Bioequivalence studies

This document explained in detail about Bioequivalence studies

1. Bioequivalence Studies
Mr. Srinivasa Reddy. Edururi

2. The main objective
Purpose of Bioequivalence studies
Phases of Bioequivalence studies


Objective

3. Introduction
Drug discovery & development
Generic Drugs
Purpose of Bioequivalence studies
Phases of bioequivalence study
Clinical
Bioanalytical
Pharmacokinetics & statistics
Regulatory submission (Module-5)

Contents

4. The life cycle of any drug involves four phases

discovery and research,

development,

regulatory review and approval, and

 commercialization/ marketing.

It takes approximately 10-12 years from the initiation and identification
of the drug target to bring the drug into the market
Drug discovery & development

5. Drug discovery & development
Discovery & Research
identify a suitable target
understand how the disease occurs at the
molecular, cellular, and genetic levels
understanding how the target plays a role
in the disease process
testing of the target against different
known and new compounds to identify
either one or several ‘lead compounds’
Drug Development
testing and optimization of the selected
compounds to identify the ‘lead structure
or candidate drug’ which might be the
most effective
Testing is done in cells (in vitro) and in
animals (in vivo) to study metabolism
(pharmacodynamics and
pharmacokinetics), safety, toxicity,
dosage, and efficacy.
clinical phase of development, the efficacy
and safety of a drug candidate
is investigated in patients
Regulatory approval
applications with all the necessary
information, including quality, preclinical and
clinical data collected during development of
the product, are submitted to the relevant
regulatory authorities
obtain approval to market the drug in their
jurisdictions (e.g., a New Drug Application
(NDA) in the US, and a Marketing
Authorisation Application (MAA) in the EU).
Marketing
marketing or commercialization of the drug
once it has been approved
The drug manufacturer must submit
marketing authorization applications in every
country or territory in which it wants to sell
the drug.
To continue evaluating the safety and efficacy
of the marketed drug, and for further
development, Phase 4 clinical trials are
conducted by the manufacturer

6. Clinical Trial Phases

7. Brand Vs. Generic drugs
Brand Name Drugs

Also called innovator drugs
Initially marketed as new
chemical entities
Supplied by one drug company
(innovator manufacturer)
Sold under companies trade
mark name
Cost is more
Generic Name Drugs
Copies of brand name drugs
Produced after the original
patent expires
May be supplied more than
one company
May be sold under active
ingredient (s) name (s)
Cost is less

8. Same active ingredient(s)
 Same route of administration
 Same dosage form
 Same strength
 Same conditions of use
Compared to brand product

What are the requirements for a
generic drug?

9. Brand Name Drug Generic Drug
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
8. Bioavailability

New Drug Vs Generic

10. Bioavailability
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.
Bioequivalence
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 study
Bioavailability / Bioequivalence

11. When do we do bioequivalence studies?

Clinical Service Form to Final Market Form

Change of formulations (capsules to tablet)

Generic Formulations

Change of Process or manufacturing site (some times)

Purpose Bioequivalence studies

12. Clinical Phase
Protocol preparation to sample transfer to Bioanalytical
Bioanalytical Phase
Method development to data transfer to Pharmacokinetics
Pharmacokinetics & Statistics
Pharmacokinetic and statistical evalusiton
Phases of Bioequivalence study

13. Single-dose, two-way crossover, fasted
Single-dose, two-way crossover, fed
Alternatives
Single-dose, parallel, fasted
Single-dose, replicate design
Multiple-dose, two-way crossover, fasted
Clinical endpoint study

Study Designs
Long Half-Life (wash-out)
Amiodarone, Etidronate

Highly Variable Drugs
Less Sensitive
Clozapine (Patient Trials)
Chemotherapy Trials
Topicals
Nasal Suspensions

14. Study Designs

15. Protocol Preparation
Ethics committee approval
Informed consent process
Selection of subjects
Test article Packaging
Drug administration
Sample collection/separation/Storage
Adverse Event Monitoring
Transfer of samples to Bioanalytical Laboratory

Clinical Phase

16. Protocol contents


♦„A document that describes the
objective(s), design, methodology,
statistical consideration and
organisation of a trial. It usually
gives the background and rationale
of the trial …“
Ref.: ICH GCP Guidance

17. Ethics committee approval

Who: An independent body (a review board or a committee,
institutional, regional, national, or supranational), constituted of
medical professionals and non-medical members.

