Guidance for Industry 

 
Bioavailability and Bioequivalence 

Studies Submitted in NDAs or INDs  

General Considerations 

 
 
 
 
 
 
DRAFT GUIDANCE 

 
This guidance document is being distributed for comment purposes only. 
 
Comments and suggestions regarding this draft document should be submitted within 60 days of 
publication in the Federal Register of the notice announcing the availability of the draft 
guidance. Submit electronic comments to http://www.regulations.gov. Submit written  
comments to the Division of Dockets Management (HFA-305), Food and Drug Administration, 
5630 Fishers Lane, rm. 1061, Rockville, MD  20852.  All comments should be identified with 
the docket number listed in the notice of availability that publishes in the Federal Register. 
 
For questions regarding this draft document contact the CDER Office of Clinical Pharmacology 
at 301-796-5008 or OCP@fda.hhs.gov. 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 

Food and Drug Administration 

Center for Drug Evaluation and Research (CDER)
  
 
March 2014 

Biopharmaceutics
 
 
   
Guidance for Industry
 
  
 
Bioavailability and Bioequivalence 

Studies Submitted in NDAs or INDs 

General Considerations  

 
 
 
 
 
 
 
Additional copies are available from: 

 
Office of Communications 

Division of Drug Information, WO51, Room 2201 

Center for Drug Evaluation and Research 

Food and Drug Administration 

10903 New Hampshire Avenue, Silver Spring, MD  20993 

http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm 

Phone: 301-796-3400; Fax: 301-847-8714 

druginfo@fda.hhs.gov 

 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 

Food and Drug Administration 

Center for Drug Evaluation and Research (CDER)
  
 
March 2014 

Biopharmaceutics 
 
   
TABLE OF CONTENTS 

 
I.   INTRODUCTION................................................................................................................. 1 
 
II.   BACKGROUND ................................................................................................................... 2 
 
A.  GENERAL ............................................................................................................................. 3 

B.  BIOAVAILABILITY ................................................................................................................ 3 

C.  BIOEQUIVALENCE ................................................................................................................ 4 

III.   METHODS TO DOCUMENT BA AND BE...................................................................... 6  

A.  PHARMACOKINETIC STUDIES ............................................................................................... 6 

B.  OTHER  APPROACHES TO SUPPORT BA/BE......................................................................... 10 

IV.   DOCUMENTING BA AND BE FOR VARIOUS DOSAGE FORMS .......................... 12  

A.  SOLUTIONS AND OTHER  SOLUBILIZED DOSAGE FORMS..................................................... 12 

B.  IMMEDIATE-RELEASE PRODUCTS....................................................................................... 12 

C.  MODIFIED-RELEASE PRODUCTS......................................................................................... 13 

D.  BATCH SIZE ....................................................................................................................... 16 

V. 	  ADDITIONAL INFORMATION ON IN VITRO APPROACHES ............................... 16 
 
A.	   IN VITRO STUDIES CONDUCTED IN SUPPORT OF A WAIVER OF AN IN VIVO BA OR BE  DATA 

REQUIREMENT ................................................................................................................... 16 

B.	    IN VITRO STUDIES CONDUCTED IN SUPPORT OF DEMONSTRATING BA OR BE .................. 17 

VI.   SPECIAL TOPICS ............................................................................................................. 20 
 
A.  ALCOHOLIC BEVERAGE EFFECTS ON MR  DRUG PRODUCTS .............................................. 20 

B.  ENANTIOMERS VERSUS RACEMATES .................................................................................. 20 

C.  DRUG PRODUCTS WITH COMPLEX MIXTURES AS THE ACTIVE  INGREDIENTS .................... 20 

D.  LONG-HALF-LIFE DRUGS .................................................................................................. 21 

E.  ORALLY ADMINISTERED DRUGS INTENDED FOR  LOCAL ACTION....................................... 21 

F.  COMBINATION/COADMINISTERED DRUG PRODUCTS ......................................................... 21 

G.  ENDOGENOUS  SUBSTANCES ............................................................................................... 22 

H.  DRUG PRODUCTS WITH HIGH INTRASUBJECT VARIABILITY .............................................. 23 

APPENDIX A: GENERAL STUDY DESIGN AND DATA HANDLING ........................... 24
  
 
  Draft  Not for Implementation  
 
Contains Nonbinding  Recommendations  
 
Guidance for Industry1  
1  Bioavailability and Bioequivalence Studies Submitted in NDAs or INDs  
2  General Considerations 
3   
4   
5   
6  This draft guidance, when finalized, will represent the Food and Drug Administration's (FDA's) current 
7  thinking on this topic.  It does not create or confer any rights for or  on any  person and does not operate to 
8  bind FDA or the public.  You can use an alternative approach if the approach satisfies the requirements of 
9  the applicable statutes and regulations. If  you want to discuss an alternative approach, contact the FDA 
10  staff responsible for implementing this guidance.  If you cannot identify  the appropriate FDA staff, call 
11  the appropriate number listed on the title page of this guidance. 
12   
13   
14   
15   
16  I.   INTRODUCTION 
17   
18  This guidance provides recommendations to sponsors and/or applicants planning to include 
19  bioavailability (BA) and bioequivalence (BE) information for drug products in investigational 
20  new drug applications (INDs), new drug applications (NDAs), and NDA supplements (referred 
21  to as the NDA BA and BE Draft Guidance).2  This guidance contains advice on how to meet the 
22  BA and BE requirements set forth in 21 CFR part 320 as they apply to dosage forms intended for 
23  oral administration.3  The guidance may also be applicable to non-orally administered drug 
24  products when reliance on systemic exposure measures is suitable to document BA and BE (e.g., 
25  transdermal delivery systems and certain rectal and nasal drug products). The guidance should 
26  be helpful for applicants conducting BA and BE studies during the IND period for an NDA and 
27  also for applicants conducting BE studies during the postapproval period for certain changes to 
                                                 
1 This guidance was developed by the Office of Clinical Pharmacology, Office of Translational Sciences, and the 
Office of New Drugs Quality Assessment, Office of Pharmaceutical Science, in the Center for Drug Evaluation and 
Research (CDER) at the U.S. Food and Drug  Administration (FDA). 
2 BA and BE information for drug  products in abbreviated new drug applications (ANDAs) and ANDA supplements  
are not the subject of this guidance.  FDA has issued a separate draft guidance on this topic entitled  Bioequivalence 
Studies with Pharmacokinetic Endpoints for Drugs Submitted Under an  ANDA (December 2013) (ANDA BE Draft 
Guidance).  The ANDA BE Draft Guidance, when  finalized, will represent FDAs current thinking  on this topic. 
Many guidances are referenced throughout this document.  The guidance referred to in this footnote, as well as 
others referenced throughout the remainder of the document, can be found on the FDA Drugs guidance  Web page at  
http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm.  We update 
guidances  periodically.  To make sure you have the most recent version of a guidance, check the FDA Drugs 
guidance Web page.  
 
3 These dosage forms include tablets, capsules, solutions, suspensions, conventional/immediate-release drug 
products, and modified (extended, delayed)-release drug  products.  
 
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28  drug products that are the subject of an NDA. 4   This guidance document is not intended to 
29  provide recommendations on studies conducted in support of demonstrating comparability or 
30  biosimilarity for biological products licensed under section 351 of the Public Health Service 
31  Act.5    
32   
33  When finalized, this guidance will revise and replace the parts of FDAs March 2003 guidance 
34  for industry on Bioavailability and Bioequivalence Studies for Orally Administered Drug 
35  Products  General Considerations (the March 2003 BA and BE Guidance) relating to BA and 
36  BE studies for INDs, NDAs, and NDA supplements.6   Since the March 2003 BA and BE 
37  Guidance was issued, FDA has determined that providing information on BA and BE studies in 
38  separate guidances according to application type will be beneficial to sponsors and applicants.  
39  Thus, FDA is issuing this NDA BA and BE Draft Guidance and, as previously noted, has issued 
40  the ANDA BE Draft Guidance for ANDA and ANDA supplements.7  
41     
42  We recognize that this guidance cannot address every issue pertaining to the assessment of BA 
43  or BE studies for INDs and NDAs, so we suggest sponsors and applicants contact the appropriate 
44  review division for guidance on specific questions not addressed by this guidance. 
45   
46  FDA's guidance documents, including this guidance, do not establish legally enforceable 
47  responsibilities. Instead, guidances describe the Agency's current thinking on a topic and should 
48  be viewed only as recommendations, unless specific regulatory or statutory requirements are 
49  cited. The use of the word should in Agency guidance documents means that something is 
50  suggested or recommended, but not required.  
51   
52  II.   BACKGROUND 
53   
                                                 
4  Bioequivalence is a statutory term reflected in the Federal Food, Drug, and Cosmetic Act (FD&C Act) in section 
505(j)  (21 U.S.C. 355(j)), which requires ANDA applicants  to demonstrate, among other things, that the  proposed  
generic product is bioequivalent to its reference listed drug.  Section 505(j)(2)(A)(iv) of the FD&C Act; see also 
section  505(j)(8) of the FD&C Act.  There is no similar statutory requirement for an  NDA applicant either under 
section 505(b)(1) or  (b)(2) of the FD&C Act to  demonstrate bioequivalence of its proposed  product to another 
product.  As a scientific matter, however, the same or a similar showing  of  the bioavailability of two  products in the 
NDA context  may be needed for the purposes of evaluating the safety  or  effectiveness of a product.  For ease of the  
reader, we refer to such evaluations  of the relative bioavailability for two or more products as an evaluation of 
bioequivalence in this guidance.  
 
