Changes to a Potency Bioassay for Biotechnology Products

Changes to a Potency Bioassay for Changes to a Potency Bioassay for

Biotechnology Products: a Regulatory Biotechnology Products: a Regulatory

PerspectivePerspective

Kathleen A. Clouse, Ph.D., DirectorKathleen A. Clouse, Ph.D., Director

Division of Monoclonal Antibodies Division of Monoclonal Antibodies

Office of Biotechnology Products Office of Biotechnology Products

Center for Drug Evaluation and ReviewCenter for Drug Evaluation and Review

Food and Drug AdministrationFood and Drug Administration

Disclaimer: Some of the views expressed in this presentation may reflect the Disclaimer: Some of the views expressed in this presentation may reflect the view of the speaker and not necessarily represent official FDA policy.view of the speaker and not necessarily represent official FDA policy.

CASSS Bioassays 2010 Symposium November 8 & 9, 2010CASSS Bioassays 2010 Symposium November 8 & 9, 2010

Biotechnology Products Biotechnology Products ––

Regulatory OrganizationRegulatory Organization

Potency Potency –– The RegulationsThe Regulations

PHS Act Section 351PHS Act Section 351 (42USC262 as amended (42USC262 as amended

by FDAMA)by FDAMA)

Approval of a biologic product is based on a Approval of a biologic product is based on a

demonstration that:demonstration that:

•• The product is safe, pure and The product is safe, pure and potentpotent•• The facility(ies) meet standards designed to The facility(ies) meet standards designed to

assure that the product continues to be safe, assure that the product continues to be safe, pure and pure and potentpotent

Potency Potency –– The RegulationsThe Regulations

“The word “The word potencypotency is interpreted to mean the is interpreted to mean the specific ability or capacity of the product (...) specific ability or capacity of the product (...) to to effect a given resulteffect a given result.” .” 21 CFR 600.3(s)21 CFR 600.3(s)

““Tests forTests for potencypotency shall consist of either shall consist of either in vitroin vitro or or in vivo tests, or both, which have been in vivo tests, or both, which have been specifically specifically designed for each productdesigned for each product so as to indicate its so as to indicate its potency in a manner adequate to satisfy the potency in a manner adequate to satisfy the interpretation of potency given by the definition in interpretation of potency given by the definition in §§600.3 of this chapter.”600.3 of this chapter.”

21 CFR 610.1021 CFR 610.10

Potency Potency -- ICH Q6BICH Q6B

•• “..for complex molecules, the physicochemical “..for complex molecules, the physicochemical information may be extensive but unable to confirm information may be extensive but unable to confirm the higherthe higher--order structure which, however, can be order structure which, however, can be inferred from the inferred from the biological activitybiological activity””

•• (Glossary)(Glossary)The measure of the biological activity using a suitably The measure of the biological activity using a suitably quantitativequantitative biological assay (also called potency assay biological assay (also called potency assay or bioassay), based on the attribute of the product or bioassay), based on the attribute of the product which which is linked to the relevant biological properties.is linked to the relevant biological properties.

•• Other procedures such as ligand and receptor binding Other procedures such as ligand and receptor binding assays assays maymay be acceptable.be acceptable.

A wellA well--designed bioassay will reflect the mechanism designed bioassay will reflect the mechanism of action of the product. (of action of the product. (Chana FuchsChana Fuchs--DMADMA))

Types of potency assaysTypes of potency assays

• Binding assays Binding assays

•• BioassaysBioassays

–– Animal based Animal based

–– Organ or tissue based Organ or tissue based

–– Cell culture basedCell culture based•• Early response (signaling pathway) Early response (signaling pathway)

•• Late response (proliferation, cytokine release)Late response (proliferation, cytokine release)

•• Multiple cell types (cellMultiple cell types (cell--cell interaction)cell interaction)

–– Biochemical Biochemical •• Enzyme activity assaysEnzyme activity assays

Change to a BioassayChange to a Bioassay

•• Change may be necessary!Change may be necessary!–– Reagents are no longer availableReagents are no longer available

–– Science (knowledge of the product) and technology Science (knowledge of the product) and technology (assay capabilities) have significantly progressed. (assay capabilities) have significantly progressed.

–– Market demand is so much better than expected that Market demand is so much better than expected that one needs a more QC friendly assay (e.g., one that can one needs a more QC friendly assay (e.g., one that can be easily transferred to many manufacturing sites).be easily transferred to many manufacturing sites).

