MONITORING METHOD PERFORMANCE

WITH BIOLOGICALS REFERENCE STANDARDS

Introduction: Reference StandardsVicky van Klinken-Manti

Assay Monitoring ToolSteven Kok

Biologics and Vaccines AnalyticsMSD Oss, The Netherlands

INTRODUCTION: Reference Standard Definitions -ICH Q6B

Generally: Reference standards are necessary for the analytical and medicinal quality control and for traceability.

Reference material is a sufficiently homogenous and stable material established to be fit for its measurement purpose: as a system suitability control, a calibrator or a comparator

The ideal Reference Material is a certified reference material with property values close to those of the test samples of interest “like vs like”

Official Reference Standard: A primary reference standard obtained from an official-recognised source, ie WHO, EDQM, USP, NIBSC, NIST etcWhere a recognized national or international standard is available, the manufacturer’s reference material and/or reference standard when used should be qualified against this standard.

For a new biological drug, it is unlikely that an international standard will be available at the time of submission.

Primary Reference Standard (PRS) (in-house): An appropriately characterized in-house material prepared by the manufacturer and is representative of the production and clinical materials. PRS is typically used to calibrate the Secondary (working) Reference Standard(s). A PRS is protocol driven, established through thorough and extensive characterization and testing and from stability data.

Secondary Reference Standard (SRS) or Working Reference Standard (WRS): SRS is an appropriately characterized in-house material that is generated from representative clinical or commercial batches. It is certified against the Primary Reference and used at product quality control testing.

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Lifecycle management of Two-Tier Reference Standards

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The NISTmAb Reference Material 8671 lifecycle management and quality plan,Schiel and Turner(2018) Analytical and Bioanalytical

Chemistry 410:2067–2078

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Assay Monitoring Tool: Content

1. Assay Monitoring Tool for BiologicsOverview of tests and labs, KeytrudaScope, Usage

2. AMT–for Method Performance Monitoring2 examples

3. AMT Extended scope: Reference Standard performance2 examples

4. Future enhancements

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Assay Monitoring Tool: Background

FDA Guidance: Analytical Procedures and Method Validation for Drugs and Biologics Final Guidance for Industry issued July 2015:

….Trend analysis on method performance should be performed at regular intervals …. If an analytical procedure can only meet the established system suitability requirements with repeated adjustments …… the analytical procedure should be reevaluated, revalidated, or amended, as appropriate…

Traditional system of Method performance monitoring: Control Charts

Keytruda: many labs (MSD, CROs, Dev labs, QC labs: DS/DP release + stab)all using 1 set of methods (e.g. some for DS + DP) and 1 SRS

Assay Monitoring Tool 1 Validated system combining System Suitability data and lab notebook entries

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Overview of Assay Monitoring Tool

The Assay Monitoring Tool (AMT) is a validated global, web-based intranet application based on Spotfire, which extracts, visualizes and performs basic statistical analysis on data from the Manufacturing Integration and Intelligence (MII) database.

MIIdatabase

• Provides instant access to the data in a form of interactive control charts• Statistical calculations, generation of validated reports

To enable early detection of trends indicating potential assay problems

AMT Scope

Method performance trending• System Suitability Test (SST) and

control sample results are monitored

• Further information as e.g. analyst, column and equipment are captured and can be used as part of an investigation to identify root causes

Out ofScope

Product trending • Release or Stability results are not

monitored

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Monitored Tests and Labs for Keytruda: DS + DP + Dev

Test # of Labs

Peptide Map (Lys-C digestion) 7Size-Exclusion Chromatography 10Capillary Electrophoresis Non-Reduced 10Capillary Electrophoresis Reduced 10Ion Exchange Chromatography (IEX) 10Hydrophobic Chromatograph (HIC) 10Protein Content assay (UV) 9Potency ELISA 5Residual protein A 4Residual Host Cell CHO protein 3Residual Exogenous DNA (QPCR) 4Osmolality 5Polysorbate-80 6Moisture 2

Data entered for IEX RunGeneral Run info: 10 itemsLab ID, Date of Test, Experiment ID, Instrument ID,HPLC Column #, SRS Lot #, SST sol. Lot #, Analyst ID,Run Status (Valid / Invalid), Run Failure Reason (A-F)

SRS results: 15 parameter valuesMean area% + RSD% area% of Main + 6 charge variantsRetention Time main peak (RSD% min.)SST Control Sample result: 1 parameter valueMean area%: Chart Var 5