5 For information on these types of studies,  see FDAs Drugs guidance Web page.  See footnote #2 for information 
on accessing this Web page. 
 
6 Revisions to the March  2003 BA and BE Guidance include (1) expansion of the section on modified-release 
products, (2) addition  of a section  on concomitant administration  of drug  products and combination  drug  products, 
(3) addition  of  a section on alcoholic beverage effects on modified-release dosage forms, (4) addition  of  an  
endogenous substance section, (5) addition of a section  on  drug  products with high intrasubject variability, and  (6) 
removal of   references to BE  studies conducted for ANDAs.  The guidance also makes other revisions for  
clarification. 
 
7  See footnote #2. 
 
 
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54  BA assessment of formulations is a component of new drug development.  The approaches of 
55  evaluating BA and BE discussed in this guidance are designed to aid FDA evaluation of the 
56  safety and effectiveness of a product that is the subject of an IND, NDA, or NDA supplement.  
57  In this endeavor, we use the totality of information available in the submission, which includes, 
58  among other things, information gathered using the principles of BE, exposure-response 
59  evaluations, and clinical trial results.  The evaluation of BE in the generic drug context, by 
60  contrast, is used to support a determination that a generic product may be substituted for its 
61  reference listed drug, and involves consideration  of different types of data permitted in an 
62  ANDA. Accordingly, the approaches discussed in this guidance may differ from similar 
63  discussions of BE in the ANDA BE Draft Guidance.  For example, this NDA BA and BE Draft 
64  Guidance recommends assessment of the effect of food on BA using the approaches set forth in 
65  FDAs 2002 guidance for industry on Food-Effect Bioavailability and Fed Bioequivalence 
66  Studies (the 2002 Food-Effect Guidance).  Fasting BE studies generally are sufficient, given the 
67  totality of information we consider in evaluating INDs, NDAs, or NDA supplements.  In 
68  contrast, we recommend in the ANDA BE Draft Guidance fed and fasting BE studies that will 
69  provide specific information to support a demonstration of BE under section 505(j) of the FD&C 
70  Act, and in turn, to support substitutability.  Even though the ANDA BE Draft Guidance revises 
71  and replaces the parts of the 2002 Food-Effect Guidance pertaining to ANDAs and ANDA 
72  supplements, this NDA BA and BE Draft Guidance does not replace the 2002 Food-Effect 
73  Guidance relating to studies for INDs, NDAs, and NDA supplements.8    
74   
75  A.   General 
76   
77  Studies to measure BA and/or establish BE of a product are important elements in support of 
78  INDs, NDAs, and NDA supplements.  Bioavailability means the rate and extent to which the 
79  active ingredient or active moiety is absorbed from a drug product and becomes available at the 
80  site of action (21 CFR 320.1(a)). BA data provide an estimate of the fraction of the drug 
81  absorbed, as well as provide information related to the pharmacokinetics of the drug.     
82   
83  Bioequivalence means the absence of a significant difference in the rate and extent to which the 
84  active ingredient or active moiety in pharmaceutical equivalents or pharmaceutical alternatives 
85  become available at the site of drug action when administered at the same molar dose under 
86  similar conditions in an appropriately designed study (21 CFR  320.1(e)).  Studies to establish 
87  BE between two products are important for certain formulation or manufacturing changes 
88  occurring during the drug development and postapproval stages.  In BE studies, the exposure 
89  profile of a test drug product is compared to that of a reference drug product. 
90   
91  B.   Bioavailability 
92   
93  BA for a given formulation provides an estimate of the relative fraction of the orally 
94  administered dose that is absorbed into the systemic circulation.  BA for orally administered drug 
95  products can be documented by comparing a systemic exposure profile to that of a suitable 
96  reference product. A profile can be generated by measuring the concentration of active 
                                                 
8 Accordingly, we are in the process of revising the 2002 Food-Effect Guidance.  
 
 
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97  ingredients and/or active moieties over time and, when appropriate, active metabolites over time  
98  in samples collected from the systemic circulation.  Systemic exposure profiles reflect both 
99  release of the drug substance from the drug product and a series of possible presystemic/systemic 
100  actions on the drug substance after its release from the drug product.   
101   
102  FDAs regulations at 21 CFR 320.25 set forth guidelines for in vivo BA studies.  As provided in 
103  this regulation, the reference product for BA studies should be a solution, suspension, or 
104  intravenous (IV) dosage form (21 CFR 320.25(d)(2) and (3)).  The purpose of conducting a BA 
105  study with an oral solution as a reference is to assess the impact of formulation on BA. 
106  Conducting a BA study with an IV reference enables assessment of the impact of route of 
107  administration on BA and defines the absolute BA of the drug released from the drug product. 
108     
109   
110  C.   Bioequivalence 
111   
112  As noted previously, both BA and BE focus on the release of a drug substance from a drug 
113  product and subsequent absorption into systemic circulation.  As a result, we recommend that 
114  approaches to determining BE generally follow approaches similar to those used for BA.  
115  Demonstrating BE involves a more formal comparative test that uses specific references with 
116  specified criteria for comparisons and predetermined BE limits for such criteria.  
117    
118  1.   Preapproval Changes 
119   
120  BE documentation can be useful during the IND period to compare (1) early and late 
121  clinical trial formulations; (2) formulations used in clinical trials and stability studies, if 
122  different; (3) clinical trial formulations and to-be-marketed drug products, if different; 
123  and (4) product strength equivalence, as appropriate.  In each comparison, the new 
124  formulation, formulation produced by the new method of manufacture, or new strength is 
125  the candidate, or test product and the prior formulation, prior method of manufacture, or 
126  prior strength is the reference product.  The decision to document BE during drug 
127  development is generally left to the judgment of the sponsor, using the principles of 
128  relevant guidances (in this guidance, see sections II.C.2, Postapproval Changes, and 
129  III.D, In Vitro Studies) to determine when changes in components, composition, and/or 
130  method of manufacture suggest that further in vitro and/or in vivo studies be performed. 
131   
132  2.   Postapproval Changes 
133   
134  In the presence of certain major changes in components, composition, manufacturing site, 
135  and/or method of manufacture after approval, FDA recommends that in vivo BE be 
136  demonstrated for the drug product after the change in comparison to the drug product 
137  before the change. Under section 506A(c)(2) of the Federal Food, Drug, and Cosmetic 
138  Act (FD&C Act) (21 U.S.C. 356a(c)(2)), certain postapproval changes that require 
139  completion of studies must be submitted in a supplement and approved by FDA before 
140  distributing a drug product made with the change. 
141   
 
 
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142  Information on the types of recommended in vitro dissolution and in vivo BE studies for 
143  immediate-release and modified-release drug products approved as NDAs for specified 
144  postapproval changes is provided in the following FDA guidances: 
145   
146     SUPAC-IR: Immediate Release Solid Oral Dosage Forms:  Scale-Up and 
147  Postapproval Changes: Chemistry, Manufacturing, and Control; In Vitro 
148  Dissolution Testing, and In Vivo Bioequivalence Documentation  
149     SUPAC-MR: Modified Release Solid Oral Dosage Forms:  Scale-Up and 
150  Postapproval Changes: Chemistry, Manufacturing, and Controls, In Vitro 
151  Dissolution Testing, and In Vivo Bioequivalence Documentation  
152   
153  3.   BE Considerations 
154   
155  BE studies are usually conducted using a crossover design.  For such studies, intrasubject 
156  variability should be considered when determining the study sample size.  In cases when 
157  a parallel design is necessary to evaluate BE, consideration should be given to total 
158  variability, including intersubject variability instead of just intrasubject variability.   
159   
160  A test product might fail to demonstrate bioequivalence because it has measures of rate 
161  and/or extent of absorption compared to the reference product outside acceptable higher 
162  or lower limits. For example, when the test product results in a systemic exposure that is 
163  significantly higher than that of the reference product, the concern is the typically limited 
164  experience from a safety standpoint for higher systemic concentrations.  When the test 
165  product has a systemic exposure that is significantly lower than that of the reference 
166  product, the concern is potentially a lack of therapeutic efficacy of the test product.  
167  When the variability of the test product is greater than the reference product, the concern 
168  relates to both safety and efficacy, because it may suggest that the performance of the test 
169  product is not comparable to the reference product, and the test product may be too 
170  variable to be clinically useful. 
171   
172  When BE is not demonstrated, the sponsor should demonstrate that the differences in rate 
173  and extent of absorption do not significantly affect the safety and efficacy based on 
174  available dose-response or concentration-response data.  In the absence of this evidence, 
175  failure to demonstrate BE may suggest that the test product should be reformulated, or 
176  the method of manufacture for the test product should be changed, or additional safety or 
177  efficacy data may be needed for the test product.  In some cases, conclusions of BE based 
178  on the peak drug concentration (C ) and area under the plasma concentration time curve 
max
179  (AUC) between the test product and the reference product may be insufficient to 
180  demonstrate that there is no difference in safety or efficacy if the systemic concentration
181  time profiles of the test product and the reference product are different (e.g., time to reach 
182  peak drug concentration (T ) is different). For example, differences in the shape of the 
max
183  systemic concentration profile between the test and reference products could imply that 
184  the test product may not produce the same clinical response as the reference product.  In 
185  such cases, additional data analysis (e.g., partial AUCs), exposure-response evaluation, or 
186  clinical studies may be recommended to evaluate the BE of the two products. 
 
