Analytical Control Strategies: from molecular understanding to CQAs, specifications and lifecycle management.

Garry B. Takle, Ph.D.Biologics and Vaccines AnalyticsMerck Manufacturing Division: MSD

Presentation Outline

• Analytical Control Strategy Concepts• Molecular/biophysical/biochemical

understanding • CQAs• Analytical Specs• Potency Method examples• Analytical Lifecycle Strategy

ICH Q10 Pharmaceutical Quality System:Analytical aspects

• Objectives– Product realization– Control– Continual Improvement

• Product Lifecycle– Pharmaceutical Development

• Analytical method development– Tech transfer

• Development through to manufacturing• Transfer of analytical methods (AMT)

– Commercial• Control of Materials• QC/QA• Release

– Discontinuation

Control Strategy Definition• Definition: A planned set of controls,

derived from current product and process understanding, that assures process performance and product quality (ICH Q10)

– Parameters and attributes related to drug substance and drug product materials and components

– Facility and equipment operating conditions

– In-process controls– Finished product specifications– Associated methods and frequency of

monitoring and control• Control strategy development is

iterative; modified throughout product lifecycle, due to changes in:

– Process– Methods– Product Understanding

Target Product Profile

Critical Quality Attributes

Risk Assessment(quality risk

Management)

Design Space(product/processUnderstanding)

Control Strategy

ContinuousImprovement

Q8(R2) Pharmaceutical development

• QbD concepts– Quality Target Product Profile (QTPP)

• Analytical Target Profile– Critical Quality Attributes (CQAs)– Manufacturing process (assay)– Identify Manufacturing Process Parameters affecting

CQAs– Control strategy

IgG1 potential heterogeneity

Fabian Higel, Andreas Seidl, Fritz Sörgel, Wolfgang FriessN-glycosylation heterogeneity and the influence on structure, function and pharmacokinetics of monoclonal antibodies and Fc fusion proteinsEuropean Journal of Pharmaceutics and Biopharmaceutics, Volume 100, 2016, 94–100

Modification Example Likely source

Glycosylation site at Asn, glycoform variation (e.g.

sialylation)Occurs during fermentation

C terminus cleavageOccurs during fermentation,

catalyzed by carboxypeptidase

C-terminal α amidation Occurs during fermentation, catalyzed by PAM

N-terminal glutamate (LC) Sequence

N-terminal glutamine (HC) Sequence

N-terminal pyroglutamate (HC) Occurs during fermentation, downstream and/or storage

Oxidation of MetOccurs during fermentation,

downstream processing, and/or storage

Deamidation of: AsnOccurs during fermentation,

downstream processing, and/or storage

McAb Post-Translational Modifications Affecting Charge Distribution - Purity

Analytical Characterization Employs Multiple Orthogonal Techniques

Intact Mass

Peptide MapLC/MS/MS

NR PeptideDisulfide Map

Free Sulfhydryl

Far UV CD

FT-IR

Near UV CD

Fluorescence

DSC

CompetitiveBinding ELISA

Cell based Potency

Biacore Kinetics& Affinity

FcRn, FcGR

ADCC / CDC

37C

55C Stressed

Forced oxidation

pH shift

HP-SEC

NR CE-SDS

R CE-SDS

DLS

SV-AUC

Particle Count -Light Obscuration

Particle Size & Morphology- MFI

HP-IEX

cIEF

Peptide MapLC-MS

HILIC-HPLCGlycan mapGal, Man, Afuco

Peptide MapLC/MS/MSAglyco

HILIC-HPLC Glycan MapSialic Acid

Intact Mass

Photostability

HP-HIC

RP-HPLC

Thorough product characterization – including variants and impurities

• Isolation and Characterization HP-IEX Species

A typical chromatogram using the semi-preparative column and collection scheme

Q6B: Specifications

• Drug Substance– Appearance and

description– Identity– Purity and

Impurities• Process related• Product related

– Potency– Quantity

• Drug Product− Appearance and

description− Identity− Purity and

Impurities− Potency− Quantity− General tests− Unique tests

Virus-Like Particle(~20,000 kDa)

