This document provides definitions and guidelines regarding bioequivalence and bioavailability testing requirements for generic drug approvals. It defines key terms like bioavailability, bioequivalence, and pharmaceutical equivalents. It outlines the types of evidence and study designs required to demonstrate bioequivalence to the reference listed drug, including in vivo blood level studies, in vitro dissolution testing, and possible clinical endpoint studies. It also discusses statistical analysis methods and factors that can impact bioequivalence studies.

1. Center for Professional Advancement
Generic Drug Approvals Course
Bioequivalence & Bioavailability
Michael A. Swit, Esq.
Vice President

2. 1

3. 2
Definitions
 Statutory
 “Bioavailability” Means The Rate And Extent To Which The
Active Ingredient Or Therapeutic Ingredient Is Absorbed And
Becomes Available To The Site Of Drug Action. §
505(j)(7)(A)
 A Generic Drug Will Be Considered To Be
“Bioequivalent” To A Listed Drug If:
 The Rate And Extent Of Absorption Of The Generic Does Not
Show A Significant Difference From The Rate And Extent Of
Absorption Of The Listed Drug. § 505(j)(7)(B)(i).
 The Extent Of Absorption Of Drug Does Not Show Significant
Difference From The Extent Of Absorption Of The Listed Drug
… If The Difference … In Rate … Is Intentional. §
505(j)(7)(B)(ii).

4. 3
Definitions …
 Regulatory
 “Bioavailability” means the rate and extent to which the
active drug ingredient or therapeutic moiety is absorbed from
a drug product and becomes available at the site of drug
action. 21 C.F.R. § 320.1(a).
 “Bioequivalence” means pharmaceutical equivalents whose
rate and extent of absorption do not show a significant
difference when administered at the same molar dose of the
therapeutic moiety under similar experimental conditions. 21
C.F.R. § 320.1(e).

5. 4
Definitions …
 Regulatory …
 “Pharmaceutical Equivalents” means drug products that
contain identical amounts of the identical active drug
ingredient, i.e., the same salt or ester of the same therapeutic
moiety, in identical dosage forms, but not necessarily
containing the same inactive ingredients, and that meet the
identical compendial or other applicable standard of identity,
strength, qaulity, and purity, including potency and, where
applicable, content uniformity, disintegration times and/or
dissolution rates. 21 C.F.R. § 320.1(c).

6. 5
Bioequivalence
 Procedures For Determining Bioavailability Or Bioequivalence.
21 C.F.R. §§ 320.21-.63.
 Considerations For Bioequivalence.
 Statutory/Regulatory.
 Proof that drug is not only pharmaceutically equivalent (same active
ingredient in same strength and dosage form), but also bioequivalent.
 Systemic bioequivalence testing is based on assumption that therapeutic
effect of A drug is A function of the concentration of the active
ingredient in the systemic circulation of A person and is thus related to its
bioavailability.
 Clinical bioequivalence is based on clinical data from the reference listed
drug (“RLD”) and generic that demonstrate the generic has the same safety
and effectiveness as the RLD. Often, a placebo is required to assure that
both drugs are superior to the placebo.

7. 6
Requirements For Submission Of Bioequivalence
Study Data.
21 C.F.R. § 320.21.
 Evidence Demonstrating Bioequivalence To Listed
Drug.
 Or Information Establishing That A Waiver Is
Appropriate

8. 7
Criteria For Waiver Of In Vivo Bioequivalence
21 C.F.R. § 320.22
 Drug Products Whose Bioequivalence Is Self Evident.
 Parenterals, ophthalmics, and otics with the same concentration of active
and inactive ingredients as the listed drug. (Qualitative and quantitative
“Q&Q”)
 Oral or topical solutions with the same concentration of active ingredient
and dosage form as the listed drug, and any difference in inactive
ingredients will not significantly affect the drug's absorption.
 DESI Drugs Without Known Or Potential Bioequivalence
Problems.
 Drug Products Whose Bioequivalence Can Be Established
Through In Vitro Evidence.
 For Good Cause, If Waiver Is Compatible With Protection
Of Public Health.

9. 8
Types Of Evidence To Establish
Bioequivalence
21 C.F.R. § 320.24.
 In Vivo Testing Of Blood Or Related Biological
Fluid.
 In Vivo Testing Of Urinary Excretion.
 In Vivo Testing To Measure Pharmacological Effect.
 Well-Controlled Clinical Trials.
 In Vitro Testing That Ensures In Vivo Bioavailability

10. 9
Components Of Bioequivalence Study
 Protocol
 Clinical Report
 Analytical Report
 Pharmacokinetic and Statistical Report
 Statistical Tables, Listings and Graphs

11. 10
Guidelines For Single Dose In Vivo
Bioavailability Study (Blood Level).
21 C.F.R. § 320.26.
 Investigational New Drug (IND) application not required.
 Protocol Example
 At least 24 healthy human volunteers (male and female).
 Age 18 - 45
 Weight –10% - +15% for frame size
 No concomitant medications allowed
 Single dose comparison.
 Fasting state of volunteers.
 10 hours
 No water/fluids ± 1 hour dosing
 Two-way crossover.
 In first leg, half receive the test (generic) product and half the listed drug.
 Adequate wash-out period (at least three times the half life of elimination
of active ingredient or metabolite)

12. 11
Key Measures During BE Study
 Samples must be collected with sufficient frequency to permit
estimate of peak concentration (CMax), area under the
concentration time curve (AUC), and time to peak (TMax).
 CMax = the observed peak drug concentration obtained directly for the
experimental data without interpolation.
 TMax = the observed time to reach peak drug concentration obtained
directly from the experimental data without interpolation.
 AUC(0-t) = area under the concentration versus time curve from time = 0
to time of last quantifiable concentration, calculated by the trapezodial
rule.
 CMax is a surrogate to indicate rate of absorption.
 AUC defines extent of absorption

13. 12
Other Key Aspects of BE Studies
 Treatment of systemic blood sample: centrifuge,
measure volume, ph, color, temperature control
 In Vitro Dissolution testing using water and acid
solutions (simulated gastric fluid) to determine that
potency of generic is within 5% of RLD.
 Validation of assay method.
 Pharmacokinetic parameters. (See Exhibits A and B.)