The composition may be as follows :-
Chairperson
1-2 basic medical scientists (preferably one pharmacologists).
1-2 clinicians from various Institutes
One legal expert or retired judge
One social scientist / representative of non-governmental voluntary agency
One philosopher / ethicist / theologian
One lay person from the community
Member Secretary

18. Ethics committee approval

Responsibility: to ensure the protection of the rights, safety and well-
being of human subjects involved in a trial.

reviewing and approving / providing favourable opinion on, the trial
protocol,
the suitability of the investigator(s), facilities, and
the methods and material to be used in obtaining and documenting
informed consent of the trial subjects.

19. Informed consent is a PROCESS
Involves
Providing all relevant information to the volunteer/ patient
The patient/ volunteer understanding the information provided
Voluntarily agreeing to participate

Informed consent is a communication process :
 between the researcher and the participant
 starts before the research is initiated
 continues throughout the duration of the study
Consent must be in a language the subject understands
Personally sign and date by subjects


What is informed consent?

20.  Objective: minimising interindividual variability in order to
detect product differences.

The inclusion and exclusion criteria should be clearly stated
in teh protocol

 General screening
The subjects should be screened for suitability by means of clinical
laboratory tests, a medical history, and a physical examination.

ECG, Chest-X ray, etc..

Selection / Enrollment of subjects

21. Age & BMI
18 years of age or older, between 18.5 and 30 kg/m
2

Gender
Males or Females

Smoking status
Smokers or Non smokers

Number of subjects (sample size calculation)
12, 24, ……

Selection / Enrollment of subjects

22.  Drug administration as per randomization
Generally administered at room temperature
Fluid for drug administration
Water
Apple sauce
Other fluids
Quantity of fluid



Drug administration
USFDA about 8 ounces (240 mL) of water
EMEA Administered with a standardised volume of fluid (at least 150 ml)
CDSCO Not Specified
Canada
The dose should be taken with water of a standard volume (150 – 250
millilitres) and at a standard temperature
Furthermore, the drugs should be administered at approximately the same
time on each study day.

23. Type of sample
Blood, urine, etc.

Sample collection at specified time points
0.0, 0.5, 1.0, 1.33, …………………..

Centrifugation & separation
Plasma
Serum
Storage of samples
Not to cause significant degradation
-30˚C or -80˚C



Sample collection/separation/Storage

24. Subject Well-being

Adverse Event documentation
Evaluation of seriousness (Mild, Moderate, severe)
Evaluation of relation to investigative drug

Follow-up of Adverse Events

Concomitant Medications

Serious Adverse Events

Hospitalization (if required)



Adverse Event Monitoring

25. Sorting of samples (subject wise)

Transfer of samples
Storage conditions shall be maintained (dry ice)




Sample transfer to Bioanalytical

26. LC-MS/MS or HPLC

Method Development

Method Validation

Routine sample analysis



Bioanalytical Phase

27. It describes the series of steps and tests performed to determine the
most effective and efficient means of quantitating the specific
compound of interest.

Parameters to be evaluated in MD
Collection of Literature
Reference standards
Selection of CC range
Tuning of molecule in MS
Selection of Internal standard
Selection of Mobile phase
Selection of column
Role of pH

Method Development

28. Literature search and prior methodology
Multiple reaction monitoring mass spectrometry (MRM)
& Optimization
Development of Sample Preparation Method
Development and optimization of the LC method
SPE Method
Validation of the method
Sample Analysis
LLE Method
Protein Precipitation
Method
Incurred Sample
Repeat Analysis
Routine Batch
Sample Analysis
Repeat
Analysis

