1

FDA recommendations for comparability studies to support

manufacturing changes

Joslyn Brunelle, PhDOffice of Biotechnology Products

OPQ/CDER/FDA

This views and opinions expressed here are my own and should not be used in place of

regulations, published FDA guidance, or discussions with the

Agency.

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Common Manufacturing changes throughout development

• New manufacturing site• Bioreactor Scale up• New master cell bank• New formulation • New purification column/resin• New equipment (filter, tubing, disposable)• Evaluate the risk (high/low)

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4

o

o

• 1o sequence• higher order

structure• post-translational

modifications• heterogeneity

Statin MW ~400 Da

Structure of complex molecules

Monoclonal Ab MW ~150,000 Da

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Post-Translational Modifications

2 x 6 x 4 x 4 x 5 x 5 x 2 = 9600K

pyro-E

O

D

GO

D

O • Methionine oxidation (2 x 2)

pyro-E• Pyro-Glu (2)

• High mannose, G0, G1, G1, G2 (5)

• Sialylation (5)

DD

• Deamidation (3 x 2)

G

D D

O

G

G

• Glycation (2 x 2)

(9600)2 ≈ 108

K • C-term Lys (2)

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Critical Quality Attributes for Biotech Products can include:

• Strength• Potency• Post-translational modifications• Isoelectric point• Aggregation• Size• Sterility• Ionic strength• Adventitious agents• Impurities (e.g., DNA, Host Cell Proteins)• Formulation components

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Critical Process Parameters (CPPs) and Quality Attributes (CQAs)CPPs

DO

pH

temperature

nutrient addition

cultivation method

CQAs

glycosylation

endogenous retrovirus production

higher level protein structure

product-related-enzyme production

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Product StructureAnd Function

Impurity Profile Adventitious Agents

QualitySafety

Efficacy

Process

Linking product characteristics to clinical parameters

• ICH Q5E guidance: Comparability of biotechnological products subject to changes in their manufacturing process

• Early development (Tox lot, Phase 1 / 2)• Phase 3 to BLA• Post-approval • Comparability protocols

• Biosimilar products are out of scope for this presentation 9

ICH Q5E• The demonstration of comparability does

not necessarily mean that the quality attributes of the pre-change and post-change product are identical, but that they are highly similar and that the existing knowledge is sufficiently predictive to ensure that any differences in quality attributes have no adverse impact on safety or efficacy of the drug product.

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ICH Q5E• Demonstration of comparability during development

depends on:– Stage of development – Availability of analytical procedures– Extent of product and process knowledge

• During early phases, comparability testing is generally not as extensive as for an approved product

• Where process changes are introduced in late stages of development and no additional clinical studies are planned to support the BLA, the comparability exercise should be as comprehensive as for an approved product

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ICH Q5E • If a manufacturer can provide assurance of comparability

through analytical studies alone, nonclinical or clinical studies with the post-change product are not warranted.

• However, where the relationship between specific quality attributes and safety and efficacy has not been established, and differences between quality attributes of the pre- and post-change product are observed, it might be appropriate to include a combination of quality, nonclinical, and/or clinical studies in the comparability exercise.

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Product Lifecycle

R&D IND Enabling Phase I/II (safety)

Development Decision

IND BLA

IV Post MarketingPhase III (efficacy)

-Tox Lots

-Limited structural characterization-Preliminary biological characterization-Limited viral clearance-Limited stability

•In depth characterization & Assay development•Additional studies to support viral clearance•Collect stability data•Manufacturing scale up

•Post marketing commitments (PMC)•Annual product review•Post-marketing surveillance (adverse events)•Annual report•Manufacturing changes•Stability

•Continue characterization & assay development.•Validated Lot release assay development•Specification setting•Collect stability data

EOP2

* Special considerations for breakthrough and orphan designations

Comparability for IND• First in human study• Tox lot should be comparable to Phase 1 clinical

material• Specify any differences between the

manufacturing process for tox lot and clinical lot • At least 1 tox lot and 1 clinical lot• Emphasis is on safety• Product and process related impurities• Place IND on hold if tox lot and clinical material

are not comparable 14

Case study #1• Tox lot manufactured using a non-clonal cell line• Clinical lot manufactured using a clonal cell line• This approach may require more rigorous

comparability data, depending on:– The product– The mechanism of action– Differences in the process between the tox

and clinical lots– Discuss with Agency

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Case study # 2• Monoclonal antibody• Change in sequence