L1 Capsomere(Pentamer)(~280 kDa)

5 × L1

L1 protein(55 or 57 kDa)

(Crystal structure coordinatescourtesy of Prof. S. C. Harrison,

Harvard University)

~ 3 nm ~ 10 nm~ 60 nm

GARDASIL®9 Based on Human Papillomavirus (HPV) Virus-Like Particle (VLP)

~72 × L1Capsomeres

• Vaccine based on HPV major capsid protein, L1, self-assembled into virus-like particles (VLPs)

• Complex structure, but well characterized

Modern Vaccines Amenable to Characterization; Robust Control Strategy Achievable

Physicochemical Properties Biological ActivityIdentity

Purity Impurities Contaminants Quantity

Primary Structure• Peptide Map (Digestion,

MALDI-MS)• Deamidation (Isoquant)• Denatured Free Thiols

Secondary Structure• CD• FTIR

Tertiary - Quarternary Structure• Native Free Thiols• Thermal Unfolding (DSC)• Morphology (TEM, CryoEM,

AFM)• Monodispersity (TEM, SEC-

HPLC)• Aggregation (DLS, Cloud

Point, SPR)

Other• Aluminum• PS-80• pH• Completeness of adsorption

Antigenicity• In Vitro Relative

Potency (sandwich ELISA)

• Solution Antigenicity (competitive ELISA – IC50)

• Epitope Mapping (SPR)

• Epitope-specific antigenicity (SPR)

• In Vivo Potency (mouse ED50)

• Purity (SDS-PAGE)

Host-Cell• Protein

(Western Blot)• Nucleic acids

Product-Related• Resistance to

Proteolysis (SDS-PAGE)

Process-Related• Protease

• Sterility• Endotoxin

• Protein concentration

Primary and Secondary

Tertiary and Quarternary Alum AdsorbedC

ompl

ex S

truc

ture

http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Scientific_Discussion/human/000703/WC500021140.pdfZhao, et al (2013) Cell Press 31(11): 654-663; http://www.tandfonline.com/doi/pdf/10.4161/hv.27316

Blue text = select release CQA tests

Release testing: Drug Substance/ Drug Product

• Drug Substance• Protein Concentration• Percent Purity/Percent Intact

Monomer • SDS-HPSEC• RNA• DLS• IVRP• Identity• Sterility• Endotoxin• Aluminum• pH • Characteristics• Completeness of Adsorption

• Drug Product• IVRP• Identity• Sterility• Endotoxin• Aluminum• pH• Completeness of

Adsoprtion• Package Identity• Volume of Fill• Syringeability

• Life cycle of a product and analytical assays• Unlikely a single method assay platform could be used between

products or even between development phases for a single product• As products or assays are better understood, assays may be added,

removed, or refined to best demonstrate product quality, consistency, and potency. As product development proceeds, an improved or more relevant assay can be developed.

Expectations for Potency Assays

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• Potency method requirements – Reflect our understanding of MOA (binding in early stage, functional CBA in later

stages)– Specific to product– Robust, fit for intended use – Linear response for an appropriate range– Control of CQA

• Stability indicating (to relevant degradation pathways)

Potency method requirements

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Three assays were developed to assess potency for an inhibitory immunomodulator based on the mechanism of action

• Competitive binding ELISA• CHO cell based competitive binding ELISA• T-cell/antigen presenting cell based Functional Assay

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Competitive Binding ELISA

• Detection of bound ligand with biotinylated AB, SA HRP and Chemiluminescent substrate

• Inhibition curve• Sample vs reference• 4-PL1 fit and PLA2 analysis• Relative potency (% of reference)

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Receptor

Ligand

Molecule

Plate Bound

ELISA detects the presence of bound ligand

14-PL: 4-parameter logistical fit2 PLA: Parallel Line Analysis

Cell Based Competitive Binding ELISA

• Receptor expressed on cell surface

• Molecule competes for binding in the presence ligand

• Detect bound ligand with biotinylated AB, SA HRP and Chemiluminescent substrate

