding existing requirements under the 
law.   FDA guidance documents, including this guidance, should be viewed only as 
recommendations, unless specific regulatory or statutory requirements are cited.   The use of 
the word should in FDA guidances means that something is suggested or recommended, but 
not required. 
 
B.  Scope (1.2) 
 
BCS-based biowaivers may be used to substantiate in vivo bioequivalence.   Examples 
include comparison between products used during clinical development through 
commercialization, post-approval changes, and applications for generic drug products in 
accordance with regional regulations. 
 
The BCS-based biowaiver is only applicable to immediate release, solid orally administered 
dosage forms or suspensions designed to deliver drug to the systemic circulation.   Drug 
products having a narrow therapeutic index are excluded from consideration for a BCS-based 
biowaiver in this guidance.   Fixed-dose combination (FDC) products are eligible for a BCS-
based biowaiver when all drug substances contained in the combination drug product meet 
the criteria as defined in sections II. (2) and III. (3) of this guidance. 
 
II.  BIOPHARMACEUTICS CLASSIFICATION OF THE DRUG SUBSTANCE (2) 
 
BCS-based biowaivers are applicable to drug products where the drug substance or 
substances exhibit high solubility and, either high permeability (BCS Class I) or low 
permeability (BCS Class III). 
 
A biowaiver is applicable when the drug substance(s) in test and reference products are 
identical.   A biowaiver may also be applicable if test and reference products contain 
different salts provided that both belong to BCS Class I (high solubility and high 
permeability).   A biowaiver is not applicable when the test product contains a different ester, 
ether, isomer, mixture of isomers, complex or derivative of a drug substance from that of the 
reference product, because these differences may lead to different bioavailabilities not 
deducible by means of experiments used in the BCS-based biowaiver concept.   Pro-drugs 
may be considered for a BCS-based biowaiver when absorbed as the pro-drug. 
 
A.  Solubility (2.1) 
 
A drug substance is classified as highly soluble if the highest single therapeutic dose is 
completely soluble in 250 milliliter (mL) or less of aqueous media over the pH range of 1.2
6.8 at 371C.   In cases where the highest single therapeutic dose does not meet this 
criterion, but the highest strength of the reference product is soluble under the 
2  Contains Nonbinding Recommendations 
 
aforementioned conditions, additional data should be submitted to justify the BCS-based 
biowaiver approach. 
 
The sponsor is expected to establish experimentally the solubility of the drug substance over 
the pH range of 1.26.8 at 371oC.   At least three pHs within this range, including buffers at 
pH 1.2, 4.5 and 6.8, should be evaluated.   In addition, solubility at the pH of lowest 
solubility of the drug substance should be evaluated if it is within the specified pH range.  
These experiments should demonstrate that solubility is maintained over relevant timeframes 
to accommodate the expected duration of absorption. 
 
Solubility should be evaluated by a method appropriate to the properties of the drug 
substance. 
 
Equilibrium solubility experiments may be performed, using a shake-flask technique or an 
alternative method, if justified.   Small volumes of solubility media may be employed if the 
available experimental apparatus will permit it.   The pH for each test solution should be 
measured after the addition of the drug substance and at the end of the equilibrium solubility 
study to ensure the solubility measurement is conducted under the specified pH.   The pH 
should be adjusted if necessary.   The experiment should be conducted over a suitable 
timeframe to reach equilibrium. 
 
Alternatively, solubility experiments where the highest therapeutic single dose is examined in 
a 250 mL volume, or a proportionally smaller amount examined in a proportionally smaller 
volume of buffer, can be considered. 
 
The lowest measured solubility over the pH range of 1.26.8 will be used to classify the drug 
substance. 
 
A minimum of three replicate determinations at each solubility condition/pH using 
appropriate compendial media is necessary to demonstrate solubility using a suitably 
validated method. 
 
