Regulatory Guidance on

BioAvailability and
BioEquivalence Studies
FDA and EMA Guidances

Outline
Overview of BA and BE studies
Methodology
PK studies
In-vitro studies

BE Comparisons
Additional considerations
General PK study design
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Bioavailability Studies

To determine the rate and process by which the active

ingredient in drug is absorbed and moves to site of action
Develop a systemic exposure profile by measuring
drug/metabolite concentration over time
Usually part of INDs (investigational new drug) and NDAs
Assess performance of formulations used with regards to safety
and efficacy
Serves as a benchmark for subsequent BE studies
Provide info on distribution, elimination, dose proportionality,
linearity, influence of enzymes and transporters

Bioequivalence Studies

To establish the absence of a significant difference in the rate

and process to which the active ingredient in drug equivalents
becomes available at the site of drug action
Compare systemic exposure profile of a test drug product vs.
reference drug product (RLD)
Active drug ingredient or active moiety in test product must
exhibit same rate and extent of absorption as RLD
Serves to document product quality
More formal comparison with pre-specified criteria and limits
If test product levels are too high compared to reference, concern
is around safety of test product
If test product levels are too low compared to reference, concern
is with therapeutic efficacy of test product
If variability of test product rises, the concern is both safety and
efficacy
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Bioequivalence Studies
Proper mapping of dose-response or concentration-response
curves are important particularly when the test drug product
has levels that are higher or lower than the RLD
Studies with doses above the recommended therapeutic doses
aim to demonstrate that the increase in plasma levels are not
accompanied by additional risk
Studies with doses below the recommended therapeutic doses
aim to demonstrate that the reduced levels of the test product
compared to the RLD are associated with adequate efficacy

Usually included in NDA, ANDA (generics), or post-approval

submissions
BE studies are a critical component of ANDA submissions for
demonstrating the BE between a pharmaceutically equivalent
generic drug product and the corresponding RLD

Methodology
Methods to establish BA/BE (in order
of preference):

PK
PD
Clinical
In vitro

PK Studies
Most commonly rely on PK measures such as AUC and Cmax
Typically cross-over design
Non-replicate recommended for immediate-release and modifiedrelease dosage forms
Replicate = at least one treatment is repeated in the same subject
Non-replicate = no treatment is repeated in the same subject

Advantages of replicate studies

Allow comparisons of within subject variances for test and
reference
Provide more info about intrinsic factors underlying formulation
performance
Reduce the number of subjects in BE study

Under fasting conditions because fasting is considered most

sensitive to detect formulation differences

PK Studies (continued)

Study population

Similar proportions of each sex in study

Include as many subjects >60 years in age for elderly studies
Statistical analysis of subgroups not recommended
Restrictions on enrollment need be based on safety
considerations
Healthy subjects, but may be appropriate to enroll patients
depending on drug/dose
(EMA) Healthy volunteers is adequate and results can be
extrapolated to populations for which product is approved in
(elderly, children, etc)
Preferably with BMI of 18.5-30kg/m2
Treatment groups should be comparable in all known variables
(e.g. age, weight, sex, race, smoking status, etc). In other
words, as homogenous as possible across treatment groups

PK Studies (continued)

Single Dose studies are recommended

They are more sensitive in assessing release of drug substance
from drug product into systemic circulation

AUC and Cmax are most common measures of BA/BE

Early exposure: partial AUC at Tmax
Peak exposure: Cmax
Total exposure: AUClast, AUCinf, AUCtau (MD)

Assume that variability arise mainly from formulation

factors
Pilot studies are commonly conducted before a full BE study
to validate analytical methods, assess variability, determine
timepoints of measurement, etc.