29. Bioanalytical method validation includes all of the procedures that
demonstrate that a particular method used for quantitative measurement of
analytes in a given biological matrix, such as blood, plasma, serum, or urine,
is reliable and reproducible for the intended use.
Full validation:
performed when implementing methods for the first time, or when additional
analytes or metabolites are added for quantitation
•Partial validation:
transfer of the bioanalytical method to another laboratory, change in
equipment, calibration concentration range, limited sample volume, another
matrix or species, change in anticoagulant, sample processing procedure,
storage conditions etc.
•Cross Validation:
Where data are obtained from different methods within and across studies
or when data are obtained within a study from different laboratories,
applying the same method, comparison of those data is needed and a cross
validation of the applied analytical methods should be carried out.
Method Validation

30. Selectivity
Carry over
Lower Limit of Quantification
Calibration Curve
Accuracy
Precision
Sensitivity
Reproducibility
Stability
Dilution integrity
Matrix effect
Recovery
Whole Blood Stability
Validation parameters

31. Calibration standards and quality control
samples (QC)
Authenticated analytical reference standard
should be used to prepare (separately)
solution of known concentration
certified reference standards
Never from a marketed drug formulation
commercially supplied reference standards
other material of documented purity
Validation
methods 31
Reference Standard

32. It is a general method for determining the concentration of a substance in an unknown
sample by comparing the unknown to a set of standard samples of known
concentration.

Instrumental response i.e., the analytical signal, changes with the concentration of the
analyte. A series of standards across a range of concentration near the expected
concentration of analyte in the unknown will give a linear plot.

The calibration curve should be prepared in the same biological matrix (e.g. plasma )
as the sample in the intended study by spiking with known concentration of the
analyte (or by serial dilution).

Calibration Curve

33. LLOQ: It is the lowest concentration of the standard curve that can be
measured with acceptable accuracy and precision.
ULOQ: The highest standard will define the upper limit of quantification
of an analytical method.
QC samples: These are standards of spiked samples with known
concentrations that provide a method of control (assessing integrity)
for each assay & they must meet the acceptance criteria related to
accuracy and precision.
This can be accomplished by analysis of replicate sets of analyte
samples of known concentration i.e., QC samples from an equivalent
biological matrix.
At a minimum, 3 concentrations representing the entire range of the
standard curve should be studied:
• low QC-within 3 times the LLOQ
• middle QC-approx. 50 to 60% of ULOQ),
• high QC- approx. 70% of ULOQ (near the upper boundary of the
CC curve).

34. Response: dependent variable
(peak,area ..)
Y (observed)

Y
X
y = ax + b
Independent variable:
exactly known
concentrations
Calibration curve
x1
y1
xn
Yn

35. Response: dependent variable
Y (observed)

Y
X
y = ax + b
Independent variable:
X
estimated concentration
^
Calibration curve
x1
y1
xn
Yn

36. x
Response
x
Response
GOOD BAD
^ ^
Calibration curve

37. Parameters FDA EMEA
Selectivity Biological matrix [plasma (at least one
heamolysed & lipemic), urine, or
other matrix] should be obtained
from at least 6 sources.
At least 6 individual sources.
Response of interfering peak
should be ≤ 20% of the LLOQ for
analyte and ≤ 5% for the IS.
Accuracy The mean value (min five
determinations) should be within 15%
of the actual value except at LLOQ,
where it should not deviate by more
than 20% (minimum Three
Concentrations)
The mean conc. (min five
determinations) should be
within 15% of the nominal values
for the QC samples, except for
the LLOQ which should be within
20% of the nominal value
(minimum four concentrations).
Precision The within & between run CV value
should be ≤ 15% for the QC samples
7 for LLOQ ≤ 20%.
The within & between run CV
value should be ≤ 15% for the
QC samples & for LLOQ ≤ 20%.
Calibration/S
tandard
Curve
It should consist of a blank sample, a
zero sample, & 6 to 8 non-zero
samples covering the expected range,
including LLOQ.
The back calculated conc. of the
CC should be within ±15% of the
nominal value & for LLOQ it
should be within ±20%.
CC/Conc.
Response
•20% deviation of the LLOQ from nominal conc.
•15% deviation of standards other than LLOQ from nominal conc.