In addition, adequate stability of the drug substance in the solubility media should be 
demonstrated.   In cases where the drug substance is not stable with >10% degradation over 
the extent of the solubility assessment, solubility cannot be adequately determined and thus 
the drug substance cannot be classified.   In addition to experimental data, literature data may 
be provided to substantiate and support solubility determinations, keeping in mind that peer 
reviewed articles may not contain the necessary details of the testing to make a judgement 
regarding the quality of the studies. 
 
B.  Permeability (2.2) 
 
The assessment of permeability should preferentially be based on the extent of absorption 
derived from human pharmacokinetic studies, e.g., absolute bioavailability or mass balance.  
High permeability can be concluded when the absolute bioavailability is 85%.   High 
permeability can also be concluded if 85% of the administered dose is recovered in urine as 
unchanged (parent drug), or as the sum of parent drug, Phase 1 oxidative and Phase 2 
conjugative metabolites.   Regarding metabolites in feces, only oxidative and conjugative 
metabolites can be considered.   Metabolites produced through reduction or hydrolysis 
should not be included, unless it can be demonstrated that they are not produced before 
3  Contains Nonbinding Recommendations 
 
absorption, e.g., by microbial action within the gastrointestinal tract.   Unchanged drug in 
feces cannot be counted toward the extent of absorption, unless appropriate data supports that 
the amount of parent drug in feces to be accounted for absorbed drug material is from biliary 
excretion, intestinal secretion or originates from an unstable metabolite, e.g., glucuronide, 
sulphate, N-oxide, that has been converted back to the parent by the action of microbial 
organisms. 
 
Human in vivo data derived from published literature (e.g., product knowledge and 
bioavailability studies) may be acceptable, keeping in mind that peer reviewed articles may 
not contain the necessary details of the testing to make a judgement regarding the quality of 
the results. 
 
Permeability can be also assessed by validated and standardized in vitro methods using Caco-
2 cells (see Annex I).   The results from Caco-2 permeability assays should be discussed in 
the context of available data on human pharmacokinetics.   If high permeability is inferred 
by means of an in vitro cell system, permeability independent of active transport should be 
proven as outlined in Annex I, Caco-2 cell permeability assay method considerations. 
 
If high permeability is not demonstrated, the drug substance is considered to have low 
permeability for BCS classification purposes. 
 
Drug Substance Stability in the Gastrointestinal Tract 
 
Additional data to document the drugs stability in the gastrointestinal tract should be 
provided if mass balance studies are used to demonstrate high permeability, unless 85% of 
the dose is recovered as unchanged drug in urine.   Demonstration of stability in the 
gastrointestinal tract is required if in vitro Caco-2 studies are used to support high 
permeability.   Stability in the gastrointestinal tract may be documented using compendial or 
simulated gastric and intestinal fluids.   Other relevant methods may be used with suitable 
justification.   Drug solutions should be incubated at 37oC for a period that is representative 
of the in vivo contact of the drug substance with these fluids, i.e., one hour in gastric fluid 
and three hours in intestinal fluid.   Drug concentrations should then be determined using a 
suitably validated method.   Significant degradation (>10%) of a drug precludes BCS high 
permeability classification. 
 
III.  ELIGIBILITY OF A DRUG PRODUCT FOR A BCS-BASED BIOWAIVER (3) 
 
A drug product is eligible for a BCS-based biowaiver provided that the drug substance or 
substances satisfy the criteria regarding solubility and permeability (BCS Class I and III), the 
drug product is an immediate-release oral dosage form with systemic action, and the drug 
product is the same dosage form and strength as the reference product.   In cases where the 
highest single therapeutic dose does not meet the high solubility criterion, but the highest 
strength of the reference product is soluble under the required conditions, BCS-based 
biowaivers can be supported based on demonstration of dose proportional pharmacokinetics 
(i.e., AUC and C ) over a dose range that includes the highest single therapeutic dose. 
max
 
Drug products with buccal or sublingual absorption are not eligible for a BCS-based 
biowaiver application.   Furthermore, the BCS-based biowaiver approach is applicable only 
when the mode of administration includes water.   If administration without water is also 
4  Contains Nonbinding Recommendations 
 
intended (e.g., orodispersible products), a bioequivalence study in which the product is dosed 
without water should be conducted. 
 