In-Vitro Studies

Dissolution test is used as a tool to identify formulation

factors that are influencing and may have a crucial effect
on the BA of the drug
Used to assess batch-to-batch quality
To establish an in vitro-in vivo correlation
Can be used to waive a BE study
Such as highly soluble, rapidly dissolving orally administered
products

Specific info is required to be included in report including

pH solubility profile
Dissolution profiles at different agitation speeds
Dissolution profiles in at least 3 different dissolution media
(varying pH)
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BE Comparisons
Recommended approach
A criterion to allow comparison
A confidence interval for the criterion
A BE limit

Log-transformation recommended
Use of an average BE criterion to compare BA measures
In vivo study is recommended for all solid oral forms
Waiver for in vivo can be granted given
The drug is in the same dosage form, but different strength
The strength is proportionally similar to the strength of product
which an in vivo study has already been conducted (typically the
highest strength)
The new strength meets an appropriate in vitro dissolution test
Oral solutions, elixirs, syrups, etc.
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BE Comparisons
Proportionally similar is defined as
All active/inactive ingredients are in exactly the same proportion
between different strengths
If not exactly proportional btw strengths, the ratios of inactive
ingredients to total weight of dosage form are within limits
defined by SUPAC-IR and SUPAC-MR guidances
For high potency drugs where the amount of active drug
substance is relatively low
the total weight of the dosage form remains nearly the same for all
strengths (within +/-10% of total weight of strength on which
biostudy was performed)
the same inactive ingredients, and/or the changes in inactive
ingredients are within limits defined by SUPAC-IR and SUPAC-MR

SUPAC-IR = Immediate Release Solid Oral Dosage Forms: Scale-Up and

Post-Approval Changes (MR= Modified Release)
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BE Comparisons
Suspensions
Both in vivo and in vitro studies are recommended

Immediate-release products: Capsules and tablets

A single-dose, fasting study is recommended
In vivo BE studies and in vitro dissolution profiles on all strengths
Specific guidance on preapproval and postapproval waivers of in
vivo BE studies

Modified-release products (delayed or extended release)

Delayed-release: coatings are intended to delay release of
medication
In vitro dissolution tests should show they are stable under acidic conditions and
drug is released only in a neutral medium

Extended release: allow for reduction in dosing frequency

Submit as NDA for first modified-release drug product following
approved immediate-release product
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BE Comparisons

(EMA) Strengths to evaluate depends on linearity of PK of active

substance
PK is considered to be linear if difference in dose-adjusted mean AUCs
is no more than 25% between different strengths
If linearity is demonstrated, it may be sufficient to establish BE with
only one strength
For drugs with linear PK, selection of a lower strength than the highest
is also acceptable to perform BE study.
e.g. if highest strength cannot be administered to healthy volunteers for
safety reasons

For drugs with non-linear PK

If more than proportional increase in AUC with increasing dose, BE study
should be conducted at the highest strength
If less than proportional increase in AUC with increasing dose, BE study
should be conducted at the highest and lowest strengths

When BE assessment at more than 2 strengths is needed, it is

acceptable to conduct 2 BE studies at the highest and lowest strengths

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Additional Considerations

Food effect studies

A single-dose, two-period, two-treatment, two-sequence crossover
study is recommended

Measurement of moieties
For BA studies: Both parent drug and its major metabolites be
measured
Measurement takes into account both concentration and activity

For BE studies: Measurement of only parent drug is generally needed

because concentration-time profile of parent drug is more sensitive
to changes in formulation than a metabolite

Long half-life drugs

For BE study: A non-replicate, single-dose, crossover study can be
conducted, provided an adequate washout period is used
If crossover is problematic, a parallel design can be used
Sample collection time be adequate to ensure completion of passage
and absorption of drug (~2-3 days)

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Additional Considerations

Long half-life drug

Cmax and truncated AUC can be used to characterize peak and
total drug exposure
For drugs with low intrasubject variability
AUC @ 72hrs can be used
For drugs with high intrasubject variability
Need to be cautious with AUC truncation

First point Cmax

Insufficient early sampling times may lead to inaccurate
measurement of true Cmax if the first point is the highest point
in a concentration-time curve
Pilot study may help avoid problem

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General PK Study Design

Under fasting conditions, unless food-effect study
(EMA) For products with specific formulation characteristics, BE
study should be performed under both fed and fasted states
Either 2 separate two-way crossover, or a four-way crossover study