14. 13
Exhibit A – Pharmacokinetics from Generic
Drug
4 8 12
Conc.(ug/ml)
Time (hr)
Subject 1
Tmax = 6 hours
Cmax = 47 ug/m/
0
0
10
20
30
40
50
0 4 8 12

15. 14
Exhibit B – Pharmacokinetic Data from
Generic and RLD
Conc.(ug/ml)
Time (hr)
4 8 12
Generic
RLD
•
•
•
•
••
•
•
••
•
•
2 6 100
Conc.(ug/ml)
0
10
20
30
40
50
60
0
0 2 4 6 8 10 12

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Multiple Dose In Vivo Biostudy Guidelines
For Multiple Dose In Vivo Bioavailability
Study (Blood Level)
21 C.F.R. § 320.27.
 Purpose is to determine steady-state levels of the
active drug ingredient or therapeutic moiety in the
body.
 Sufficient blood or urine samples necessary to establish
maximum and minimum blood concentrations on 2 or
more consecutive days.

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Clinical Bioequivalence Study
 Submission of protocol for review or reliance on
guidance
 Dosage Forms -- At present time, clinical trials in
patients is about only methodology available to establish
bioequivalence where dosage form makes systemic
blood level studies not possible or unreliable.
 Oral drugs that are not systemically absorbed. (e.g., Sucralfate)
 Most topically administered drugs
 Intrauterine
 Surgical antibacterial scrubs

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Clinical Bioequivalence Study
 Must show RLD and generic are superior to placebo
 Must show RLD and generic are equivalent as to clinical effect.
 Example – Draft 1990 Guidance For Performance Of A Bioequivalence
Study For Topical Antifungal Products requires:
 Placebo – normally generic without active ingredient
 RLD – usually from single lot of RLD manufacturer
 Generic drug
 Clinical study that is probably identical to or very similar to study used by
RLD to obtain approval
 IND required
 Measurements
 Mycological
 Clinical
 Therapeutic (combination of mycological and
clinical)

19. 18
Statistical Evaluation Required For Any
Bioequivalence Tests
 Average Bioequivalence Method
 Systemic Blood Level Studies
 90% Confidence Interval Using The Two One-Sided T-Test. CMax
And AUC = 80% - 125%.
 July 1992 Guidance: Statistical Procedures For Bioequivalence Studies
Using A Standard Two-Treatment Crossover Design
 Clinical Study
 Statistical model will be determined by FDA usually in guidance
 Statistical analysis to demonstrate RLD and generic are
bioequivalent

20. 19
Collateral Issues for BE Studies
 Guidance On Food-Effect Bioavailability And Fed Bioequivalence Studies
(Dec. 2002) (see www.fda.gov/cder/guidance/5194fnl.pdf)
 Retention Of Bioequivalence Samples. 21 C.F.R. § 320.63.
 OGD does not distinguish between systemic blood level studies and
clinical studies so retention samples must be held by clinical investigator or
third party and not sponsor.
 See 21 C.F.R. § 320.38 and § 320.63,
www.fda.gov/cder/ogd/retention_samples.htm.
 See also August 2002 Draft Guidance for Industry, Handling and
Retention of BA and BE Testing Samples
www.fda.gov/cder/guidance/4843dft.pdf.

21. 20
BE Guidances
 FDA has available approximately 70 guidances for
bioequivalence tests, including:
 Design of study.
 Number of subjects to use.
 Identification of reference product.
 Duration of study.
 Collection times.
 Suggested methods for assay.
 Dissolution testing methodology and specifications

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Factors That Can Impact BE Study
 Physicochemical Properties of Drug.
 Physiological Factors.
 Biopharmaceutical Factors.
 Formulation Factors.
 Pharmaceutical Factors.
 Analytical Control.
 Statistical Analysis and Acceptance Criteria.

23. 22
Settled Bioequivalence Study Parameters Are
Binding On FDA
 Provision added by Food And Drug Administration Modernization Act Of
1997 (FDAMA)
 Applicant to provide written request to FDA for determination of
acceptable biostudy
 Purpose is to reach agreement on the design and size of
bioavailability and bioequivalence studies needed for approval
 Agreements regarding study parameters reached between FDA and
the applicant are binding
 Cannot be directly or indirectly changed by field or compliance personnel
 Cannot be changed after testing begins, with two exceptions:
 When applicant agrees to changes in writing
 When director of reviewing division determines that A substantial scientific
issue essential to determining the safety or effectiveness of the drug has been
identified

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Challenging BE Issues
 Racemic Drugs, Metabolites
 Gender and Age (Including Pediatrics).
 Long Shelf-Life Drugs.
 Locally Acting Drug Products.
 Estrogenic Drugs
 Narrow Therapeutic Index Drugs