In order for a drug product to qualify for a BCS-based biowaiver, criteria with respect to the 
composition (excipients) and in vitro dissolution performance of the drug product should be 
satisfied.   The drug product acceptance criteria are described in sections III.A. (3.1) and 
III.B. (3.2) below. 
 
A.  Excipients (3.1) 
 
Ideally, the composition of the test product should mimic that of the reference product.  
However, where excipient differences exist, they should be assessed for their potential to 
affect in vivo absorption.   This should include consideration of the drug substance properties 
as well as excipient effects.   To be eligible for a BCS-based biowaiver, the sponsor should 
justify why the proposed excipient differences will not affect the absorption profile of the 
drug substance under consideration, i.e., rate and extent of absorption, using a mechanistic 
and risk-based approach.   The decision tree for performing such an assessment is outlined in 
Figures 1 and 2 in Annex II. 
 
The possible effects of excipients on aspects of in vivo absorption such as solubility, 
gastrointestinal motility, transit time, and intestinal permeability, including transporter 
mechanisms, should be considered.   Excipients that may affect absorption include sugar-
alcohols, e.g., mannitol, sorbitol, and surfactants, e.g., sodium lauryl sulfate.   The risk that a 
given excipient will affect the absorption of a drug substance should be assessed 
mechanistically by considering: 
 
  The amount of excipient used 
 
  The mechanism by which the excipient may affect absorption 
 
  Absorption properties (rate, extent, and mechanism of absorption) of the drug 
substance 
 
The amount of excipients that may affect absorption in the test and reference formulations 
should be addressed during product development, such that excipient changes are kept to a 
minimum.   Small amounts included in the tablet coating, or levels below documented 
thresholds of effect for the specific drug substance, are of less concern. 
 
By definition, BCS Class I drugs are highly absorbed, and have neither solubility nor 
permeability limited absorption.   Therefore, they generally represent a low risk group of 
compounds in terms of the potential for excipients to affect absorption, compared to other 
BCS classes.   Consideration of excipient effects for BCS Class I drug products should focus 
on potential changes in the rate or extent of absorption.   For example, if it is known that the 
drug has high permeability due to active uptake, excipients that can inhibit uptake 
transporters are likely to be of concern.   For BCS Class I drugs that exhibit slow absorption, 
the potential for a given excipient to increase absorption rate should also be considered. 
 
For BCS Class I drugs, qualitative and quantitative differences in excipients are permitted, 
except for excipients that may affect absorption, which should be qualitatively the same and 
quantitatively similar, i.e., within  10% of the amount of excipient in the reference product.  
5  Contains Nonbinding Recommendations 
 
Additionally, the cumulative difference for excipients that may affect absorption should be 
within  10%. 
 
BCS Class III drug substances are considered to be more susceptible to the effects of 
excipients.   These drugs are not considered highly permeable and may have site-specific 
absorption, so there are a greater number of mechanisms through which excipients can affect 
their absorption than for BCS Class I drugs.   For BCS Class III drugs, all of the excipients 
should be qualitatively the same and quantitatively similar (except for film coating or capsule 
shell excipients).   Excipients that may affect absorption should be qualitatively the same and 
quantitatively similar, i.e., within  10% of the amount of excipient in the reference product, 
and the cumulative difference for these excipients should be within  10%.   This is defined 
in Table 1.   Examples of acceptable differences in excipients are shown in Annex II.  
Differences in colorants and flavoring may be permitted when these constitute very small 
amounts of the formulation. 
 
It is recognized that there are limitations to the application of Table 1, e.g., difficulty in 
determining the film coat weight for the reference product.   Table 1 is provided as a target to 
give clarity to sponsors.   Deviations from this will require appropriate justification, based on 
the principles described above. 
 