38. Parameters FDA EMEA
LLOQ

The analyte response at the LLOQ
should be at least 5 times the
response compared to blank response
& analyte peak response reproducible
with a precision of 20% and accuracy
of 80-120%.
The analyte signal of the LLOQ
sample should be at least 5
times the signal of a blank
sample.

Freeze and
Thaw
Stability
Analyte stability should be
determined after three freeze and
thaw cycles.

The number of cycles in the
freeze-thaw stability should
equal or exceed that of the
freeze/thaw cycles of study
samples.
Short-Term
Temperature
Stability:
3 aliquots of each of the low and high conc. should be thawed at Room
temp and kept at room temperature from 4 to 24 hours and analyzed.
Long-Term
Stability
The storage time in a long-term
stability evaluation should exceed the
time between the date of first sample
collection and the date of last sample
analysis.
stability at each temperature at
which study samples will be
stored should be determined.

39. Parameters EMEA
Stock
Solution
Stability
Should be evaluated at RT for at least 6 hr.
If the stock solutions are refrigerated or frozen for the relevant period,
the stability should be documented
Post-
Preparative
Stability
The stability of the drug and the IS in the processed samples should be
assessed over the anticipated run time for the batch size
Whole Blood
Stability
Should demonstrate sample integrity from the time of blood sample is
drawn to the time the plasma/serum is obtained.
Recovery Recovery of the analyte need not be 100%, but the extent of recovery of
an analyte and of the IS should be consistent, precise, and reproducible.
Big Batch Should demonstrate accuracy and precision of QC samples with at least
one run in a size equivalent to prospective study samples analytical run.
Carry-over

Carry over in the blank sample following the high concentration standard
should not be greater than 20% of the LLOQ and 5% for the IS.
Dilution
Integrity
Accuracy and precision should be within the set criteria, i.e. within ±15%.
Dilution integrity should cover the dilution applied to the study samples
Matrix Effect

at least 6 lots of blank matrix(heamolysed & hyperlipidaemic) from
individual donors. The CV should not be greater than 15 %.

40. Analysis of study samples
An analytical run consists of the
•Blank sample (processed matrix sample without analyte and without IS)
•A zero sample (processed matrix with IS)
•Calibration standards at a minimum of 6 concentration levels
•At least 3 levels of QC samples (low, medium and high) in duplicate (or at
least 5 % of the number of study samples, whichever is higher)
•Study samples to be analyzed.

Possible reasons for reanalysis of study samples and criteria to select the
value to be reported should be predefined in the protocol, study plan or
SOP, before the actual start of the analysis of the samples.
Reanalysis of study samples

41. Differences for instance in protein binding, back-conversion of known and
unknown metabolites, sample inhomogeneity or concomitant medications,
may affect the accuracy and precision of the analyte in such samples during
processing and storage.

•10% of the samples should be reanalyzed in case the number of samples
is less than 1000 samples.

•5% of the number of samples exceeding 1000 samples.

The concentration obtained for the initial analysis and the concentration
obtained by reanalysis should be within 20% of their mean for at least 67% of
the repeats.
Incurred samples reanalysis

42. the pharmacokinetic analysis, where pharmacokinetic
parameters derived from the concentrations are
calculated;

Statistics: is the study of the collection, organization,
analysis, interpretation, and presentation of data

Pharmacokinetics & Statistics

43. Comparison

44. AUCt The area under the curve (AUC - calculated by the linear trapezoidal rule) from
time zero up to the sampling time for which the last measured concentration
was equal to or larger than LOQ.
AUCinf The AUC from time zero to infinity estimated by adding to AUCt a value equal to
Clast/Kel, where Clast is the measured last quantifiable concentration (occurring
at time Tlast) and Kel is the apparent elimination rate constant (see below).
Cmax The maximum observed concentration of the analyte over the sampling
interval.
Tmax The sampling time at which Cmax occurred.
Kel Apparent first-order terminal elimination rate constant estimated as the
absolute value of the slope of the leastsquares regression line fitted through
the ln(concentration)-time data pairs located on the terminal linear phase of the
ln(concentration)-time profile
Thalf The apparent terminal elimination half-life of the analyte,
calculated as ÷
Pharmacokinetic parameters

45. The area under the curve (AUC - calculated by the linear trapezoidal rule)
from time zero up to the sampling time for which the last measured
concentration was equal to or larger than LOQ.
Sample time point deviations

AUCt-Area under the curve

46. Cmax-The maximum observed concentration of the analyte over the sampling interval.

Tmax-The sampling time at which Cmax occurred.



