BIOEQUIVALANCE STUDIES :
PROTOCOL AND REGULATORY
Executive, Regulatory Affairs
SQUARE Pharmaceuticals Ltd.
Regulatory Definition (21 CFR 320.1(a)):
“Bioavailability means the rate and extent to which the active
ingredient or active moiety is absorbed from a drug product and
becomes available at the site of action.”
Regulatory Definition (21 CFR 320.1(e)):
“Bioequivalence means the absence of a significant difference in the
rate and extent to which the active ingredient or active moiety in
pharmaceutical equivalents or pharmaceutical alternatives becomes
available at the site of drug action when administered at the same
molar dose under similar conditions in an appropriately designed
Pharmaceutical equivalents are 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.
Pharmaceutical alternatives are drug products that contain the identical
therapeutic moiety, or its precursor, but not necessarily in the same amount
or dosage form or as the same salt or ester.
Therapeutic equivalents are drug products that contain the same active
substance or therapeutic moiety and, clinically show the same efficacy and
Identified by the Regulatory Authorities as
“Designated Reference Product”
Usually the Global Innovator’s Product
Protected by a patent
Marketed under manufacturers brand name
Clinical efficacy & safety profile is well documented in extensive
All generics must be Bioequivalent to it
For Oral solid forms for systemic action:
a) The test product should usually originate from a batch of at least 1/10 of
production scale or 100,000 units, whichever is greater, unless otherwise justified.
b) The production of batches used should provide a high level of assurance that the
product and process will be feasible on an industrial scale. In case of a production
batch smaller than 100,000 units, a full production batch will be required.
c) The characteristics and specification of critical quality attributes of the drug
product, such as dissolution, should be established from the test batch, i.e. the
clinical batch for which Bioequivalence has been demonstrated.
d) Samples of the product from additional pilot and/or full scale production batches,
submitted to support the application, should be compared with those of the BE
study test batch, and show similar in vitro dissolution profiles when employing
suitable dissolution test condition.
e) Comparative dissolution profile testing should be undertaken on the first three
f) If full scale production batches are not available at the time of submission, the
applicant should not market a batch until comparative dissolution profile testing
has been completed.
For other immediate release pharmaceutical forms for systemic action, justification of
the representative nature of the test batch should be similarly established.
REQUIREMENT OF BA & BE STUDIES
To establish equivalence between:
• Early & late clinical trial formulations
• Formulations used in clinical trial & stability studies, if different
• Clinical trial formulations & to-be-marketed drug product
• Any other comparisons, if appropriate
ANDA for a generic drug product
Change in components, composition, &/or manufacturing process
Change in dosage form (capsules to tablet)
NDA VS. ANDA REVIEW PROCESS
NDA Requirements ANDA Requirements
1. Chemistry 1. Chemistry
2. Manufacturing 2. Manufacturing
3. Controls 3. Controls
4. Labeling 4. Labeling
5. Testing 5. Testing
6. Animal Studies
7. Clinical Studies 6. Bioequivalence
DESIGN AND CONDUCT OF STUDIES
I. Pharmacokinetic Studies
II. Pharmacodynamic Studies
III. Comparative Clinical Studies
IV. Dissolution Studies
a) Study b) Study c) Selection d) Study e) Statistical
Design Population Criteria Condition evaluation
The basic design of an in-vivo bioavailability study
is determined by the following:
• What is the scientific question(s) to be
• The nature of the reference material and the
dosage form to be tested.
• The availability of analytical methods.
• Benefit-risk ratio considerations in regard to
testing in humans.
The number of subjects to be included in the study based
on an appropriate Sample size which is estimated by:
• Pilot experiment
• Previous studies
• Published data
The number of subjects recruited should be sufficient to
allow for possible withdrawals or removals (dropouts) from
Significance level desired, usually 0.05
Power of the study, normally 80% or more
Minimum 12 subjects, unless ethical justification
if the drug product is intended for use in both sexes, the
sponsor attempt to include similar proportions of males and
females in the study.
SELECTION OF SUBJECTS
Healthy adult volunteers
Age: 18years or older
Age/Sex representation corresponding to therapeutic & safety profile
Non-smokers/without a history of alcohol or drug abuse Medical history/Clinical Lab
test values must be within normal ranges
Weight within normal limits→ BMI (18.5-30 kg/m2)
Women: Women should be required to give assurance that they are neither
pregnant, nor likely to become pregnant until after the study.
Drug use intended in Elders, the sponsor attempt to include as many subjects of
60yrs of age or older as possible.