•• Change is possible!!! Change is possible!!! –– Even after the BLA has been approved.Even after the BLA has been approved.

http://bongoking.files.wordpress.com/2009/02/don_t_panic_button-758852.jpg

Application of a BioassayApplication of a Bioassay

•• Indicates biological activities specific/relevant to the Indicates biological activities specific/relevant to the product (mechanism of action) product (mechanism of action) (“…to effect a given result”)(“…to effect a given result”)

•• Clinical correlateClinical correlate (if possible)(if possible)

•• Product release (“…Product release (“…to confirm higher order structure”/activity)to confirm higher order structure”/activity)

•• Stability program Stability program (ideally (ideally -- stability indicating)stability indicating)

•• Manufacturing changes Manufacturing changes –– ComparabilityComparability

•• QbD QbD –– One would want good bioassays to enable a better One would want good bioassays to enable a better understanding of changes as one moves within the design space.understanding of changes as one moves within the design space.

Change to a BioassayChange to a Bioassay

•• The degree of data needed to support a change will The degree of data needed to support a change will depend upon depend upon –– The development stage of the productThe development stage of the product

–– The type(s) of assay(s) being exchangedThe type(s) of assay(s) being exchanged

–– Product specific requirementsProduct specific requirements

•• A new assay should have a justifiable advantage over the A new assay should have a justifiable advantage over the old assay. e.g.:old assay. e.g.:–– Stability indicatingStability indicating

–– Reflects Mechanism of ActionReflects Mechanism of Action

–– Distinguishes product variantsDistinguishes product variants

–– Detects degradation productsDetects degradation products

–– Accuracy, precision, sensitivity, etc.Accuracy, precision, sensitivity, etc.

–– More “QC friendly”More “QC friendly”

Lack of correlation on stability between a Lack of correlation on stability between a binding binding assay and a bioassayand a bioassay

Lack of correlation on stability between a Lack of correlation on stability between a binding binding assay and a bioassayand a bioassay

1 2 3

4

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50

100

150

Months at elevated temperatureMonths at elevated temperature% a

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BioassayBioassayELISAELISA

From Anthony Mire-Sluis

Guidance for Industry: Comparability Protocols Guidance for Industry: Comparability Protocols --Protein Drug Products and Biological ProductsProtein Drug Products and Biological Products--

Chemistry, Manufacturing, & Controls Chemistry, Manufacturing, & Controls

Information (September 2003)Information (September 2003)

(section V.C)(section V.C)

“A comparability protocol for changing an analytical “A comparability protocol for changing an analytical

procedure should provide the plan for validation of the procedure should provide the plan for validation of the

changed analytical procedure and indicate whether the changed analytical procedure and indicate whether the

protocol will be used to modify the existing analytical protocol will be used to modify the existing analytical

procedure (i.e., retaining the same principle), or to procedure (i.e., retaining the same principle), or to

change from one analytical procedure to another.”change from one analytical procedure to another.”

Guidance for Industry; Comparability Protocols Guidance for Industry; Comparability Protocols --

Protein Drug Products and Biological ProductsProtein Drug Products and Biological Products--


Information (September 2003) Information (September 2003)

“We recommend that you design the comparability protocol “We recommend that you design the comparability protocol

to demonstrate that the proposed changes in the analytical to demonstrate that the proposed changes in the analytical

procedures procedures improveimprove or do not significantly changeor do not significantly change

analytical procedure characteristics that are relevant to the analytical procedure characteristics that are relevant to the

type of analytical procedure, its validation, and intended type of analytical procedure, its validation, and intended

use (e.g., accuracy, precision, specificity, detection limit, use (e.g., accuracy, precision, specificity, detection limit,

quantitation limit, linearity, range).”quantitation limit, linearity, range).”




Information (September 2003)Information (September 2003)

“You should identify in the plan any statistical analyses that “You should identify in the plan any statistical analyses that

you will perform and whether you intend to perform product you will perform and whether you intend to perform product

testing to compare the two procedures. The need and plan testing to compare the two procedures. The need and plan

for providing product testing to compare the two procedures for providing product testing to compare the two procedures

could vary depending on the extent of the proposed change, could vary depending on the extent of the proposed change,

type of product, and type of test (e.g., chemical, biological).”type of product, and type of test (e.g., chemical, biological).”