Cmax & Tmax
Time Concent
0.00 0.0
0.08 0.0
0.17 4820.5
0.25 5502.5
0.33 5009.9
0.42 4578.0
0.50 4305.4
0.67 3894.7
0.83 3433.7
1.00 3258.7
1.25 2766.6
1.50 2646.8
1.75 2257.1
2.00 1916.2
2.33 1653.4
2.67 1369.6
3.00 1085.1
4.00 703.1
5.00 389.5
6.00 242.8
7.00 154.6
8.00 101.4
9.00 62.5
10.00 49.0
Cmax 5502.5
0.25, 5502.5
0.0
500.0
1000.0
1500.0
2000.0
2500.0
3000.0
3500.0
4000.0
4500.0
5000.0
5500.0
6000.0
0.001.002.003.004.005.006.007.008.009.0010.0011.0012.00
Concentration

Time
Single dose-Profile
Cmax
Tmax

47. The AUC from time zero to infinity estimated by adding to AUCt a value equal to Clast/Kel,
where Clast is the measured last quantifiable concentration (occurring at time Tlast) and Kel is
the apparent elimination rate constant (see below).

AUCinf-Area under the curve
Time Concent AUCt
0.00 0.0
0.08 0.0 0.0
0.17 4820.5 216.9
0.25 5502.5 412.9
0.33 5009.9 420.5
0.42 4578.0 431.5
0.50 4305.4 355.3
0.67 3894.7 697.0
0.83 3433.7 586.3
1.00 3258.7 568.9
1.25 2766.6 753.2
1.50 2646.8 676.7
1.75 2257.1 613.0
2.00 1916.2 521.7
2.33 1653.4 589.0
2.67 1369.6 513.9
3.00 1085.1 405.0
4.00 703.1 894.1
5.00 389.5 546.3
6.00 242.8 316.2
7.00 154.6 198.7
8.00 101.4 128.0
9.00 62.5 82.0
10.00 49.0 55.8
9982.6 KelClastAUCtAUC /inf 
Time Concent AUCt
0.00 0.0
0.08 0.0 0.0
0.17 4820.5 216.9
0.25 5502.5 412.9
0.33 5009.9 420.5
0.42 4578.0 431.5
0.50 4305.4 355.3
0.67 3894.7 697.0
0.83 3433.7 586.3
1.00 3258.7 568.9
1.25 2766.6 753.2
1.50 2646.8 676.7
1.75 2257.1 613.0
2.00 1916.2 521.7
2.33 1653.4 589.0
2.67 1369.6 513.9
3.00 1085.1 405.0
4.00 703.1 894.1
5.00 389.5 546.3
6.00 242.8 316.2
7.00 154.6 198.7
8.00 101.4 128.0
9.00 BLQ 50.7
10.00 BLQ 0.0
9895.6

48. To determine it, we need to calculate slope of the straight line having y-intercept = B. if natural log are used
the rate constant of elimination (b) = negative slope of this straight line.

A key parameter for calculation of AUCinf and Thalf

Calculation from Excel

SLOPE(known_y's, known_x's)

Manual calculation Formaula




Kel-Calculation

49. Conce Time Ln Conc
0.0 0.00 #NUM!
0.0 0.08 #NUM!
4820.5 0.17 3.7
5502.5 0.25 3.7
5009.9 0.33 3.7
4578.0 0.42 3.7
4305.4 0.50 3.6
3894.7 0.67 3.6
3433.7 0.83 3.5
3258.7 1.00 3.5
2766.6 1.25 3.4
2646.8 1.50 3.4
2257.1 1.75 3.4
1916.2 2.00 3.3
1653.4 2.33 3.2
1369.6 2.67 3.1
1085.1 3.00 3.0
703.1 4.00 2.8
389.5 5.00 2.6
242.8 6.00 2.4
154.6 7.00 2.2
101.4 8.00 2.0
62.5 9.00 1.8
49.0 10.00 1.7
slope -0.17834
Kel-Calculation
Ln Concentration