– 2 amino acids in heavy chain– Product was comparable according to

analytical testing • End of Phase 1• The starting dose in the next clinical trial

was lowered in order to evaluate safety in a few patients

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Case Study # 3• Manufacturing changes during Phase 1 / 2

may require additional viral clearance studies if small scale model does not adequately represent the new process

• Quality attributes (purity & potency) may be comparable, but…

• Partial hold, cannot initiate studies with post-change lots until updated viral clearance study (safety)

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Case Study # 4• IgG1 monoclonal antibody • Oncology indication (multiple trials in Phase 1 / 2)• Pre- and post-change drug substance and drug product are not

comparable– particularly with respect to the carbohydrate profile– batches have approximately 12% higher G0, 10% lower G1 and

1% lower G2• CDC activity declined in forced degradation studies • It is not understood why CDC activity in post-change lots is sensitive

to thermal stress, but remains unaltered in pre-change lots • Additional non-clinical studies were performed to compare toxicity,

PK, and efficacy• Clinical study will be performed in myeloma patients to assess the

safety and PK; the outcome of the clinical study will inform the future direction of clinical development 18

Comparability for IND• End of Phase 2 (EOP2) • Encourage sponsors to implement

manufacturing changes prior to the initiation of pivotal clinical studies

• Clinical experience (safety and efficacy) is linked to product used in pivotal studies

• Manufacturing changes after pivotal studies are higher risk

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Regulatory filing• IND quality information amendment• Cover letter should adequately summarize the

extent of manufacturing changes• Clearly specify the date when post-change

product will be introduced into clinical studies• Type C meeting to discuss comparability

exercise expectations for your product depending on the type of process change and stage of development

• Minor changes may be annual reportable20

Expectations depend on:• Phase of development• Extent of changes• Number and availability of pre-change

clinical lots• Characterization of pre-change lots• General product knowledge, Target

product profile• Analytical methods

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• Lower probability to adversely impact safety & efficacy– Improvements in impurity and stability profile are

encouraged• Higher probability to adversely impact safety &

efficacy– New impurity – Glycosylation changes– Sialic acid content could impact PK (half-life)– Significant increase or decrease in potency could

impact dosing and safety (i.e. narrow therapeutic index drugs) 22

Analytical comparability

• Release testing (purity & potency)• Extended characterization *• Stability studies: real-time, accelerated/

stressed conditions *• If analytical comparability cannot be

shown, then nonclinical and/or clinical studies may be needed

(*) may be very limited if very early in development23

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Common Analytical Methods for Biotechnology Products

Quality Attribute Tests

Identity Western blot, ELISA, N-terminal sequencing, Peptide map, CE

Purity (Impurities) SDS-PAGE, IEF, IEX, CE, SEC-HPLC, RP-HPLC

Potency Bioassay (cell based or animal based), Binding assay

Strength(Protein concentration)

Absorbance at 280 nm, Bradford, HPLC, ELISA

Safety Bioburden, Endotoxin, Appearance, DNA, Host Cell Protein, Sterility, Pyrogenicity

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Extended Characterization• Primary structure

– Amino acid composition– N-terminal sequence analysis– C-terminal variants analysis– Peptide mapping

• Higher order structure– Disulfide linkage analysis– Circular Dichroism spectroscopy

• Biophysical characterization– UV spectral profile– Differential scanning calorimetry

• Charge profile– Isoelectric focusing

• Glycosylation– Site specific glycan composition

and occupancy– Glycan mapping by HPAE-PAD– Glycan structure determination by

MS/MS fragmentation analysis– Glycan linkage analysis– Monosaccharide composition– Sialic acid

• Molecular weight distribution– MALDI-TOF mass spectrometry– SDS-PAGE– Size exclusion HPLC

• Biological activity– Specific activity and enzyme

kinetics– Cellular Uptake & internalization– Cell based bioassay– Binding assay

Analytical comparability• One lot pre-change & one lot post-change may be sufficient

at early stage of development

• One lot is not sufficient at later stages of development, such as when manufacturing changes are made:– middle of Phase 3– after pivotal clinical study and prior to BLA– post-approval changes – Three pre-change lots & three post-change lots

is typical26

Analytical comparability• Do I need to set acceptance criteria for my

comparability exercise? • If you use “meets release specifications”

as criteria, are the specifications too wide?