Teratogenic Drugs→ Male volunteers
Highly toxic drugs: Patients with concerned disease (stable) eg. Cancer
Selection of Blood Sampling
For at least three elimination half-lives (cover >80% of AUC)
• Absorption phase : 3-4 points
• Around Tmax : 3-4 points
• During elimination : 4 points
Intervals not longer than the half-life of the drug
If urine tested, collect it for at least 5 half-lives
The lot Numbers of both reference
listed products and the expiration date
for the reference product would be
The drug content of the test product
cannot differ from that of reference
product by more than ± 5%.
The sponsor can include a statement
of the composition of the test product
and if possible a side-by-side
comparison of the compositions of the
test and reference product.
Samples of the test and reference
listed product must be retained for 5
Fasting or fed conditions
• A single dose study should be conducted after an overnight fast (at least 10 hours),
with subsequent fast of 4 hours following dosing.
• For multiple dose fasting state studies, when an evening dose must be given, two
hours of fasting before and after the dose is considered acceptable.
• Drug recommended with food
• Modified release product
• Assessment of Cmax and Tmax difficult with fasting state study
Requires consumption of a high fat food, 30 minutes before dosing
Provide 800-1000 kcals with about 50% of calories derived from fat.
Fat- 500-600, Proteins - 150, Carbohydrate- 250 Kcal
Ethnic & cultural variation considered
Specified in protocol
In general BE Study conducted under
1. Where the SmPC recommends
intake of the reference medicinal
product on an empty stomach or
irrespective of food intake, BE study
conducted under fasting condition.
2. Where SmPC recommends intake of
RMP only in fed state, BE Study
conducted under fed state.
3. For specific formulation both study
recommended, unless product taken
only in fasting or fed state.
Where information is required in both Fasting and fed state, it is acceptable to
a. Two separate two-way cross-over OR,
b. A four way cross-over study
STEADY STATE/ MULTIPLE DOSE STUDIES
Long elimination half life→ Accumulation in the body
Toxic drugs requiring multiple dose therapy
Some Modified-release drugs
Drugs inducing own metabolism
Drugs showing non-linear pharmacokinetics
• Difficult to conduct
• Longer monitoring
• Longer exposure to drug
CHARACTERISTICS TO BE MEASURED
Evaluations of BA/BE will be based upon the measured concentrations of the active
drug substance(s) in the biological matrix.
The measurements of an active or inactive metabolite may be necessary:
a)where the concentrations of the drug(s) may be too low to accurately measure in
the biological matrix, (b) limitations of the analytical method, (c) unstable drug(s), (d)
drug(s) with a very short half-life or (e) in the case of prodrugs.
Racemates should be measured using an achiral assay method.
The plasma-time concentration curve is mostly used to assess the rate and extent of
absorption of the study drug. This include Cmax, Tmax, AUC0-t and AUC0-∞.
Pharmacokinetic Parameters measured are:
• Plasma conc. and time points
• Subject, period, sequence, treatment
• Cmax, Tmax, AUC0-t ,AUC0-∞
• Partial AUC only for early exposure of drug content.
For steady state studies:
• Degree of fluctuation
Measurement of Individual enantiomers in BE
Studies is recommended if
1. Exhibit different Pharmacodynamic
2. Exhibit different Pharmacokinetics
3. Primary efficacy and safety activity resides
with minor enantiomers
4. Nonlinear absorption is present
Primary concern of bioequivalence is to limit Consumer’s & Manufacturer’s risk
• Cmax & AUC analysed using ANOVA
• Tmax analysed by non-parametric methods
Use natural log transformation of Cmax and AUC
Calculate 90% confidence interval for this GMR for Cmax
Similarly calculate GMR for AUC
Criteria for bioequivalence
• To establish Bioequivalence, the calculated 90% confidence interval for AUC and
Cmax should fall within the bioequivalence range, usually 80-125%.
• Tighter limits for permissible differences in bioavailability may be required for
drugs that have:
I A narrow therapeutic index.
II A serious, dose-related toxicity.
III A steep dose/effect curve, or
IV A non-linear pharmacokinetics within the therapeutic dose range.
I. Two-Period Crossover Design
2 formulations, even number of subjects,
randomly divided into 2 equal groups
First period , each member of one group receive a single dose of the test
formulation; each member of the other group receive the standard formulation
Subjects Period 1 Period 2
1-8 T S
9-16 S T
II. Latin Square Design
More than two formulations
A group of volunteers will receive formulations in the sequence
III. Balance Incomplete Block Design (BIBD)
More than 3 formulations, Latin square design will not be
Because each volunteer may require drawing of too many blood
If each volunteer expected to receive at least two formulation,
then such a study can be carried out using BIBD
IV. Parallel-Group Design
Even number of subjects in two groups
Each receive a different formulation
No washout necessary
For drugs with long half life
Treatment A Treatment B
V. Replicate Crossover-study design
For highly variable drugs
Allows comparisons of within-subject variances
Reduce the number of subjects needed
Four-period, two-sequence, two-formulation design
Three-sequence, three-period, single-dose, partially replicated
Period 1 2 3
Group 1 T R T
Group 2 R T R
VI. Pilot Study
If the sponsor chooses, in a small number of subjects
To assess variability, optimize sample collection time intervals
& provide other information, Validate analytical methodology.