Table 1: Criteria Expected to Demonstrate Quantitative Similarity for Products 
Containing BCS Class III Drugs. 
Within the context of quantitative similarity, differences in excipients for drug products containing 
BCS Class III drugs should not exceed the following targets: 
Percent of the Amount of Excipient in the 
Excipient Class 
Reference 
Excipients which may affect absorption   
Per excipient:  10% 
Sum of differences:  10% 
Percent Difference Relative to Core 
 
Weight* (w/w) 
All excipients:   
Filler  10% 
Disintegrant   
Starch  6% 
Other  2% 
Binder  1% 
Lubricant   
Stearates  0.5% 
Other  2% 
Glidant   
Talc  2% 
Other  0.2% 
6  Contains Nonbinding Recommendations 
 
Total % change permitted for all excipients (including 
10% 
excipients which may affect absorption): 
*Note: Core does not include tablet film coat or capsule shell. 
 
BCS-based biowaivers are applicable to FDCs which are the same dosage form and strength.  
FDC formulations containing only BCS Class I drugs should meet criteria regarding 
excipients for a BCS Class I drug.   FDC formulations containing only BCS Class III drugs, 
or BCS Class I and BCS Class III drugs, should meet criteria regarding excipients for a BCS 
Class III drug. 
B.  In Vitro Dissolution (3.2) 
When applying the BCS based biowaiver approach, comparative in vitro dissolution tests 
should be conducted using one batch representative of the proposed commercial 
manufacturing process for the test product relative to the reference product.   The test 
product should originate from a batch of at least 1/10 of production scale or 100,000 units, 
whichever is greater, unless otherwise justified.   During a (clinical) development phase, 
smaller batch sizes may be acceptable, if justified.   The comparative in vitro dissolution 
experiments should use compendial apparatus and suitably validated analytical method or 
methods. 
The following conditions should be employed in the comparative dissolution studies to 
characterize the dissolution profile of the product: 
  Apparatus: paddle or basket. 
  Volume of dissolution medium: 900 mL or less (it is recommended to use the volume 
selected for the quality control (QC) test). 
  Temperature of the dissolution medium: 371C. 
  Agitation:   paddle apparatus - 50 revolutions per minute (rpm). 
  basket apparatus - 100 rpm. 
  At least 12 units of reference and test product should be used for each dissolution 
profile determination. 
  Three buffers: pH 1.2, pH 4.5, and pH 6.8.   Pharmacopoeial buffers should be 
employed.   Additional investigation may be required at the pH of minimum 
solubility (if different from the buffers above). 
  Organic solvents are not acceptable, and no surfactants should be added. 
  Samples should be filtered during collection, unless in situ detection methods are 
used. 
  For gelatin capsules or tablets with gelatin coatings where cross-linking has been 
demonstrated, the use of enzymes may be acceptable, if appropriately justified. 
When high variability or coning is observed in the paddle apparatus at 50 rpm for both 
reference and test products, the use of the basket apparatus at 100 rpm is recommended.  
Additionally, alternative methods (e.g., the use of sinkers or other appropriately justified 
approaches) may be considered to overcome issues such as coning, if scientifically 
substantiated.   All experimental results should be provided. 
7  Contains Nonbinding Recommendations 
 