• Statistical methods can be included, but are not necessary

• Most reviewers prefer to review your actual data!

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Analytical comparability• “comparable to reference standard” is not

well defined, so primary data (gels & chromatograms) should be provided

• Side-by-side testing not always required, but may be preferable if significant assay variability is observed – Prior to method validation– Extended characterization methods

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Changes to analytical methods• If new analytical methods are implemented

during development, the older methods may be included in the comparability exercise

• Retain samples can be re-tested when new and improved methods are implemented

• Retain samples will tell you whether a “new impurity” identified by a new method is entirely new or whether it was present all along in the clinical material (but not detectable by the old method)

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When your manufacturing experience contains multiple lots

• Compare comparability data to historical lot release, include trend charts

• Within specification, but out of trend, is there a concern?

• Provide justification for how lots were chosen for the comparability exercise– Avoid “cherry picking” (i.e. using certain pre-

change lots that are more comparable to your post-change lots)

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Stability testing • Real time temperature

– Limited data at the time of submission

• Accelerated/Stressed stability– More likely to discover changes when the products

are challenged– Shorter time course– Choice of stressed conditions defined during forced

degradation/characterization studies– Include conditions that result in incremental changes

over time– Include stability indicating assays 31

Regulatory filing post-approval• 21 CFR 601.12

– An applicant must inform the FDA about each change in the product, process, quality controls, equipment, facilities, responsible personnel, or labeling established in the approved license application

• Prior Approval Supplement (PAS): changes requiring supplement submission and approval prior to distribution of the product made using the change– Major changes have substantial potential to have an adverse

effect on a product’s identity, strength, quality, purity, or potency as they may relate to its safety or effectiveness

– Manufacturing site, scale, specifications, formulations

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Regulatory filings• CBE-0/CBE-30: changes requiring supplement

submission at least 30 days prior to distribution of the product made using the change– Moderate potential to have adverse effect– Like-for-like change of equipment– Add alternate QC testing site

• Annual report– Minimal potential to have adverse effect– Simple change in floor plan

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PAS - comparability study• Include at least three drug substance lots Process A commercial scale and

three lots Process B commercial scale • Release and extended characterization testing• Real-time, accelerated, and stressed stability data• Release testing and 3 months real time and accelerated stability data on at

least one lot of drug product• All process validation data for Process B 10,000L scale• Trending of all historical data (both process A and B clearly labeled)

including release, characterization, stability data, and an evaluation of process comparability for upstream and downstream process parameters

• Provide evidence that the assay you currently have in place to measure host cell proteins provides a suitable measure of HCP under the new process

• A pre-approval inspection for the Process B manufacturer may also be required

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Case study # 5 • Switching from vial to pre-filled syringe late

in development is not just a simple comparability exercise, additional clinical experience is needed, including:

• Human factor studies• Stability data to establish expiry• Extractable/leachable studies for licensure• Additional clinical studies also necessary

for a switch from IV to SC administration 35

• Animal studies– PK/PD/biodistribution– Toxicity– Tissue cross reactivity study

• Clinical studies– PK/PD– Safety and efficacy

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Case study # 6• Pivotal clinical studies were complete• Manufacturing changes:

– Upstream: Serum removed from media, change from roller bottle to bioreactor

– Downstream: New purification site & scheme • Analytical comparability cannot be demonstrated:

– Inadequate release testing = no historical data for pre-change lots

– Reference standards were changed multiple times and linkage to original clinical material was lost

– Lack of retain samples37

Case study # 6 cont.• Clinical comparability: differences

observed in PK study– Bioequivalence study in healthy volunteers

did not met criteria (80-120%)– Post-change product has 30% lower exposure – PD marker has high subject variability

• Additional clinical trial (efficacy) needed for approval

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Comparability protocols• Typically used in Post-approval setting• Extensive knowledge of process and

product• Protocol approved for a specific change• Protocol submitted to Agency as PAS• Acceptance criteria tighter than release• Stability (real time, accelerated/stressed)• Reduced reporting category (CBE-30)

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Acknowledgements• Michele Dougherty• Gibbes Johnson

• Sarah Kennett• Susan Kirshner• Juhong Liu• Marjie Shapiro• Sean Fitzsimmons• Chana Fuchs• Cristina Ausin