Highest marketed strength be administered as a single unit

Adequate washout period (>5 half lives)
Drug content of test product cannot differ from reference
product by more than 5%
(EMA) At least 12 or more subjects in BE study
Collection of blood samples is recommended serum or plasma
in most cases
(EMA) Urine is acceptable where it is not possible to reliably
measure plasma concentration-time profile
should be collected over at least 3 times the half-life

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General PK Study Design

Sampling frequency and schedule
12-18 samples, including pre-dose, per subject per dose is
recommended
Sampling should continue for at least 3 or more half lives of drug
At least 3-4 samples be obtained during terminal log-linear phase
to obtain accurate estimate of elimination rate constant (z) from
linear regression
Actual clock time when samples are drawn as well as elapsed time
related to drug administration be recorded
(EMA) Sampling schedule should be long enough to ensure that
AUC0-t covers at least 80% of AUCinf

(EMA) The bioanalytical method for analyzing PK samples

should have a LLOQ of 1/20 of Cmax or lower
Analysis of samples should be conducted without info on
treatment
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General PK Study Design

PK info recommended for submission

Plasma concentrations and timepoints

Subject, period, sequence, treatment
AUC0-t, AUCinf, Cmax, Tmax, z, t1/2, (EMA) Residual Area

Intersubject, intrasubject, and/or total variability

(steady-state studies) Cmin, Cav, degree of fluctuation, swing
Partial AUC, if appropriate
(EMA) For urine, Ae(0-t) and Rmax, if applicable, should be
collected
Non-compartmental methods should be used for determination
of PK parameters

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General PK Study Design

Statistical info to be provided for AUC0-t, AUCinf, Cmax

Geometric mean
Arithmetic mean
Ratio of means
Confidence intervals

Log transformation be applied

Rounding off of confidence interval values are not
recommended.
To pass 90% CI limit of 80 to 125, value should be at least
80.00 and not more than 125.00

(EMA) For products with narrow therapeutic index,

acceptance interval for AUC should be tightened to 90111%

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General PK Study Design

(EMA) For highly variable drug products (HVDP), the

acceptance criteria for Cmax can be widened to a
maximum of 69.84-143.19%
For CI to be widened, must demonstrate that the within subject
variability for Cmax of the reference compound is >30%
Does not apply to AUC where acceptance range should remain at 80125%

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General PK Study Design

(EMA) Two-Stage Design

Two-Stage approach is acceptable for demonstrating BE
An initial group of subjects is treated and data analyzed
If BE is not demonstrated, an additional group is recruited and
the results from both groups are combined in a final analysis

Overall type I error must be preserved

Stopping criteria needs to be clearly defined prior to study
Plan to use two-stage approach must be pre-specified in
protocol along with adjusted alphas to be used for each
analysis
A term for stage should be included in the ANOVA model

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General PK Study Design

GLM or linear mixed effects model is recommended
Model should include factors such as sequence, subjects nested
in sequences, period, and treatment

(EMA) Fixed effect ANOVA model only should be used

Formulation comparisons are primarily based on paired t test
Only patients who have data from both Test and Reference
period will be included

If there is no missing data the two models are equivalent

If there is missing data there can be differences
Less data points will be included in the method recommended
by EMA

Internally, mixed effects model is recommended as a default

approach. it was found that the mixed effects model will
have more power

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References

Guidance for Industry Bioavailability and Bioequivalence Studies for Orally

Administered Drug Products General Considerations. US Department of Health and
Human Services, Food and Drug Administration Center for Drug Evaluation and
Research (CDER), March 2003.
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Gu
idances/ucm070124.pdf
Guideline on the Investigation of bioequivalence. European Medicines Agency.
Committee for MEDICINAL Products for Human Use. January 2010. http
://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2010/01/WC
500070039.pdf
Statistical Approaches to Establishing Bioequivalence. US Department of Health and
Human Services, Food and Drug Administration Center for Drug Evaluation and
Research (CDER), January 2001.
http://www.fda.gov/downloads/Drugs/Guidances/ucm070244.pdf

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