Information (September 2003) Information (September 2003)

“When you use the new revised analytical procedure for “When you use the new revised analytical procedure for

release or process control, you should not delete a test or release or process control, you should not delete a test or

relax acceptance criteria that we approved in your relax acceptance criteria that we approved in your

application, unless and until FDA informs you that the application, unless and until FDA informs you that the

approved acceptance criteria are no longer required.”approved acceptance criteria are no longer required.”

Changing BioassaysChanging Bioassays

• New assay should have comprehensive validation studies as scientifically appropriate. Accuracy, precision, specificity, detection limit, quantitation limit, linearity, and range should be comparable or better.

• Show a correlation between the current bioassay and the new one. Overlapping data using both assays is required to support assay change and provide link between assays, non-clinical and clinical data.

Case StudiesCase Studies

Case Study 1: Switching BioassaysCase Study 1: Switching BioassaysChange in analytical procedures assessed using stressed conditions

Samples and Conditions Mean %CV Mean %CV

Control, Normal Storage Temperature 96 8 99 5

Elevated Temperature – short term 86 8 71 4

Light Protection 99 6 97 2

High Light Exposure 70 5 47 3

0% Oxidizing Agent 103 8 100 4

5% Oxidizing Agent 94 7 80 4

Acidic pH Conditions 97 4 86 1

Basic pH Conditions 90 5 77 3

Assay#1 Assay #2

Case Study 2Case Study 2

•• Bioassay change from cell proliferation assay to Bioassay change from cell proliferation assay to gene expression assaygene expression assay

•• New assay validated for system suitability, assay New assay validated for system suitability, assay acceptance, accuracy, repeatability, intermediate acceptance, accuracy, repeatability, intermediate precisions, sample location effects, linearity/range, precisions, sample location effects, linearity/range, and specificity.and specificity.

•• No change in the approved release specification.No change in the approved release specification.

•• Potency assay comparison was done using:Potency assay comparison was done using:–– Multiple lots, including different strengths and formulations.Multiple lots, including different strengths and formulations.

–– Real time stability samplesReal time stability samples

–– Accelerated stability samplesAccelerated stability samples

–– Stressed samples (Stressed samples (photophoto--exposed, UV induced dimerization, enriched aggregates, exposed, UV induced dimerization, enriched aggregates, proteolyzed samples, simulated potencyproteolyzed samples, simulated potency--low and high % spikedlow and high % spiked))


•• Statistical analysis was provided to establish the Statistical analysis was provided to establish the equivalence of the two bioassays.equivalence of the two bioassays.

•• Paired tPaired t--test analysis of the release data was test analysis of the release data was included to demonstrate that the old and new included to demonstrate that the old and new methods are not significantly different. (FDA methods are not significantly different. (FDA statisticians restatisticians re--analyze data with various statistical analyze data with various statistical tools to assess validity of sponsor’s conclusions).tools to assess validity of sponsor’s conclusions).

•• Potency values of new assay ranged within that of Potency values of new assay ranged within that of the old assay.the old assay.


•• FDA had concerns regarding the suitability of the gene FDA had concerns regarding the suitability of the gene expression assay for testing potency of this product.expression assay for testing potency of this product.

–– Not clear if gene expression assay captured all biological activitiesNot clear if gene expression assay captured all biological activitiesof the protein measured by the cell proliferation assayof the protein measured by the cell proliferation assay

•• Product acts through the JAKProduct acts through the JAK--STAT pathways. Activation of signaling STAT pathways. Activation of signaling pathways by cognate ligands to the receptor also show that alternative protein pathways by cognate ligands to the receptor also show that alternative protein kinases can become activated by the product, leading to cell proliferation. kinases can become activated by the product, leading to cell proliferation. Downstream element driving gene expression pathway was not the only Downstream element driving gene expression pathway was not the only pathway involved.pathway involved.

–– Dose response curves identifying EC50 values for each assay show that Dose response curves identifying EC50 values for each assay show that the the gene expression assay is approximately 10 times less sensitivegene expression assay is approximately 10 times less sensitiveto the product than the cell proliferation assayto the product than the cell proliferation assay

–– There was no systematic study performed to compare the extent to which There was no systematic study performed to compare the extent to which the each assay detects product degradation (rate of sample decay)the each assay detects product degradation (rate of sample decay)

–– The The new method exhibited a lower average potencynew method exhibited a lower average potency (i.e., systematic (i.e., systematic bias) of 8bias) of 8--10% in stability samples (real time, accelerated & stressed 10% in stability samples (real time, accelerated & stressed samples). Using larger dataset corrected this apparent bias. Sponsor samples). Using larger dataset corrected this apparent bias. Sponsor tested bracketing strength of 72 DS and DP release samples. tested bracketing strength of 72 DS and DP release samples.