 
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187   
188  III.     METHODS TO DOCUMENT BA AND BE 
189   
190  Under FDAs regulations, applicants must use the most accurate, sensitive, and reproducible 
191  method available to demonstrate BA or BE of a product (21 CFR 320.24(a)).  As noted in 21 
192  CFR 320.24, several in vivo and in vitro methods can be used to measure BA and to establish 
193  BE. These include, in general order of preference, pharmacokinetic (PK) studies, in vitro tests 
194  predictive of human in vivo BA (in vitro-in vivo correlation), pharmacodynamic (PD) studies, 
195  studies with clinical benefit endpoints, and other in vitro studies. In addition, where in vivo data 
196  are appropriate to demonstrate BA, our regulations provide guidelines on specific types of in 
197  vivo BA studies (see 21 CFR 320.25 through 320.29).  This guidance predominantly focuses on 
198  the use of PK studies to document BA or BE.   
199   
200  A.   Pharmacokinetic Studies 
201   
202  1.   General Considerations 
203   
204  FDAs regulations generally define BA and BE in terms of rate and extent of absorption 
205  of the active ingredient or moiety to the site of action.9  For in vivo studies, the 
206  regulations also provide for use of PK measures in an accessible biological matrix such as 
207  blood, plasma, and/or serum to indicate release of the drug substance from the drug 
208  product into the systemic circulation.10  BA and BE frequently rely on PK measures such  
209  as AUC to assess extent of systemic exposure and C and T  to assess rate of systemic 
max   max
210  absorption. PK-based comparisons to describe relative BA or make BE determinations 
211  are predicated on an understanding that measuring the active moiety or ingredient at the 
212  site of action is generally not possible and on an assumption that some relationship exists 
213  between the efficacy/safety and concentration of the active moiety and/or its important 
214  metabolite(s) in the systemic circulation.  A typical study is conducted as a crossover 
215  study. The crossover design reduces variability caused by patient-specific factors, thereby 
216  increasing the ability to discern differences because of formulation. 
217   
218  2.   Pilot Study  
219   
220  If the sponsor chooses, a pilot study in a small number of subjects can be carried out 
221  before proceeding with a full-scale BA or BE study. The pilot study can be used to 
222  validate analytical methodology, assess PK variability, determine sample size to achieve 
223  adequate power, optimize sample collection time intervals, and determine the length of  
224  the washout period needed between treatments.  For example, for conventional 
225  immediate-release products, careful timing of initial samples may avoid a subsequent 
226  finding in a full-scale study that the first sample collection occurs after the C  For  
max.
227  modified-release products, a pilot study can help determine the sampling schedule needed 
                                                 
9 21 CFR 320.1(a) and  (e). 
10 See, e.g., 21  CFR 320.24(b)(1)(i).  If serial measurements of the drug  or its metabolites in plasma, serum, or blood  
cannot be accomplished,  then measurement  of urinary  excretion can be used.   
 
 
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228  to assess lag time and dose dumping.  The results of a pilot study can be used as the sole 
229  basis to document BA or BE provided the studys design and execution are suitable and a 
230  sufficient number of subjects have completed the study. 
231   
232  3.   Full-Scale Study 
233   
234  General recommendations for a standard BA or BE study based on PK measurements are 
235  provided in Appendix A. Nonreplicate crossover study designs are recommended for BA 
236  and BE studies of immediate-release and modified-release dosage forms.  However, 
237  sponsors and/or applicants have the option of using replicate designs for BE studies.  
238  Replicate crossover designs are used to allow estimation of (1) within-subject variance 
239  for the reference product, or for both the test and reference products, and (2) the subject 
240  by formulation interaction variance component.  This design accounts for the inter
241  occasion variability that may confound the interpretation of a BE study as compared to a 
242  non-replicate crossover approach. The recommended method of analysis for nonreplicate 
243  or replicate studies to evaluate BE is average BE, as discussed in section IV.  
244  Recommendations for conducting and evaluating replicate study designs can be found in 
245  the FDA guidance for industry Statistical Approaches to Establishing Bioequivalence.  
246   
247  4.   Study Population 
248   
249  Subjects recruited for BA or BE studies should be 18 years of age or older and capable of 
250  giving informed consent.  In general, BA and BE studies should be conducted in healthy 
251  volunteers if the product can be safely administered to this population. A study in healthy 
252  volunteers is likely to produce less PK variability compared with that in patients with 
253  potentially confounding factors such as underlying and/or concomitant disease and 
254  concomitant medications. Male and female subjects should be enrolled in BA and BE 
255  studies unless there is a specific reason to exclude one sex. Such exclusions could be 
256  related to the drug product being indicated in only one sex or a greater potential for 
257  adverse reactions in one sex compared to the other.  For example, oral contraceptives are 
258  evaluated in female subjects because the indication is specific to females.  If a drug has  
259  the potential to be a teratogen, the drug product should be evaluated in male subjects.  
260  Female subjects enrolled in the study should not be pregnant at the beginning of the study 
261  and should not become pregnant during the study.  In some instances (e.g., when safety 
262  considerations preclude use of healthy subjects), it may be necessary to evaluate BA and 
263  BE in patients for whom the drug product is intended.  In this situation, sponsors and/or 
264  applicants should attempt to enroll patients whose disease process is expected to be stable 
265  for the duration of the study. 
266   
267  5.   Single-Dose and Multiple-Dose (Steady State) Testing 
268   
269  This guidance generally recommends single-dose PK studies to assess BA and BE 
270  because they are generally more sensitive than steady-state studies in assessing rate and 
271  extent of release of the drug substance from the drug product into the systemic 
272  circulation.  
 
 
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273   
274  FDAs regulations at 21 CFR 320.27 provide guidelines on the design of a multiple-dose 
275  in vivo BA study. This regulation also identifies instances in which multiple-dose BA  
276  studies may be required: 
277   
278  i.   There is a difference in the rate of absorption but not in the extent of absorption. 
279  ii.   There is excessive variability in bioavailability from subject to subject.  
280  iii.   The concentration of the active drug ingredient or therapeutic moiety, or its 
281  metabolite(s), in the blood resulting from  a single dose is too low for accurate 
282  determination by the analytical method.  
283  iv.   The drug product is an extended-release dosage form.11  
284   
285  We recommend that if a multiple-dose study design is performed, appropriate dosage 
286  administration and sampling be carried out to document attainment of steady state. 
287   
288  6.   Bioanalytical Methodology 
289   
290  We recommend that sponsors ensure that bioanalytical methods for BA and BE studies 
291  be accurate, precise, specific, sensitive, and reproducible.  A separate FDA guidance, 
292  Bioanalytical Method Validation,  is available to assist sponsors in validating 
293  bioanalytical methods.12  
294   
295  7.   Administration Under Fasted/Fed Conditions 
296   
297  The BA or BE study should be conducted under fasting conditions (after an overnight fast 
298  of at least 10 hours) except when tolerability issues are anticipated with fasting.  In these 
299  cases, we recommend that applicants conduct only a fed study.  A separate FDA 
300  guidance, Food-Effect Bioavailability and Fed Bioequivalence Studies is available to 
301  assist sponsors. 
302   
303  8.   Moieties to Be Measured 
304   
305  The active ingredient that is released from the dosage form  or its active moiety and, when 
306  appropriate, its active metabolites13 should be measured in biological fluids collected in 
307  BA studies. 
308   
309  Measurement of the active ingredient or the active moiety, rather than metabolites, is 
310  generally recommended for BE studies because the concentration-time profile of the 
311  active ingredient or the active moiety is more sensitive to changes in formulation 
312  performance than that of the metabolite, which is more reflective of metabolite formation, 
313  distribution, and elimination.  The following are instances when an active metabolite(s) 
314  should be measured. 
                                                 
11 21 CFR 320.27(a)(3). 