To qualify for a BCS-based biowaiver for BCS Class I drug substances, both the test product 
and reference product should display either very rapid (85% for the mean percent dissolved 
in  15 minutes) in vitro dissolution characteristics, or rapid (85% for the mean percent 
dissolved in  30 minutes) and similar in vitro dissolution characteristics (i.e., based on f2 
comparison), under all of the defined conditions.   In cases where one product has rapid 
dissolution and the other has very rapid dissolution, similarity of the profiles should be 
demonstrated as below. 
For the comparison of dissolution profiles, where applicable, the similarity factor f2 should 
be estimated by using the following formula: 
f2 = 50  log {[1 + (1/n) n (R - T)2]-0.5  100} 
t=1 t t
In this equation, f2 is the similarity factor, n is the number of time points, R(t) is the mean 
percent reference drug dissolved at time t after initiation of the study, and T(t) is the mean 
percent test drug dissolved at time t after initiation of the study. 
The evaluation of the similarity factor is based on the following conditions: 
  A minimum of three time points (zero excluded). 
  The time points should be the same for the two products. 
  Mean of the individual values for every time point for each product. 
  Not more than one mean value of 85% dissolved for either of the products. 
  To allow the use of mean data, the coefficient of variation should not be more than 
20% at early time-points (up to 10 minutes) and should not be more than 10% at other 
time points. 
Two dissolution profiles are considered similar when the f2 value is 50.   When both test 
and reference products demonstrate that 85% of the labeled amount of the drug is dissolved 
in 15 minutes, comparison with an f2 test is unnecessary, and the dissolution profiles are 
considered similar.   When the coefficient of variation is too high, f2 calculation is 
considered inaccurate and a conclusion on similarity in dissolution cannot be made. 
To qualify for a BCS-based biowaiver for BCS Class III drug substances, both the test 
product and reference product should display very rapid (85% for the mean percent 
dissolved in 15 minutes) in vitro dissolution characteristics under the defined conditions. 
For FDC formulations, dissolution profiles should meet the criteria for all drug substances in 
the FDC to be considered.   FDC formulations containing only BCS Class I drugs should 
meet dissolution criteria for a BCS Class I drug.   FDC formulations containing only BCS 
Class III drugs should meet dissolution criteria for a BCS Class III drug.   For FDCs 
containing both BCS Class I and BCS Class III drugs, the dissolution criteria for the 
applicable BCS class for each component should be applied. 
For products with more than one strength, the BCS approach should be applied for each 
strength, i.e., it is expected that test and reference product dissolution profiles are compared 
at each strength. 
 
IV.  DOCUMENTATION (4) 
 
8  Contains Nonbinding Recommendations 
 
The sponsor should provide complete information on the critical quality attributes of the test 
drug substance(s) and drug product, and as much information as possible for the reference 
product, including, but not limited to: polymorphic form and enantiomeric purity; and any 
information on bioavailability or bioequivalence problems with the drug substance(s) or drug 
product, including literature surveys and sponsor-derived studies.   All study protocols and 
reports should be provided.   Information on validated test methods should be appropriately 
detailed according to current regulatory guidances and policies. 
 
The reporting format should include tabular and graphical presentations showing individual 
and mean results and summary statistics.    
 
The report should include all excipients, their qualitative, and, where appropriate, quantitative 
differences between the test and reference products. 
 
A full description of the analytical methods employed, including validation and qualification 
of the analytical parameters, should be provided.   A detailed description of all test methods 
and media, including test and reference batch information [unit dose (strength and assay), 
batch number, manufacturing date and batch size where known, expiration date] should also 
be provided.   The dissolution report should include a thorough description of experimental 
settings and analytical methods, including information on the dissolution conditions, such as 
apparatus, deaeration, filtration during sampling, volume, etc. 
 
In addition, complete information with full description of the methods applied should be 
provided for the Caco-2 cell permeability assay method, if applicable (see Annex I). 
 
V.  GLOSSARY (5) 
 
AUC: Area under the concentration versus time curve  
BCS: Biopharmaceutics Classification System 
C : Maximum concentration  
max
FDC: Fixed-dose combination 
QC: Quality control 
rpm: Revolutions per minute 
   
9  Contains Nonbinding Recommendations 
 
ANNEX I: CACO-2 CELL PERMEABILITY ASSAY METHOD CONSIDERATIONS 
Permeability assays employing cultured Caco-2 epithelial cell monolayers derived from a 
human colon adenocarcinoma cell line are widely used to estimate intestinal drug absorption 
in humans.   Caco-2 cells undergo spontaneous morphological and biochemical enterocytic 
differentiation, express cell polarity with an apical brush border, tight intercellular junctions, 
and several active transporters as in the small intestine.   Due to a potential for low or absent 
expression of efflux (e.g., P-gp, BCRP, MRP2) and uptake (e.g., PepT1, OATP2B1, MCT1) 
transporters, the use of Caco-2 cell assays as the sole data in support of high permeability for 
BCS classification is limited to passively transported drugs (see Assay Considerations). 
Method Validation 
 