•• Product available in 2 presentations; lyophilized vials and Product available in 2 presentations; lyophilized vials and liquid PFSliquid PFS

•• Assay change from a cell survival/proliferation assay to an Assay change from a cell survival/proliferation assay to an apoptosis assay measuring Caspase activation (a more apoptosis assay measuring Caspase activation (a more proximal signaling event).proximal signaling event).

•• The specification for bioassay potency was not changed The specification for bioassay potency was not changed as the test sample biological activity is determined by as the test sample biological activity is determined by comparison to reference standard. comparison to reference standard.

•• New assay has enhanced precision over the old assay New assay has enhanced precision over the old assay and required less plates to meet system suitability criteria.and required less plates to meet system suitability criteria.


•• An SOP for the assay was submitted An SOP for the assay was submitted

•• Assay optimization for multiple parameters were presented. (reagent & ligand incubation Assay optimization for multiple parameters were presented. (reagent & ligand incubation

times, well size, cell number, cell culturing procedures prior to the assay etc.). Critical times, well size, cell number, cell culturing procedures prior to the assay etc.). Critical

reagents are qualified on a batchreagents are qualified on a batch--byby--batch basis for use in the assay.batch basis for use in the assay.

•• System and assay acceptance criteria were set for the overall assay & individual samples System and assay acceptance criteria were set for the overall assay & individual samples

based on based on

•• max to min signal ratios max to min signal ratios

•• goodness of fit (R2) of the reference standard and sample curvesgoodness of fit (R2) of the reference standard and sample curves

•• curve geometry of standards, controls and samples, e.g., slopes; ratios of asymptotes, etc.curve geometry of standards, controls and samples, e.g., slopes; ratios of asymptotes, etc.

•• Assay was validated for: accuracy (spike recovery), intermediate precision, repeatability, Assay was validated for: accuracy (spike recovery), intermediate precision, repeatability,

reproducibility, absence of matrix effects, linearity, range, robustness and instrumentreproducibility, absence of matrix effects, linearity, range, robustness and instrument--toto--

instrument ruggedness. instrument ruggedness.

•• Comparability of the two assays was demonstrated in a sideComparability of the two assays was demonstrated in a side--byby--side analysis of:side analysis of:

–– Lot release samples, ten drug substance, ten lyophilized drug product and ten liquid drug product.Lot release samples, ten drug substance, ten lyophilized drug product and ten liquid drug product.

–– Approximately 50 stability samples (Approximately 50 stability samples (--2020ooC, 2C, 2--88ooC, 25C, 25ooC and 40C and 40ooC).C).

–– Photo, heat, and trypsin stressed samples (multiple samples of each).Photo, heat, and trypsin stressed samples (multiple samples of each).

•• All comparisons met the study statistical analysis acceptance criteria.All comparisons met the study statistical analysis acceptance criteria.

Changing Bioassays Changing Bioassays -- SummarySummary•• Identify whether the new bioassay fulfills the needs based Identify whether the new bioassay fulfills the needs based

on mechanism(s) of action and use.on mechanism(s) of action and use.

•• Validate the new assayValidate the new assay

•• Test samples sideTest samples side--byby--side in old and new assay side in old and new assay

•• Determine if new assay can detect changes in potencyDetermine if new assay can detect changes in potency

•• Include variety of samples in the assessment, as Include variety of samples in the assessment, as appropriate, e.g.:appropriate, e.g.:–– Multiple lots of product; In process materialsMultiple lots of product; In process materials

–– Temperature accelerated and stressed samplesTemperature accelerated and stressed samples

–– Stability samples; Photostability samplesStability samples; Photostability samples

–– Samples exposed to pH extremesSamples exposed to pH extremes

–– Product variants (glycosylation variants, oxidized variants, etc)Product variants (glycosylation variants, oxidized variants, etc)

–– AggregatesAggregates

–– Samples from freeze/thaw studiesSamples from freeze/thaw studies

–– Proteolytic degradantsProteolytic degradants

AcknowledgementsAcknowledgements

Chana Fuchs CDER/OBP/DMA

Kathy Lee CDER/OBP/DTP

Barry Cherney CDER/OBP/DTP

Ingrid Markovic CDER/OBP/DTP

Kurt Brorson CDER/OBP/DMA

Carla Lankford CDER/OBP/DMA

Documents

Changes to a Potency Bioassay for Biotechnology Products