12 See also  21 CFR 320.29. 

13 See 21 CFR 320.24(b)(1)(i). 

 
 
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315   
316     Measurement of a metabolite(s) is necessary when the active ingredient or the active 
317  moiety concentrations are too low to allow reliable analytical measurement in blood, 
318  plasma, or serum.  In this case, the metabolite should be measured in lieu of the active 
319  ingredient or active moiety. We recommend that the confidence interval approach be 
320  applied to the metabolite data obtained from these studies.   
321   
322     Measurement of a metabolite(s) is necessary in addition to the active ingredient or  
323  active moiety if the metabolite is formed by presystemic metabolism and contributes 
324  meaningfully to efficacy and/or safety.  The confidence interval approach should be 
325  used for all moieties measured.  However, the BE criteria are only generally applied 
326  to the active ingredient or active moiety.  Sponsors should contact the appropriate 
327  review division to determine which moieties should be measured.  
328   
329  9.   Pharmacokinetic Measures of Systemic Exposure  
330   
331  This guidance recommends that systemic exposure measures be used to evaluate BA and 
332  BE. Exposure measures are defined relative to peak, partial, and total portions of the 
333  plasma, serum, or blood concentration-time profile, as describe here: 
334   
335     Peak Exposure 
336   
337  We recommend that peak exposure be assessed by measuring the C  obtained directly 
max
338  from the systemic drug concentration data without interpolation.  The T  can provide 
max
339  important information about the rate of absorption.  The first point of a concentration
340  time curve based on blood and/or plasma  measurements is sometimes the highest 
341  concentration, which raises a question about the measurement of true C  because of 
max
342  insufficient early sampling times.  A carefully conducted pilot study may help to avoid 
343  this problem.  Collection of an early time point between 5 and 15 minutes after dosing 
344  followed by additional sample collections (e.g., two to five) in the first hour after dosing 
345  may be sufficient to assess early peak concentrations.  If this sampling approach is 
346  followed, we consider the data to be adequate, even when the highest observed 
347  concentration occurs at the first time point.  
348   
349     Total Exposure (Extent of Absorption) 
350   
351  For single-dose studies, we recommend that the measurement of total exposure be: 
352   
353  -  Area under the plasma, serum, or blood concentration time curve from time zero 
354  to time t (AUC ), where t is the last time point with a measurable concentration.  
0-t
355    
356  -  Area under the plasma, serum, or blood concentration time curve from time zero 
357  to time infinity (AUC ), where AUC  = AUC + C/ . C is the last 
0- 0- 0-t  t z t
358  measurable drug concentration and   is the terminal or elimination rate constant 
z
359  calculated according to an appropriate method. 
 
 
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360   
361  -  For drugs with a long half-life, truncated AUC can be used (see section VII.D, 
362  Long-Half-Life Drugs). 
363   
364  For steady-state studies, we recommend that the measurement of total exposure be the 
365  area under the plasma, serum, or blood concentration time curve from time zero to time  
366  tau over a dosing interval at steady state (AUC ), where tau is the length of the dosing 
0-tau
367  interval.  
368   
369     Partial Exposure  
370   
371  For orally administered drug products, BA and BE can generally be demonstrated by 
372  measurements of peak and total exposure.  For certain classes of drugs and under certain 
373  circumstances (e.g., to assess onset of an analgesic effect), an evaluation of the partial 
374  exposure could be used to support the performance of different formulations by providing 
375  further evidence of therapeutic effect. This guidance recommends the use of partial AUC 
376  as a partial exposure measure. The time to truncate the partial area should be related to a 
377  clinically relevant PD measure. We also recommend that sufficient quantifiable samples 
378  be collected to allow adequate estimation of the partial area.  For questions on the 
379  suitability of the PD measure or use of partial exposure in general, we recommend that 
380  sponsors and/or applicants consult the appropriate review division.  
381   
382  10.   Comparison of PK measures in BE studies 
383   
384  An equivalence approach is recommended for BE comparisons.  The recommended 
385  approach relies on (1) a criterion to allow the comparison, (2) a confidence interval for 
386  the criterion, and (3) a BE limit.  Log-transformation of exposure measures before 
387  statistical analysis is recommended.  This guidance recommends use of an average BE  
388  criterion to compare systemic exposure measures for replicate and nonreplicate BE 
389  studies of both immediate- and modified-release products.  For additional information on 
390  data analysis, refer to Appendix A and to the FDA guidance for industry on Statistical  
391  Approaches to Establishing Bioequivalence. 
392   
393  B.   Other Approaches to Support BA/BE 
394   
395  In certain circumstances, other approaches are recommended to support a demonstration of 
396  BA/BE. Below are some general considerations regarding these other approaches.  Sponsors 
397  should consult FDAs guidances for industry for additional information on these methods as 
398  well.14    
399   
400  1.  In Vitro Tests Predictive of Human In Vivo BA 
401  1.    
                                                 
14  See footnote 2. 
 
 
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402  In vitro-in vivo correlation (IVIVC) is an approach to describe the relationship 
403  between an in vitro attribute of a dosage form (e.g., the rate or extent of drug 
404  release) and a relevant in vivo response (e.g., plasma drug concentration or 
405  amount of drug absorbed).  This model relationship facilitates the rational 
406  development and evaluation of extended-release dosage forms.  Once an IVIVC is 
407  validated, the in vitro test serves as a surrogate for BA and/or BE testing, as well 
408  as a tool for formulation screening and setting of the dissolution/drug-release 
409  acceptance criteria.  
410   
411  Specifically, in vitro dissolution/drug-release characterization is encouraged for 
412  all extended-release product formulations investigated (including prototype 
413  formulations), particularly if in vivo absorption characteristics are being defined 
414  for the different product formulations.  Such efforts may enable the establishment 
415  of an IVIVC.  When an IVIVC or association is established (21 CFR 
416  320.24(b)(1)(ii)), the in vitro test can serve not only as a quality control 
417  specification for the manufacturing process, but also as an indicator of how the 
418  product will perform in vivo.  
419   
420  Additional information on the development and validation of an IVIVC can be 
421  found in the FDA guidance for industry Extended Release Oral Dosage Forms: 
422  Development, Evaluation, and Application of In Vitro/In Vivo Correlations.  
423   
424  2.  Pharmacodynamic Studies 
425   
426  PD studies are not recommended for orally administered drug products when the 
427  drug is absorbed into systemic circulation and a PK approach can be used to 
428  assess systemic exposure and evaluate BA or BE.  PK endpoints are preferred 
429  because they are generally the most accurate, sensitive, and reproducible 
430  approach. However, in instances where a PK endpoint is not possible, a well
431  justified PD endpoint can be used to demonstrate BA or BE.  
432   
433  3.  Comparative Clinical Studies 
434   
435  Clinical endpoints can be used in limited circumstances, for example, for orally 
436  administered drug products when the measurement of the active ingredients or 
437  active moieties in an accessible biological fluid (PK approach) or PD approach is 
438  not possible.  Because these circumstances  do not occur very often, use of this 
439  approach is expected to be rare.  
440   
441  4.  In Vitro Studies 
442   
443  Under certain circumstances, BA and BE can be evaluated using in vitro 
444  approaches (e.g., dissolution/drug-release testing) during the preapproval and 
445  postapproval phases (see 21 CFR 320.24(b)(5) and (6)).   For example, orally 
446  administered drugs that are highly soluble and highly permeable, and for which 
 
 
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447  the drug product is rapidly dissolving, documentation of BE using an in vitro 
448  approach (dissolution/drug-release studies) may be appropriate based on the 
449  Biopharmaceutics Classification System.15  
450   
451  The following FDA guidances provide recommendations on the development of 
452  dissolution methodology, setting specifications, and the regulatory applications of 
453  dissolution testing:  
454   
455     Dissolution Testing of Immediate-Release Solid Oral Dosage Forms 
456   
457     Extended-Release Oral Dosage Forms: Development, Evaluation, and 
458  Application of In Vitro/In Vivo Correlations 
459   
460  In addition, we recommend that sponsors consult other FDA guidances for 
461  additional information on when in vitro data may be appropriate to demonstrate 
462  BA or BE of a product. 
463   
464  IV.   DOCUMENTING BA AND BE FOR VARIOUS DOSAGE FORMS  
465   
466  This section summarizes the recommendations for documenting BA and BE studies based on the 
467  specific dosage forms and whether these evaluations occur preaapproval or postapproval. 
468   
469  A.   Solutions and Other Solubilized Dosage Forms  
470   
471  For oral solutions, elixirs, syrups, tinctures, or other solubilized forms, in vivo BA and/or BE  are 
472  generally self-evident and a requirement of in vivo data for a product may be waived (21 CFR 
473  320.22(b)(3)). In such instances, the applicant would be deemed to have complied with and 
474  fulfilled any requirement for in vivo data.16 Although a comparative study is not necessary, 
475  characterization of the pharmacokinetics of the drug is required (21 CFR 314.50(d)(3)).   In 
476  addition, in vivo BE studies that compare different solution formulations are waived based on the 
477  assumptions that release of drug substance from  the drug product is self-evident and that the 
478  solutions do not contain any excipients that significantly affect drug absorption.  However, there 
479  are certain excipients that may alter the BA (e.g., sorbitol may reduce the BA of drugs, and 
480  vitamin E may enhance the BA) in amounts sometimes used in oral liquid dosage forms.  In this 
481  case, evaluation of in vivo BA and/or BE may be required.  
482   
483  B.   Immediate-Release Products 
484   
485  Included in this discussion are capsules, tablets (including conventional, buccal, chewable, orally 
486  disintegrating, and sublingual dosage forms), and suspensions.  
                                                 
15 See the FDA  guidance for industry on  Waiver of  In Vivo Bioavailability and Bioequivalence Studies for 
Immediate-Release Solid  Oral Dosage Forms Based on  a Biopharmaceutics Classification  System. This document 
provides complementary information on the Biopharmaceutics Classification System (BCS).   
16 See 21 CFR 320.22(b)(3).   
 