R-
T-
Slope= -0.17834

50. Kel-Calculation
Ln Concentration

R-
T-
Slope= -0.17834

51. The AUC from time zero to infinity estimated by adding to AUCt a value equal to Clast/Kel,
where Clast is the measured last quantifiable concentration (occurring at time Tlast) and Kel is
the apparent elimination rate constant (see below).

AUCinf-Area under the curve
Time Concent AUCt
0.00 0.0
0.08 0.0 0.0
0.17 4820.5 216.9
0.25 5502.5 412.9
0.33 5009.9 420.5
0.42 4578.0 431.5
0.50 4305.4 355.3
0.67 3894.7 697.0
0.83 3433.7 586.3
1.00 3258.7 568.9
1.25 2766.6 753.2
1.50 2646.8 676.7
1.75 2257.1 613.0
2.00 1916.2 521.7
2.33 1653.4 589.0
2.67 1369.6 513.9
3.00 1085.1 405.0
4.00 703.1 894.1
5.00 389.5 546.3
6.00 242.8 316.2
7.00 154.6 198.7
8.00 101.4 128.0
9.00 62.5 82.0
10.00 49.0 55.8
9982.6 KelClastAUCtAUC /inf 
Time Concent AUCt
0.0 0.00
0.0 0.08 0.0
4820.5 0.17 216.9
5502.5 0.25 412.9
5009.9 0.33 420.5
4578.0 0.42 431.5
4305.4 0.50 355.3
3894.7 0.67 697.0
3433.7 0.83 586.3
3258.7 1.00 568.9
2766.6 1.25 753.2
2646.8 1.50 676.7
2257.1 1.75 613.0
1916.2 2.00 521.7
1653.4 2.33 589.0
1369.6 2.67 513.9
1085.1 3.00 405.0
703.1 4.00 894.1
389.5 5.00 546.3
242.8 6.00 316.2
154.6 7.00 198.7
101.4 8.00 128.0
62.5 9.00 82.0
49.0 10.00 55.8
AUCt 9982.6
Kel 0.41073
C*/Kel 119.3012
AUCinf 10101.9
AUCinf = 9982.6+49/0.41073
= 10101.9

52. Thalf-Half-life
The period of time required for the concentration or amount of drug in the body to be reduced
by one-half

Usually consider the half life of a drug in relation to the amount of the drug in plasma.

A drug’s plasma half-life depends on how quickly the drug is eliminated from the plasma.

Formula

Thalf = Ln (2) / Kel
or
Thalf = 0.693 / Kel

Kel = 0.41073

Thalf = 0.693/0.41073
= 1.69





Time Ln Conce
6.00 2.4
7.00 2.2
8.00 2.0
9.00 1.8
10.00 1.7
slope -0.17834
Kel 0.41073
Thalf (T1/2) 1.69

53. USFDA
The calculated 90% confidence interval for AUC and Cmax should l fall within the
bioequivalence range, usually 80-125%.
EMEA
In studies to determine bioequivalence after a single dose, the parameters to be analysed
are AUC(0-t), or, when relevant, AUC(0-72h), and Cmax.

For these parameters the 90% confidence interval for the ratio of the test and reference
products should be contained within the acceptance interval of 80.00-125.00%.
CDSCO
The calculated 90% confidence interval for AUC and Cmax should l fall within the
bioequivalence range, usually 80-125%.
Canada
The 90% confidence interval of the relative mean AUC
T of the test to reference product should be
within 80.0 % to 125.0 % inclusive.

The relative mean maximum concentration (C
max) of the test to reference product should be between
80.0 % to 125.0 % inclusive.

Acceptable Limits

54. T/R (%) 80% 125%
Demonstrate BE
Fail to Demonstrate BE
Fail to Demonstrate BIE
Demonstrate BIE Demonstrate BIE
Acceptable Limits