• Inhibition curve• Sample vs reference• 4-PL fit and PLA analysis• Relative potency (% of reference)

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Receptor

Ligand

Molecule

Cell surface

ELISA detects the presence of bound ligand

Functional Cell-based Assay

• Receptor/ligand interaction leads to inhibition of cytokine production

• Molecule blocks receptor/ligand engagements and removes inhibition of cytokine production and re-activates the T-cells

• Detection of cytokine by ELISA• Sample vs reference• 4-PL fit and PLA analysis• Relative potency (% of reference)

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Receptor

T-Cell surface

Molecule

APC Cell surface

Ligandcascade of events lead to increased cytokine production that is quantified

□ Reference material, o isotype control, ∆ cell control

Establishment of System Suitability Acceptance Criteria

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Upper Asymptote (UA)

Lower Asymptote (LA)

RS Slope

Control/Sample SlopeRoot Sum of SquaresError (RSSE)

UA/LA Ratio

Side-by-side release results, 3-way analysis

• Geometric mean potency +/- 95% confidence limits. • Similar results are obtained with all three assays and all geometric

mean potencies are within 80 to 120% of reference

Potency Assay lyo Reagent controlCritical reagent Time post

reconstitutionTime post initialreading (min)

Nominal/expected protein concentration (ug/mL)

Actual determined protein concentration

Assay pass rate (%)

Vial A 5 days0 100 66.6

34.527 100 88.431 100 94.2

Critical reagent Time post reconstitution

Time between readings (min)

Nominal/expected protein concentration (ug/mL)

Actual determined protein concentration

Assay pass rate (%)

Vial B1 day

0 100 73.8

0 and 33

2 100 79.66 100 53.28 100 41.410 100 53.7

2 days0 100 122.42 100 84.9

Assay Lifecycle Strategy

• Legacy Vaccines Panel• Phase 1: Scope Definition and General Assay Review

– Excel Workbook approach– 34 method attributes

• Phase 2: Method Improvement• Phase 3: Continuous Review

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Phase 1 General Assay Review:Strategy & Outcomes

Strategy/Principles

• Definition of scope and timelines.• “Divide and Conquer” – define broad analytics commercialization team.• Collaboration with QC.• Comprehensive concurrent approach vs linear.• Standardized Numeric Output (Workbook).• Drop Down menus – Pre-populated – Simple.• Rapid Metrics Generation: brings reagent, personnel, equipment,

validation, performance monitoring etc. issues into focus.• Real-Time Status/Progress Report• Outcomes:

– Completed in 6 months – Completed Workbooks and Final Report archived.– Definition and prioritization of Phase 2 efforts

Phase 2• 2nd Gen methods

– Invalid rate reduction (potency method)– Lead time reduction (rapid micro method)

• Derisk reagents– Dual source– Reagent quality

• Instruments– Redundancy– Obsolescence– Replacement

• Method precision improvements– Reduce testing burden.

• Replace animal model based methods.• Industry and regulatory expectations

– Validation

Phase 3 Continuous Review

• Validation status• Assay performance monitoring• Regulatory strategy/change control• Process performance monitoring• Comparability

Opportunities – PAT/nRTRTUse of biomarkers for process optimization.

– Protein, nucleic acids, metabolites– Newer technologies and real time measurements– Numerical score associated with optimal

potency/yield– Increased process understanding and control– Higher yields

FDA Emerging Technology Draft Guidance (2015)

nRTRT

• Overall E2E release strategy.• MAMs • Longest lead time assays – DS.

– In vitro/in vivo assays– Mycoplasma/micro

Acknowledgements

• Athena Nagi• Peg Criswell• Jennifer Dashnau• Gargi Maheshwari• Parimal Desai• The entire Biologics and Vaccines Analytics

(BVA) team• Other MSD colleagues (too many to list)