 
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487   
488  1.   Preapproval Changes 
489   
490  For BA and BE studies, we recommend a single-dose, fasting study be performed.  Under 
491  certain circumstances, multiple-dose BA studies (see section III.A.5) and/or food effect 
492  studies may be necessary (See the FDA guidance for industry Food-Effect Bioavailability 
493  and Fed Bioequivalence). Unconventional dosage forms (buccal, chewable, orally 
494  disintegrating, and sublingual dosage forms) should be administered according to 
495  intended label use/instructions.  In addition, a BA study may be needed with the 
496  unconventional dosage form swallowed intact to assess the impact of accidental 
497  swallowing of the intact product. Sampling should adequately capture the T  and C  
max max 
498  in addition to total exposure. 
499   
500  We recommend that in vitro dissolution be evaluated for all orally administered products.  
501  In vitro dissolution test conditions could be the same or different for unconventional 
502  compared to conventional dosage forms.  If differences in dissolution data exist, they 
503  should be discussed with the appropriate review division.  
504   
505  2.   Postapproval Changes 
506   
507  Information on the types of in vitro dissolution and in vivo BE studies needed for 
508  approved immediate-release drug products when postapproval changes are made is 
509  provided in an FDA guidance for industry entitled SUPAC-IR: Immediate Release Solid 
510  Oral Dosage Forms Scale-Up and Postapproval Changes: Chemistry, Manufacturing, 
511  and Controls, In Vitro Dissolution Testing, and In Vivo Bioequivalence Documentation. 
512  We recommend that for postapproval changes, the in vitro or in vivo comparison be made 
513  between the post-change and pre-change products. 
514   
515  C.   Modified-Release Products  
516   
517  Modified-release (MR) products include extended-release (controlled-release, sustained-
518  release)17 and delayed-release products. 
519   
520  Extended-release (ER) products are dosage forms that are designed to extend or prolong the 
521  release of active ingredient or active moiety from the drug product and may allow a reduction in 
522  dosing frequency as compared to when the drug is administered in an immediate-release (IR) 
523  dosage form. These drug products can be developed to reduce fluctuations in plasma  
524  concentrations when compared to an IR product. ER products can be capsules, tablets, granules, 
525  pellets, or suspensions. 
526   
527  Delayed-release (DR) drug products are dosage forms that release active ingredient or active 
528  moiety at a time later than immediately after administration (i.e., these drug products exhibit a 
529  lag time in quantifiable plasma concentrations).  Typically, coatings (e.g., enteric coatings) are 
                                                 
17  For the purpose of this guidance, the terms extended, controlled, and  sustained are used interchangeably. 
 
 
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530  used to delay the release of the drug substance until the dosage form has passed through the 
531  acidic medium of the stomach.  Generally, DR products are treated as IR products.  However, if 
532  the DR product has complex release characteristics, the relevant review division should be 
533  contacted for additional guidance. 
534   
535  If the drug product is an ER product, the following recommendations apply. 
536   
537  1.   Preapproval: BA and BE Studies 
538   
539  FDAs regulations at 21 CFR 320.25(f) address the purpose of a BA study for an 
540  extended-release product, which is to determine if certain delineated conditions are met.18     
541  This regulation also provides that the reference material(s) for such a bioavailability  
542  study shall be chosen to permit an appropriate scientific evaluation of the extended 
543  release claims made for the drug product.19  Appropriate reference products may include 
544  (1) a solution or suspension of the active drug ingredient or therapeutic moiety, (2) a 
545  currently marketed non-controlled-release drug product containing the same active drug 
546  ingredient or therapeutic moiety and administered according to the dosage 
547  recommendations in the labeling of the non-controlled release drug product, and (3) a 
548  currently marketed ER drug product subject to an approved full NDA containing the 
549  same active drug ingredient or therapeutic moiety and administered according to the 
550  dosage recommendations in the labeling of currently marketed ER product.20  
551   
552  In general, the PK profile of the ER product may not match that of the approved IR 
553  product (e.g., T  is different) or, in some cases, to another ER product. In such a case, 
max
554  establishing similar PK profiles using C  and AUC may not be sufficient to show that 
max
555  the ER product is bioequivalent to the IR product.  Thus, additional safety or efficacy 
556  studies or PK/PD assessments may be recommended. This guidance recommends that the 
557  following BA studies and food effect BA studies be conducted for an ER drug product 
558  submitted as an NDA for the scenarios described below: 
559   
560  New ER formulation comparison to an already-approved IR product  
561   
562     For drugs with linear pharmacokinetics over the therapeutic dose range:  A fasting 
563  study should be conducted comparing the ER product administered as a single 
564  dose at the highest strength to the IR reference administered over the least 
565  common time interval to achieve equivalent total dose as for the ER product.21  If 
                                                 
18 21 CFR 320.25(f)(1). 
19 21 CFR 320.25(f)(2). 
20  21  CFR 320.25(f)(2)(i), (ii), and (iv).  We recommend  that a sponsor seeking to  use as a reference product a 
currently marketed extended release drug  product  subject to  an approved full new drug application containing the 
same active drug ingredient or therapeutic moiety and administered according to the dosage recommendations in the  
labeling proposed for the extended release drug  product, under 21  CFR 320.25(f)(2)(iii), consult with the Agency 
before commencing such a study.   
21 For example, when a 150-milligram (mg) ER product administered  once daily (QD) is being  developed that gives 
an approved 50-mg IR  reference product administered three times a day (TID) or a 75-mg product administered two 
times a day (BID), a comparison of the 150-mg ER product administered as a single  dose could be compared to  
 
 
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566  for safety reasons the highest strength cannot be used, a lower strength may be 
567  acceptable.   
568   
569     For drugs with nonlinear pharmacokinetics over the therapeutic dose range:  At a 
570  minimum, a single dose of the highest and lowest strengths of the ER product 
571  should be compared to their corresponding IR references administered over the 
572  ER dosing interval. If the relative BA  of intermediate ER strengths cannot be 
573  inferred based on the above studies, a single-dose fasting study for the 
574  intermediate strength(s) of the ER product should be compared to the 
575  corresponding IR reference administered over the ER dosing interval. 
576   
577     When the ER strengths are not proportionally similar in composition, a single
578  dose fasting dosage strength equivalence assessment study22 or a dosage strength 
579  proportionality study23 for the ER product should be conducted. 
580   
581     A single-dose food-effect study should be conducted on the highest ER strength 
582  (see the 2002 Food-Effect Guidance). 
583   
584     A steady state study should be conducted on the highest strength of the ER 
585  product compared to an approved IR reference dosed to achieve equivalent total 
586  dose as for the ER product. 
587   
588  New ER product (ER ) comparison to an approved ER product (ER ) with a different 
new old
589  dosing interval (i.e., where ER  and ER  have unequal dosing intervals) 
new old
590   
591     The recommendations are the same as outlined in the previous section 
592  (Development of a new ER formulation given an already approved IR product) 
593  except for the choice of the reference product.  In this case, the reference product 
594  could be either the approved ER  or IR product. 
old
595   
596  New ER product (ER ) comparison to an approved ER product (ER ) with the same  
new old
597  dosing interval 
598   
599     A single-dose fasting BE study on the highest strength of the ER  product 
new
600  compared to the ER  product.  If ER  and ER  are of different strength, then 
old new old
 
either the 50-mg IR reference product administered TID or 75-mg IR  reference product administered BID.  In this  
case, the least common time  interval is 24  hours.  
 
22 If three strengths, 10, 25, and 50 mg, are being developed for a new ER  dosage form, the dosage strength  
equivalence study should be conducted using 510  mg, 225  mg, and 150  mg  to  achieve constancy of dose.  
 
23 If three strengths, 10, 25, and 50 mg, are being developed for a new ER  dosage form, the dosage strength  
proportionality study should  be conducted using  110 mg, 125 mg, and  150 mg to achieve constancy of dose and  
the dosage strength  proportionality study should  be conducted using  110 mg, 125 mg, and 150 mg. 
 