• Immediate-release products: careful timing of initial samples→ avoid a
subsequent finding that the first sample collection, occured after the plasma
Can be appropriate, provided its design & execution are suitable &
sufficient number of subjects have completed the study
In vitro studies ,i.e. dissolution studies can be used in place of
in vivo bioequivalence under certain conditions ,called
1. The drug product differs only in strength of active
substance, provided the following condition hold ;
a) Pharmacokinetics are linear.
b) The qualitative composition is same.
c) The ratio between active substance and excipient is same.
d) Both product are produced by same manufacturer at same
site with same manufacturing process.
2. The drug has been slightly reformulated or manufacturing
method has been slightly modifies by same manufacturer
in ways that can be argues irrelevant for BA.
3. The product meets following requirement :
a) The product is in form of solution or solublised form ( elixir,
b) The product contain active ingredient in same conc. as
c) The product contain no excipient known to significantly affect
absorption of active ingredient.
d) The product is administered by inhalation as gas or vapor.
e) The product is for oral administration but not intended for
absorption ( antacid or radio opaque medium ).
f) The product is intended for topical administration
(ointment, creams, gels etc,) for local effect.
Measurement of effect on a Patho-physiological process as a function of
time, after administration of 2 different products
1. Quantitative analysis in plasma or urine not possible with sufficient
accuracy & sensitivity
2. Drug concentrations are not surrogate endpoints e.g. Topical
formulations without systemic absorption
3. In situations of ‘Superiority Claims’
In case only Pharmacodynamic data is collected→ other methods tried &
why they were unsuitable should be mentioned.
COMPARATIVE CLINICAL STUDIES
• Both pharmacokinetic & pharmacodynamic parameters not
properly measurable or not feasible
• Mention which methods were tried & found unsuitable
Statistical principles to be considered:
• No. of patients→ Variability of assessed parameters &
acceptance range Much higher than BE studies
FOLLOWING CRITICAL POINTS NEED TO BE
DEFINED IN ADVANCE, ON CASE TO CASE BASIS:
Clinical end points (Target parameters)→ intensity & onset of
Size of equivalence range→ case-to-case basis (dependings on
natural course of disease, efficacy of available treatments, target
Statistical confidence interval
Placebo included when appropriate
Safety end-points in some cases
1. Suitable to confirm unchanged product quality with minor changes in
formulation / manufacturing after approval→ SUPAC ( Scale-Up & Post-
2. Different strengths of drug manufactured by same manufacturer
• Qualitative composition is same
• Ratio of active ingredients & excipients is same
• Method of manufacture is same
• BE study has been performed on 1 strength
• Linear pharmacokinetics
3. Assess batch-to-batch quality
More than 1 batch of each formulation tested
Design should include:
Individually testing of at least 12 dosage units of each batch
→Mean & Individual results with Sd or SE
Measurement of percentage of content released at suitably
spaced time points(eg. At 10, 20 & 30 mins or appropriate
for complete dissolution)
Dissolution profile in atleast 3 aqueous media with pH
range of 1.0-6.8 Or 1.0-8.0 wherever necessary
Conduct tests on each batch with same apparatus & on
same or consecutive days
Dissolution testing should be carried out in:
USP Apparatus I at 100 rpm or Apparatus II at 50 rpm
using 900 ml of the following dissolution media:
• 0.1N HCl or Simulated Gastric Fluid USP without enzymes
• a pH 4.5 buffer
• a pH 6.8 buffer or Simulated Intestinal Fluid USP without
For capsules and tablets with gelatin coating
• Simulated Gastric and Intestinal Fluids USP (with enzymes) can
• With respect to the conduct of bioequivalence/bioavailability studies
following important documents must be maintained:
A) Clinical Data :
• All relevant documents as required to be maintained for compliance
with GCP Guidelines .
B) Details of the analytical method validation including the following:
a. System suitability test
b. Linearity range
c. Lowest limit of quantization
d. QC sample analysis
e. Stability sample analysis
f. Recovery experiment result
C) Analytical data of volunteer plasma samples which should
include the following:
a. Validation data of analytical methods used,
b. Chromatograms of all volunteers,
c. Inter-day and intra-day variation of assay results,
d. Details including chromatograms of any repeat analysis
e. Calibration status of the instruments .
D) Raw data
E) All comments of the chief investigator regarding the data of
the study submitted for review.
F) A copy of the final report