Data entered for Osmolality RunGeneral Run info: 7 itemsLab ID, Date of Test, Experiment ID, Instrument ID, Analyst ID, Run Status T (Valid / Invalid), Run Failure Reason (A-F)

Reference Solution results: 2 parametersAccuracy of 200/300 mOsmol/kgRSD for reference solution (RSD% mOsmol/kg)

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AMT for Method Performance MonitoringExample for 1 lab: SEC

AMT

Data is within control limits, method performance is under control

Nelson rule 2: 9 points in a row on the same

side of the central line (mean shift) obtained in

4 labs x 6 runs

1 lab selected onlyRefSt

Validatedexport

UCL

LCL

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AMT for Method Performance MonitoringExample for 7 labs: CESDS-NR

AMT

Data is within control limits, method performance is under control

• CMO 1, • CMO 2, • MSD Lab 1, • MSD Lab 2, • MSD Lab 3, • MSD Lab 4, • MSD Lab 5

obtained in4 labs x 6 runs

Note: ≠ SSTlimits in method

UCL

LCL

Data gathered using AMT: Valuable data on SRS and Control Samples

Examples of data gathered for previous SRS; in use Dec 2016 – Jun 2020:

- HP-IEX: 375 successful runs, in 9 labs

Data on Area % of Main peak + 6 charge variants in SRS

- Potency ELISA: 2054 successful runs, in 5 labs

Data on Potency % CS vs SRS, EC50, Curve Slopes,

3 batches Control Sample, numerous Ligand lots, PD-1 coating lots, etc

Trending: Lab vs instrument/analyst, Lab vs Lab, Lab vs time, Lab vs instrument, Reference

method / lab performance SRS / Control Sample performance 10

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Extended Scope 1: Evaluation of Reference Standards

T= 0

T= 1

yr

T= 2

yr

T= 3

yr

Traditional stability-evaluationRe

spon

se

100%

L Spec

U Spec

Number of runs based on σ of method

Performed in 1 lab, Stored in 1 location

Influence of reagents, equipment, etc unknown

Evaluation Control Sample for IEX

Used as SST solution in IEX method: Sample with elevated Chart Variant%

100 µL single use vials. Yearly re-evaluation

Method description:

SRS12

SST Control sampleSRS (lower)

Re-evaluation based on comparing results from runs in 5 labs with runs of Qualification

2.4

2.6

2.8

3.0

3.2

3.4

3.6

μ₀=3.000

LCL=2.400

UCL=3.600

Evaluation Control Sample for IEX

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decl value + 3σ• actual result duringRelease or Stability testingDS or DP (178 runs, 5 labs)

decl value - 3σ

obtained inqualification

(≥ 6 runs)

1 year

Data is within limits, Control sample can be re-qualified

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Extended Scope 2: SRS Stability Monitoring

T= 0

T= 1

yr

T= 2

yr

T= 3

yr

Traditional Stability Monitoring of SRSSR

S vs

PRS

100%

L Spec

U Spec

Number of runs based on σ of method

Performed in 1 lab, Stored in 1 location

Influence of reagents, equipment, etc unknown

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SRS Stability monitoring: IEX; yearly Re-evaluation

% P

eak

Area

1 year

obtained inqualification

(4 labs x 6 runs)

• CMO 1, • CMO 2, • MSD Lab 1,• MSD Lab 2, • MSD Lab 3

No trends visible for any peak over all labs,SRS can be re-qualified

Note; trending done for many tests

+ 3σ

- 3σ

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SRS Stability monitoring: Potency: CS vs SRS

T= 0

T= 1

year

T= 2

yea

r

T= 3

yea

r

Actual results CS

vs S

RS

Performed in 5 labs, Stored in 5 location

Influence of reagents, equipment, people,etc included

No trends visible, SRS can be re-qualified

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SRS Stability monitoring: Potency: combinationT=

0

T= 1

year

T= 2

yea

r

T= 3

yea

r

Dec

201

6

Oct

2017

Aug

2018

Jun

2019

Apr 2

020

Traditional Stability Monitoring of SRS Stability Monitoring of SRS with AMT

SRS

vs P

RS

CS v

s SRS

(Pot

ency

%)

• CRO 1• MSD Lab 1• MSD Lab 2• MSD Lab 3• MSD Lab 4

• T=0, • T= 2yr <-15°C, • T= 2yr <-60°C

No trends visible, SRS can be re-qualified

Future enhancements AMT

- All digital data entry (from digital notebooks)

- When digital; more data

- Enhanced data analysis

- New system “PDM”;

combining Product + Analytical performance data

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Thank You