 
 
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601  comparison of ER  versus ER  should be made based on dose using the highest 
new old
602  strengths.  
603   
604     A single-dose, food-effect study should be conducted on the highest ER  
new 
605  strength.  
606   
607     When the ER  strengths are not proportionally  similar in composition, a single
new
608  dose fasting dosage strength equivalence assessment study or a dosage strength 
609  proportionality study24 for the ER  product should be conducted. 
new
610   
611     In some cases, BE between the new and old ER products may not be sufficient to 
612  ensure that there is no difference in safety or efficacy if the PK profiles of the two 
613  ER products do not match (e.g., T  is different). Additional data analysis or 
max
614  clinical studies may be needed to ensure that the two products are clinically 
615  equivalent. 
616   
617  2.    Postapproval Changes 
618   
619  Information on the types of in vitro dissolution and in vivo BE studies for ER drug 
620  products approved in the presence of specific postapproval changes are provided in an 
621  FDA guidance for industry SUPAC-MR:  Modified Release Solid Oral Dosage Forms:  
622  Scale-Up and Postapproval Changes: Chemistry, Manufacturing, and Controls; In Vitro 
623  Dissolution Testing, and In Vivo Bioequivalence Documentation. We recommend that 
624  for postapproval changes, the in vitro or in vivo comparison be made between the post
625  change and pre-change products. 
626   
627  D.   Batch Size   
628   
629  For pivotal BE studies, the test batch should be representative of the production batches. 
630  Therefore, the size of the test batch should be at least 10% of the planned production batch size, 
631  or a minimum of 100,000 units, whichever is larger. 
632   
633  V.   ADDITIONAL INFORMATION ON IN VITRO APPROACHES  
634   
635  A.  In Vitro Studies Conducted in Support of a Waiver of an In Vivo BA or BE 
636  Data Requirement 
637   
638  As discussed above, FDAs regulations contemplate that if in vivo BA or BE data are required 
639  for a product, a sponsor may seek a waiver of that requirement under certain circumstances.25   
                                                 
24 21 CFR 320.21(b) (giving applicants the option  of submitting information that would  permit FDA to  waive the 
submission of evidence demonstrating in vivo bioequivalence) and 320.21(f) (requiring that the information 
submitted in support of a waiver request shall meet the criteria set forth in   320.22). 
25 21 CFR 320.21(b) (giving applicants the option  of submitting information that would  permit FDA to  waive the 
submission of evidence demonstrating in vivo bioequivalence) & 320.21(f) (requiring that the information 
submitted in support of a waiver request shall meet the criteria set forth in   320.22.) 
 
 
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640  For example, in some instances, in vivo BA or BE is self-evident based on certain characteristics 
641  of the drug product (21 CFR 320.22(b)), and therefore, any in vivo data requirement has been 
642  deemed to have been met.  In other delineated circumstances, an  in vivo BA or BE data 
643  requirement may be waived, and in vitro data may be accepted in lieu of in vivo data (21 CFR 
644  320.22(d)). For example, an in vivo data requirement may be waived for different strengths of 
645  an immediate-release drug product under 21 CFR 320.22(d)(2) when (1) the drug product is in 
646  the same dosage form, but in a different strength; (2) this different strength is proportionally 
647  similar in its active and inactive ingredients to another drug product for which the same 
648  manufacturer has obtained approval; and (3) the new strength meets an appropriate in vitro test 
649  as outlined in the regulation.26  In addition, for waiving higher strengths, linearity of the 
650  pharmacokinetics over the therapeutic dose range should be demonstrated. 
651   
652  This guidance defines proportionally similar in the following ways: 
653   
654     All active and inactive ingredients are in exactly the same proportion between different 
655  strengths (e.g., a tablet of 50-mg strength has all the inactive ingredients, exactly half that 
656  of a tablet of 100-mg strength, and twice that of a tablet of 25-mg strength). 
657   
658     For high-potency drug substances (where the amount of active drug substance in the 
659  dosage form is relatively low), (1) the total weight of the dosage form remains nearly the 
660  same for all strengths (within  10 % of the total weight of the strength on which a BE 
661  was performed), (2) the same inactive ingredients are used for all strengths, and (3) the 
662  change in any strength is obtained by altering the amount of the active ingredients and 
663  one or more of the inactive ingredients.   
664   
665     Bilayer tablets are considered to be one formulation even though they consist of two 
666  separate layers with different compositions.  In assessing the proportional similarity of  
667  the different strengths, all components of both layers should be proportionally similar.  
668  The fact that only one layer is proportionally similar and the other is not clearly indicates 
669  that the products (whole tablet) are not proportionally similar. This is relevant because 
670  there can be interactions between the different tablet layers, which can differ across 
671  different strengths because of the different size of the layers and the varying amounts of 
672  excipients present in each layer.  
673   
674  Exceptions to the above definitions may be possible if adequate justification is provided and 
675  discussed with the appropriate review division. 
676   
677  B.  In Vitro Studies Conducted in Support of Demonstrating BA or BE 
678    
 
 
26 See also  21 CFR 322.22(d)(3) and  (4) for additional bases for waiver.  Also, FDA, for good cause, may waive a 
requirement for the submission  of evidence of in vivo bioavailability or  bioequivalence if waiver is compatible with 
the protection  of the public health.  For full NDAs, FDA may defer a requirement for the submission of evidence of 
in vivo bioavailability if deferral is compatible with  the protection of  the public health (21  CFR 320.22(e)). 
 
 
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679  FDA may determine that in vitro data are the most accurate, sensitive, and reproducible method 
680  to demonstrate BA or BE in other contexts (21 CFR 320.24(b)(5) and (6)).27  Below we provide 
681  additional guidance on the conduct of such studies.   
682   
683  1.  Immediate-Release Formulations (Capsules, Tablets, and Suspensions) 
684   
685  In vitro data can be used to compare formulations of drug products under certain 
686  circumstances.  If an applicant seeks to demonstrate the BA or BE of immediate-release 
687  formulations for capsules, tablets, and suspensions using in vitro data, FDA recommends 
688  that sponsors generate dissolution profiles for all strengths using an appropriate 
689  dissolution method.  If the dissolution results indicate that the dissolution characteristics 
690  of the product are not dependent on the pH and product strength, dissolution profiles in 
691  one medium are usually sufficient to support demonstrating BE.  Otherwise, dissolution 
692  data in at least three media (e.g., pH 1.2, 4.5, and 6.8) are recommended.  The f  test 
2
693  should be used to compare profiles from the different strengths of the product (see FDA 
694  guidance for industry, Dissolution Testing of Immediate Release Solid Oral Dosage 
695  Forms). An f  value > 50 indicates a sufficiently similar dissolution profile to support a 
2
696  biowaiver. For an f  value < 50, discussion with the appropriate review division is 
2
697  recommended to determine whether an in vivo study is needed.  The f  approach is not 
2
698  suitable for rapidly dissolving drug products (e.g., > 85% dissolved in 15 minutes or less).  
699   
700     Over-encapsulation of clinical trial formulations  
701   
702  During the course of drug development, sponsors sometimes have to blind the 
703  formulations that they use in the clinical trials.  In certain situations, the only difference  
704  between the to-be-marketed and clinical trial formulations is that the dosage form is put 
705  into a capsule. This over-encapsulation is done mainly for blinding purposes.  It may be 
706  possible to support bioequivalence of the to-be-marketed and clinical trial formulations 
707  using in vitro data only, provided that no other excipients are added to the capsule and the 
708  dissolution profiles are comparable in three media:  pH 1.2, pH 4.5 and pH 6.8.  
709   
710     Scale-up and postapproval changes 
711   
712  Certain formulation changes in components and composition, scale-up, manufacturing 
713  site, manufacturing process, or equipment can be made postapproval.  Depending on the 
714  possible impact of the manufacturing change on the release of the active ingredient from  
715  the formulation and its BA, certain manufacturing changes for IR products can be 
716  approved based solely on similarity of the dissolution profiles between the postchange 
717  and prechange formulations.  Information on recommendations for using in vitro 
718  dissolution and in vivo BE studies for immediate-release drug products in such 
719  circumstances is provided in FDAs guidance for industry on SUPAC IR:   Immediate-
720  Release Solid Oral Dosage Forms: Scale-Up and Post-Approval Changes: Chemistry, 
721  Manufacturing, and Controls; In Vitro Dissolution Testing, and In Vivo Bioequivalence 
                                                 