The suitability of the Caco-2 cell assays for biopharmaceutics classification system (BCS) 
permeability determination should be demonstrated by establishing a rank-order relationship 
between experimental permeability values and the extent of drug absorption in human 
subjects using zero, low (<50%), moderate (5084%), and high (85%) permeability model 
drugs.   A sufficient number of model drugs are recommended for the validation to 
characterize high, moderate, and low permeability (a minimum 5 for each), plus a zero-
permeability marker; examples are provided in Table 2.   Further, a sufficient number 
(minimum of 3) of cell assay replicates should be employed to provide a reliable estimate of 
drug permeability.   The established relationship should permit differentiation between low, 
moderate, and high permeability drugs. 
Caco-2 cell monolayer integrity should be confirmed by comparing transepithelial electrical 
resistance (TEER) measures and/or other suitable indicators, before and after an experiment.  
In addition, cell monolayer integrity should be demonstrated by means of compounds with 
proven zero permeability (see Table 2). 
 
Reporting of the method validation should include a list of the selected model drugs, along 
with data on extent of absorption in humans (mean, standard deviation, coefficient of 
variation) used to establish suitability of the method, permeability values for each model drug 
(mean, standard deviation, coefficient of variation), permeability class of each model drug, 
and a plot of the extent of absorption as a function of permeability (mean  standard 
deviation or 95% confidence interval) with identification of the high permeability class 
boundary and selected high permeability model drug used to classify the test drug substance. 
In addition, a description of the study method, drug concentrations in the donor fluid, 
description of the analytical method, and equation used to calculate permeability, should be 
provided.   Additionally, information on efflux potential, e.g., bidirectional transport data, 
should be provided for a known substrate. 
 
Assay Considerations 
 
Passive transport of the test compound should be demonstrated.   This may be verified using 
a suitable assay system that expresses known efflux transporters, e.g., by demonstrating 
independence of measured in vitro permeability on initial drug concentration, e.g., 0.01, 0.1, 
and 1 times the highest strength dissolved in 250 milliliter (mL), or on transport direction 
(efflux ratio, i.e., ratio of apparent permeability (P ) between the basolateral-to-apical and 
app
apical-to-basolateral directions <2 for the selected drug concentrations). 
10  Contains Nonbinding Recommendations 
 
Efflux ratio = P /P . 
appBLAP appAPBL
Functional expression of efflux transporters should be verified by using bidirectional 
transport studies demonstrating asymmetric permeability of selected efflux transporter 
substrates, e.g., digoxin, vinblastine, rhodamine 123, at non-saturating concentrations. 
The test drug substance concentrations used in the permeability studies should be justified.  
A validated Caco-2 method used for drug permeability determinations should employ 
conditions established during the validation and include a moderate and a high permeability 
model drug in the donor fluid along with the test drug as internal standards to demonstrate 
consistency of the method.   The choice of internal standards should be based on 
compatibility with the test drug, i.e., they should not exhibit any significant physical, 
chemical, or permeation interactions.   The permeability of the internal standards may be 
determined following evaluation of the test drug in the same monolayers or monolayers in the 
same plate, when it is not feasible to include internal standards in the same cell culture well 
as the test drug permeability evaluation.   The permeability values of the internal standards 
should be consistent between different tests, including those conducted during method 
validation.   Acceptance criteria should be set for the internal standards and model efflux 
drug.   Mean drug and internal standards recovery at the end of the test should be assessed.  
For recoveries <80%, a mass balance evaluation should be conducted, including 
measurement of the residual amount of drug in the cell monolayer and testing apparatus. 
Evaluation of the test drug permeability for BCS classification may be facilitated by selection 
of a high permeability internal standard with permeability in close proximity to the 
moderate/high permeability class boundary.   The test drug is considered highly permeable 
when its permeability value is equal to or greater than that of the selected internal standard 
with high permeability. 
Information to support high permeability of a test drug substance (mean, standard deviation, 
coefficient of variation) should include permeability data on the test drug substance, the 
internal standards, in vitro gastrointestinal stability information, and data supporting passive 
transport mechanism. 
 