27 In such instances, no waiver under 21 CFR  320.21 and 320.22 is  necessary.  
 
 
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722  Documentation.  The same principles described in the guidance can be applied to 
723  pre-approval changes in which the to-be-marketed formulation differs from the clinical 
724  trial formulation.  
725   
726  2.  Modified-Release Formulations 
727   
728  The use of in vitro data may be acceptable for modified-release drug products for which 
729  specific postapproval changes are sought is delineated in the FDA guidance for industry 
730  SUPAC-MR: Modified Release Solid Oral Dosage Forms: Scale-Up and Postapproval 
731  Changes: Chemistry, Manufacturing, and Controls; In Vitro Dissolution Testing, and In 
732  Vivo Bioequivalence Documentation. The same principles described in the guidance may 
733  also apply to preapproval changes.  Additional considerations for use of in vitro data are 
734  described below. 
735   
736     Beaded capsules: lower/higher strength 
737   
738  For ER beaded capsules where the strength differs only in the number of beads 
739  containing the active moiety, a single-dose, fasting BA or BE study, as appropriate, 
740  should be carried out on the highest strength. In vivo BA or BE of one or more lower 
741  strengths can be demonstrated based on dissolution profile comparisons, with an in vivo 
742  BA or BE study only on the highest strength (unless safety reasons preclude the 
743  administration of the highest strength to healthy volunteers). The dissolution profiles for 
744  each strength should be generated using the recommended dissolution method.  If the 
745  dissolution method has not been finalized, dissolution profiles should be generated in at 
746  least three media (e.g., pH 1.2, 4.5, and 6.8). In vivo BE studies for higher strengths may 
747  not be necessary based on (1) clinical safety and/or efficacy data on the proposed dose 
748  and the need for the higher strength, (2) linearity of pharmacokinetics over the 
749  therapeutic dose range, and (3) the same dissolution procedures being used for all 
750  strengths with similar dissolution results.  The f  test can be used to demonstrate similar 
2
751  profiles among the different strengths of the product.   
752   
753     MR dosage forms: lower strength 
754   
755  For MR dosage forms, when the drug product is in the same dosage form but in a 
756  different strength and when (1) the drug exhibits linear pharmacokinetics, (2) the various 
757  strengths are proportionally similar in their active and inactive ingredients28 and (3) the 
758  drug-release mechanism is the same, an in vivo BA or BE determination of one or more 
759  lower strengths can be demonstrated based on dissolution profile comparisons, with an in 
760  vivo BA or BE study only on the highest strength.  The dissolution profiles for each 
761  strength should be generated using the recommended dissolution method.  If the 
762  dissolution method has not been finalized, dissolution profiles should be generated in at 
                                                 
28 If the formulations of all the strengths are not compositionally proportional, in  vitro data can  be submitted  for the 
middle strength(s) if the following data are acceptable: (1) BA or BE data, as appropriate, for both the highest and 
the lowest strengths, and (2) in  vitro multimedia dissolution comparison  profiles using f2 evaluation.    
 
 
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763  least three media (e.g., pH 1.2, pH 4.5, and pH 6.8).  The dissolution profile should be 
764  generated on the test and reference products of all strengths using the same dissolution 
765  test conditions. 
766   
767  VI.   SPECIAL TOPICS  
768   
769  A.   Alcoholic Beverage Effects on MR Drug Products 
770   
771  The consumption of alcoholic beverages may affect the release of a drug substance from an MR 
772  formulation.  The formulation may lose its MR characteristics, leading to more rapid drug release 
773  and altered systemic exposure.  This more rapid drug release may have deleterious effects on the 
774  drug's safety and/or efficacy. 
775   
776  In vitro assessments of the drug release from  the drug product using media with various alcohol 
777  concentrations should be conducted.  Based on the results of the in vitro assessments, an in vivo 
778  BA study of the drug product when administered with alcohol may be needed.    
779   
780  B.   Enantiomers versus Racemates 
781   
782  During development of a racemic drug product, the racemate should be measured in BA studies.  
783  It may also be important to measure the individual enantiomers of the racemate to characterize 
784  the pharmacokinetics of the enantiomers.  For the development of a specific enantiomer, chiral 
785  inversion should be assessed. 
786   
787  Measurement of the racemate using an achiral assay is recommended for BE studies.  
788  Measurement of individual enantiomers in BE studies is recommended only when all of the 
789  following conditions are met:  (1) the enantiomers exhibit different PD characteristics, (2) the 
790  enantiomers exhibit different PK characteristics,  (3) primary efficacy and safety activity resides 
791  with the minor enantiomer, and (4) nonlinear absorption is present (as expressed by a change in 
792  the enantiomer concentration ratio with change in the input rate of the drug) for at least one of 
793  the enantiomers.  In such cases, we recommend that BE criteria be applied to the enantiomers 
794  separately. 
795   
796  C.   Drug Products With Complex Mixtures as the Active Ingredients 
797   
798  Certain drug products may contain complex drug substances (i.e., active moieties or active 
799  ingredients that are mixtures of multiple synthetic and/or natural source components).  Some or 
800  all of the components of these complex drug substances may not be fully characterized with 
801  regard to chemical structure and/or biological activity.  Quantification of all active or potentially 
802  active components in BA and BE studies may not be possible.  In such cases, we recommend 
803  that BA and BE studies be based on a select number of components.  Criteria for component 
804  selection typically include the amount of the moiety in the dosage form, plasma or blood levels 
805  of the moiety, and biological activity of the moiety.  When PK approaches are infeasible to 
806  assess rate and extent of absorption of a drug substance from a drug product, PD, clinical, or in 
807  vitro approaches may be appropriate.  
 
 
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808   
809  D.   Long-Half-Life Drugs 
810   
811  In a BA or PK study involving an IR  oral product with a long half-life ( 24 hours), adequate 
812  characterization of the half-life should include blood sampling over a long period of time.  For 
813  BA or BE determination of a drug product containing a drug with a long half-life, a nonreplicate, 
814  single-dose, crossover study can be conducted, provided an adequate washout period is used.  If 
815  the crossover study is problematic, a study with a parallel design can be used.  For either a 
816  crossover or parallel study, we recommend that the sample collection time be adequate to ensure 
817  completion of gastrointestinal transit (approximately 2 to 3 days) of the drug product and 
818  absorption of the drug substance. C  and a suitably truncated AUC can be used to characterize 
max
819  peak and total drug exposure, respectively. For drugs that demonstrate low intrasubject 
820  variability in distribution and clearance, a truncated AUC (e.g., AUC ) can be used in place 
0-72 hr
821  of AUC or AUC . For drugs that demonstrate high intrasubject variability in distribution and 
0-t  0-
822  clearance, AUC truncation should not be used.  In such cases, we recommend that sponsors 
823  and/or applicants consult the appropriate review division. 
824   
825  E.   Orally Administered Drugs Intended for Local Action  
826   
827  Documentation of BA and BE when the drug substance produces its effects by local action in the 
828  gastrointestinal tract can be achieved either by using pharmacokinetics, an acceptable PD end 
829  point, clinical efficacy and safety studies, and/or suitably designed and validated in vitro studies, 
830  as appropriate. For such cases, we recommend that sponsors and/or applicants consult the 
831  appropriate review division. Additional safety studies may also be recommended to characterize 
832  the local safety of the product. The in vitro studies should reflect important clinical effects or 
833  should be more sensitive to changes in product performance compared to a clinical study.  To 
834  ensure comparable safety, additional studies with and without food may help to understand the 
835  degree of systemic exposure that occurs following administration of a drug product intended for 
836  local action in the gastrointestinal tract.  
837   
838  F.   Combination/Coadministered Drug Products 
839   
840  Two or more active ingredients can be formulated as a single drug product, which is referred to 
841  as a combination drug product.  Generally, the purpose of an in vivo BA study involving a 
842  combination drug product is to compare the rate and extent of absorption of each active drug 
843  ingredient or therapeutic moiety in the combination drug product to the rate and extent of 
844  absorption of each active drug ingredient or therapeutic moiety administered concurrently in 
845  separate single-ingredient preparations (21 CFR 320.25(g). 
846   
847  For the purpose of defining BA or determining BE when required, this guidance recommends 
848  that the following studies be conducted for a combination drug product: 
849   
850     A two-treatment, single-dose, fasting study of the combination drug product versus 
851  single-ingredient drug products administered concurrently as a single treatment or an 
852  approved combination product containing the same active ingredients.  This study should 
 
 
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853  use the highest strength of the combination product with matching doses of individual 
854  drug products.   
855   
856     Certain alternative study designs may also be acceptable depending on the specific 
857  situation. For instance, in the case of a combination product consisting of two 
858  components, a three-treatment study design comparing the combination drug product 
859  versus single-ingredient drug products administered separately may be appropriate. 
860   
861     A single-dose, food-effect study on the combination drug product. 
862   
863  BE studies for the combination product should include the measurement of systemic 
864  concentrations of each active ingredient.  The confidence interval approach should be applied to 
865  each measured entity of the combination drug product and its reference product.  
866   
867  In specific cases, drug products are given in combination (not co-formulated) with the objective 
868  of increasing the exposure of one of the drugs (subject drug).  The second drug is not intended to 
869  have a therapeutic effect and is given only to increase the systemic exposure of the subject drug.  
870  When both the subject and second drug are new molecular entities, the BA of each should be 
871  assessed separately. If a BE study is needed for the subject drug for any reason, the subject drug 
872  should be administered with the second drug for both test and reference products.  The 
873  corresponding PK results, including confidence intervals for BE criteria, should be applied to the 
874  subject drug. It is not necessary to measure the concentrations of the second drug.  BE studies 
875  that are needed for the second drug should be conducted only with the second drug; the subject 
876  drug is not dosed with the second drug.  When the combination includes a new molecular entity 
877  and an approved product, only the BA of the new molecular entity should be assessed.  It is 
878  assumed that the BA of the approved product has been previously evaluated.    
879   
880  G.   Endogenous Substances 
881   
882  Drug products can be developed that contain compounds that are endogenous to humans (e.g., 
883  testosterone). When the endogenous compounds are identical to the drug that is being 
884  administered, determining the amount of drug released from the dosage form and absorbed by 
885  each subject is difficult. In most cases, it is important to measure and approximate the baseline 
886  endogenous levels of the compound in blood (plasma) and subtract these levels from the total 
887  concentrations measured from each subject after the drug product is administered.  In this way, 
888  an estimate of actual drug availability from  the drug product can be achieved, and therefore BA 
889  and BE can be assessed. Endogenous substances may have homeostatic processes that affect 
890  their production and therefore impact their systemic concentrations. To reduce the complication 
891  of these homeostatic processes and to potentially avoid the need for baseline correction, an 
892  alternative approach might be to enroll patients in BA and BE studies with low or no production 
893  of the endogenous substances instead of healthy volunteers. 
894   
895  Baseline concentrations of the endogenous substance produced by the body are measured in the 
896  time period prior to study drug administration.  Depending on the proposed indication, 
897  subtraction of the time-averaged baseline or time-matched baseline from the post-dose 
 
 
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898  concentration for each subject may be recommended.  When the endogenous levels are 
899  influenced by diet, strict control of the dietary intake of the compound prior to and during the 
900  study may also be appropriate.  To achieve a stable baseline, subjects should be housed at the 
901  clinic for a sufficient time prior to the study and served standardized meals with similar content 
902  of the compound to that of the meals served on the PK sampling day. 
903   
904  In either case, baseline concentrations should be determined for each dosing period, and baseline 
905  corrections should be period-specific.  If a negative plasma concentration value results after 
906  baseline correction, this should be set to 0 prior to calculating the baseline-corrected AUC.  
907  Pharmacokinetics and statistical analysis should be performed on both uncorrected and corrected 
908  data as appropriate.  Because of the complexities associated with endogenous compounds, we 
909  recommend that sponsors and/or applicants contact the appropriate review division for additional 
910  guidance. 
911   
912  H.   Drug Products With High Intrasubject Variability  
913   
914  In addition to the traditional approach and the use of average BE using replicate designs, the use 
915  of a reference-scaled BE approach using a replicate design can be considered.  This approach 
916  should be reserved for drugs that demonstrate a high intrasubject variability (30%). The 
917  reference-scaled average BE approach adjusts the BE limits of highly variable drugs by scaling 
918  to the within-subject variability of the reference product in the study and imposes a limit of 0.8 to 
919  1.25 on the geometric mean ratio.29  The appropriate review division should be consulted when 
920  planning the use of the reference-scaled BE approach. 
921   
922 
                                                 
29  For general principles of the reference-scaled  approach, refer to Davit B, Conner D. Reference-Scaled Average 
Bioequivalence Approach.  In: Kanfer I, Shargel L, Eds. Generic Drug Product Development  International 
Regulatory Requirements For Bioequivalence.   Informa Healthcare, 2010:271-272. 
 
 
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923  APPENDIX A:  GENERAL STUDY DESIGN AND DATA HANDLING 

924 
  
925  The following general approaches are recommended, recognizing that the elements can be 

926  adjusted for certain drug substances and drug products. 

927 
  
928  Study conduct 

929 
  
930     The BA or BE study should be conducted under fasting conditions (after an overnight fast of 

931  at least 10 hours). If the BA or BE study needs to be conducted with food, a separate FDA 

932  guidance Food-Effect Bioavailability and Fed Bioequivalence Studies is available to assist
  
933  sponsors. 

934 
  
935     The test and reference products should be administered with about 8 ounces (240 milliliters) 

936  of water to an appropriate number of subjects. 

937  

938     Generally, the highest marketed strength should be administered as a single unit.  If 

939  warranted, to achieve sufficient bioanalytical sensitivity multiple units of the highest strength 

940  can be administered, provided the total single dose remains within the labeled dose range and 

941  the total dose is safe for administration to the study subjects. 

942  

943     An adequate washout period (e.g., 5 half-lives of the moieties to be measured) should 

944  separate each treatment. 

945  

946     The lot numbers of both test and reference listed products and the expiration date for the 

947  reference product should be stated. We recommend that the assayed drug content of the test 

948  product batch not differ from the reference product by more than +/- 5 percent.  The sponsor 

949  should include a statement of the composition of the test product and, if possible, a side-by
950  side comparison of the compositions of test and reference listed products.  In accordance 

951  with 21 CFR 320.38, and 21 CFR 320.63, samples of the test and reference listed product 

952  must be retained for at least 5 years. For additional information, please refer to the FDA 

953  guidance for industry on Handling and Retention of Bioavailability and Bioequivalence 

954  Testing Samples. 
 
955 
  
956     Before and during each study phase, we recommend that subjects (1) be allowed  water as 

957  desired except for 1 hour before and after drug administration, (2) be provided standard 

958  meals no less than 4 hours after drug administration, and (3) abstain from alcohol for 24 

959  hours before each study period and until after the last sample from each period is collected. 
 
960 
  
961  Sample collection and sampling times 

962 
  
963     We recommend that under normal circumstances, blood, rather than urine or tissue, be used.  

964  In most cases, drug or metabolites are measured in serum or plasma.  However, in certain 

965  cases, such as when an assay of sufficient sensitivity cannot be developed for plasma, whole 

966  blood may be more appropriate for analysis.  We recommend that blood samples be drawn at 

 
 
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967  appropriate times to describe the absorption, distribution, and elimination phases of the drug.  

968  For most drugs we recommend that 12 to 18 samples, including a pre-dose sample, be 

969  collected per subject per dose. This sampling should continue for at least three or more 

970  terminal elimination half-lives of the drug to capture 90 percent of the relevant AUC.  For 

971  multiple-dose studies, sampling should occur across the dose interval and include the 

972  beginning and the end of the interval. The exact timing for sample collection depends on the 

973  nature of the drug and the rate of input from the administered dosage form.  The sample 

974  collection should be spaced in such a way that the maximum concentration (C ) of the drug 

max
975  in the blood and terminal elimination rate constant ( ) can be estimated accurately. 
 
z
976 
  
977  Three or more samples should be obtained during the terminal log-linear phase to obtain an 

978  accurate estimate of   from linear regression.  We recommend recording the actual clock 

z
979  time when samples are drawn, as well as the elapsed time related to drug administration. 

980 
  
981  Subjects with pre-dose plasma concentrations 

982 
  
983     If the pre-dose concentration is  5 percent of C  value in that subject, the subjects data 

max
984  without any adjustments can be included in all PK measurements and calculations.  We 

985  recommend that if the pre-dose value is > 5 percent of C , the subject should be dropped 

max
986  from all PK evaluations.  The subject data should be reported and the subject should be 

987  included in safety evaluations. 

988 
  
989  Data deletion because of vomiting 

990 
  
991     We recommend that data from subjects who experience emesis during the course of a study 

992  for immediate-release products be deleted from  statistical analysis if vomiting occurs at or 

993  before 2 times median T .  For modified-release products, subjects who experience emesis 

max
994  at any time during the labeled dosing interval should not be included in PK analysis. 

995 
  
996  Data submission and analysis 
 
997 
  
998  The following PK information is recommended for submission: 

999 
  
1000     Plasma concentrations and time points. 

1001     Subject, period, sequence, treatment.  

1002     Intersubject, intrasubject, and/or total variability, if available. 
 
1003     For single-dose studies: AUC , AUC , C , T ,   and t . 

0-t 0-inf max max z, 1/2
1004     For steady-state studies:  AUC  C  T  C  (lowest concentration in a dosing 

0-tau, maxss, max, minss
1005  interval), C  (concentration at the end of the dosing interval), C  (average 

trough avss
1006  concentration during a dosing interval), degree of fluctuation [(C -C )/C ], swing 

max min avss
1007  [(C -C )/C ]. C  should be measured for several dosing intervals to assess 

maxss minss minss trough
1008  whether steady-state was achieved. 

 
 
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1009     In addition to the above information, clearance and volume of distribution should be 
1010  reported for BA studies. 
1011   
1012  In addition, we recommend that the following statistical information be provided for AUC  
0-t, 
1013  AUC  and C : 
0-, max
1014   
1015     Geometric means 
1016     Arithmetic means 
1017     Geometric mean ratios 
1018     90 percent Confidence intervals (CI) 
1019   
1020  We also recommend that logarithmic transformation be provided for measures used for BE 
1021  demonstration.  An FDA guidance for industry, Statistical Approaches to Establishing 
1022  Bioequivalence, is available. 
1023   
1024  Rounding off of confidence interval values 
1025   
1026  We recommend that applicants not round off CI values; therefore, to pass a CI limit of 80 to 125 
1027  percent, the value should be at least 80.00 percent and not more than 125.00 percent. 
1028   
1029   
 
 
26