Table 2.   Examples of Model Drugs for Permeability Assay Method Validation 
 
Group  Drug 
High Permeability   Antipyrine  
(f  85%)  Caffeine 
a
Ketoprofen 
Naproxen 
Theophylline 
Metoprolol 
Propranolol 
Carbamazepine 
Phenytoin 
Disopyramide 
Minoxidil 
Moderate Permeability  Chlorpheniramine 
(f  = 50-84%)   Creatinine 
a
11  Contains Nonbinding Recommendations 
 
Group  Drug 
Terbutaline 
Hydrochlorothiazide 
Enalapril 
Furosemide 
Metformin 
Amiloride 
Atenolol 
Ranitidine 
Low Permeability  Famotidine 
(f < 50%)   Nadolol 
a 
Sulpiride 
Lisinopril 
Acyclovir 
Foscarnet 
Mannitol 
Chlorothiazide 
Polyethylene glycol 400 
Enalaprilat 
Zero Permeability  FITC-Dextran 
  Polyethylene glycol 4000 
Lucifer yellow 
Inulin 
Lactulose 
Efflux Substrates  Digoxin 
  Paclitaxel 
Quinidine 
Vinblastine 
 
   
12  Contains Nonbinding Recommendations 
 
ANNEX II: FURTHER INFORMATION ON THE ASSESSMENT OF EXCIPIENT 
DIFFERENCES 
 
Figure 1.   BCS Class I Drug Substances 
 
 
 
   
13  Contains Nonbinding Recommendations 
 
Figure 2.   BCS Class III Drug Substances 
 
 
   
14  Contains Nonbinding Recommendations 
 
EXAMPLES OF ACCEPTABLE DIFFERENCES IN EXCIPIENTS 
Example 1: BCS Class I Biowaiver 
 
The formulation of the test product is qualitatively the same as that of the reference product.  
Additionally, it contains sorbitol, an excipient with known or suspected effects on drug 
absorption.   The amount of sorbitol in the test formulation is within the permitted range of 
45 milligrams (mg) to 55 mg based on the amount of sorbitol in the reference formulation 
(i.e., 50 mg  10%). 
Amount (mg) 
Component  Amount (mg) Test 
Reference 
Drug substance   100  100 
Microcrystalline cellulose (filler)  100  95 
Sorbitol (filler)  50  55 
HPMC (binder)  10  10 
Talc (glidant)  5  5 
Total   265  265 
 
   
15  Contains Nonbinding Recommendations 
 
 
Example 2: BCS Class III Biowaiver 
 
The test formulation is qualitatively the same as the reference formulation.  Additionally, it 
contains sorbitol, an excipient with known or suspected effects on drug absorption.  The 
amount of sorbitol in the test formulation is within the permitted range of 9 mg to 11 mg 
based on the amount of sorbitol in the reference formulation (i.e., 10 mg  10%).  
Differences in the amount of other excipients are within the criteria outlined in Table 1, 
Section III.A. (3.1). 
Reference Product  Test Product 
Absolute 
% 
Proportion  Proportion  difference 
Component 
Composition  relative to  Composition  relative to  relative to 
(mg)  core weight  (mg)  core weight 
core 
(%w/w)  (%w/w) 
weights 
Drug substance   100  49.3%  100  46.5%  -- 
Lactose 
monohydrate  85  41.9%  97  45.1%  3.2% 
(filler) 
Sorbitol (filler)  10  4.9%  9  4.2%  0.7% 
Croscarmellose 
sodium  6  3.0%  7  3.3%  0.3% 
(disintegrant) 
Magnesium 
stearate  2  1.0%  2  0.9%  0.1% 
(lubricant) 
 
Total   203  100%  215  100% 
Total 
        4.3% 
change: 
 
 
16