Application of a Mass Spectrometry Based Multi-Attribute-Method (MAM) for QC Release and Stability Testing of Protein Therapeutics

Tamer Eris, Principal Scientist Process DevelopmentAmgen, Thousand Oaks, CACASSS CMC Strategy Forum July 18th, 2016

For Internal Use Only. Amgen Confidential. 2

Key Drivers for a Multi Attribute Method (MAM)

• MAM allows for direct monitoring of relevant product quality attributes rather than indirect measurement by conventional methods

• MAM has capability for new peak detection

Attribute focused Process Development

and Quality Control

• MAM has potential for reduced number of assays for process development, product disposition, and improving cycle time

• Potential for modality independent , platform based method

Reduce Cost of Development and

Quality Control

• Application of MAM method to process development and characterization embraces QBD principles

Quality-By-Design (QBD)

For Internal Use Only. Amgen Confidential. 3

Identification and Selection of the Critical Quality Attributes is Key for Application of MAM

Target Product Profile Product Quality Attribute Assessments

Quality Target Product Profile (QTPP)

• Indication & use

• Dosage & administration

• Tolerability

• Dosage forms & strength

• Efficacy

• Safety/side effects

• Value & access

• Attribute definition

• Product quality attribute

assessment

• Potential impact for

safety/efficacy

• Critical quality attribute

selection

• Attribute range

determination

• Attribute focused

molecule & process

design & development

The QTPP guides product quality characteristics prospectively

Definition: A prospective summary of the quality characteristics of a drug product that ideally will be achieved to ensure the desired quality, taking into account safety and efficacy of the drug product

The quality target product profile forms the basis of design for the development of the product.

Considerations for the Quality Target Product Profile (QTPP) could include:

• Intended Use in Clinical Setting, Route of Administration, Dosage Form, Delivery Systems

• Dosage Strength(s)

• Container Closure System

• Therapeutic Moiety Release or Delivery and Attributes affecting Pharmacokinetic Characteristics Appropriate to the Drug Product Dosage Form being Developed

• Drug Product Quality Criteria (e.g. sterility, purity, stability, and drug release) Appropriate for the Intended Marketed Product

Drug Substance and Dug Product are in Scope of QTPP

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QTPP: PQA Ranges can be designed into the Product during Development

Category DS Attributes Target Range Ranges Achieved

Strength Concentration 126 – 154 mg/mL 131 – 149 mg/mL

Quality

Asp Isomerization ≤ 2% 0.1 – 0.5%

Trp Oxidation ≤ 5% 0.1%

Met Oxidation ≤ 5% 0.3 – 0.9%

Met Oxidation ≤ 5% 0.4%

Met Oxidation 1% – 7% 2.5 – 4.1%

Met Oxidation ≤ 5% 0.7 – 1.6%

High Mannose Glycans 2% – 12% 6.2 – 8.5%

Protein Dimer/Oligomers (SEC HMW) ≤ 1% 0.4 – 0.6%

Protein Fragmentation (rCE LMW+MMW) ≤ 1% < 0.6%

Glycation (LC K) ≤ 5% 0.8 – 1.5%

Hydroxylysine (HC K) ≤ 2% < 0.1%

Hydroxylysine (HC K) ≤ 4% 1.0 – 2.0%

Osmolality 250 – 350 mOsm/kg 301 – 312 mOsm/kg

Polysorbate 80 0.005% – 0.015% 0.009 – 0.013%

pH 4.9 – 5.5 5.1 – 5.2

Safety

Host Cell Protein ≤ 100 ppm 20 – 49 ppm

Residual Protein A < 6 ppm < 1 ppm

Endotoxin ≤ 0.25 EU/mg ≤ 0.0022 EU/mg

Bioburden ≤ 10 CFU/10 mL 0

MAM is a Peptide Map based on Mass Spectrometry that measures several Attributes in a single Assay

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• Denature the sample• Reduce and alkylate• Desalt• Digest with trypsin • Inject the digest• LC/MS analysis

DenatureProteins

Protein Sample Total Ion Chromatogram

Extracted Ion Chromatograms

• Quantitation of modification of a peptide in CDR of the monoclonal antibody by reduced and alkylated Lys-C map

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Area (Modified pep.Peak)% Modified pep. =

[Area(Modified pep. Peak)+Area (Unmodified pep. Peak)]x100

Quantification of Targeted attribute using Conventional Peptide Map with UV Detection

Modified peptide

Unmodified peptide

Conventional Peptide Map is Used for …

Applications for Conventional maps:• Targeted quantification

• Sequence verification

• Sequence variants

• PTM and glycan characterization

• Degradation pathways 8

UV Profile

Challenges for Conventional map:• Purity of peaks under UV trace

• Insufficient selectivity

Confirm Identity by MS

Principles of Mass Spectrometry for Mass Determination and Relative Quantitation

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Calculate the monoisotopic mass of peptides:

MS spectra of modified peptide @ 33.27 min

Calculate the peak area of modified peptide

%𝑴𝑴𝑴𝑴𝑴𝑴 =𝑷𝑷𝑷𝑷𝑷𝑷𝑷𝑷 𝑨𝑨𝑨𝑨𝑷𝑷𝑷𝑷𝑴𝑴𝑴𝑴𝑴𝑴

𝑷𝑷𝑷𝑷𝑷𝑷𝑷𝑷 𝑨𝑨𝑨𝑨𝑷𝑷𝑷𝑷𝑴𝑴𝑴𝑴𝑴𝑴 + 𝑷𝑷𝑷𝑷𝑷𝑷𝑷𝑷 𝑨𝑨𝑨𝑨𝑷𝑷𝑷𝑷𝑼𝑼𝑼𝑼𝑼𝑼𝑴𝑴𝑴𝑴

300 400 500 600 700 800 900 10000

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

838 839 840 841 842 843m/z

0

20

40

60

80

100

Rela

tive

Abun

danc

e

2+

3+

0.5

2+

Calculate the peak area of unmodified peptide

𝑴𝑴𝑴𝑴𝑼𝑼𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴 𝑴𝑴𝑷𝑷𝑴𝑴𝑴𝑴 = (𝑼𝑼/𝒛𝒛 − 𝑯𝑯+) × 𝒛𝒛

m/z

31 32 33 34 35Time (min)

0

1000

1000

1000

1000

100

Rel

ativ

e Ab

unda

nce

0100

0100

0

100

Quantification of Deamidated Peptides without MS Resolution requires Chromatography

10 05/01/2013

GFYPSDIAVEWESNGQPENNYK

Iso Asp-393

Asp-398Asp-397

NativeIso Asp-398

Iso Asp-397 Asp-393

All Control and Data Analysis Components are in a Single Compliant Package

Chromeleon 7.2 SR2CFR Title 21 Part 11 Compliant Package

New Peak Detection

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Attribute Quantitation

Instrument Control

Chromeleon 7.2 SR 2.0

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2

4

Software navigation

panel

Preview of integration of the selected peptide

Data Analysis quantification-

Pinpoint

Isotopic fingerprinting

/peptide I.D

1 2 3 4

12

Software: New Peak Detection

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Binary comparison of test sample and a reference standard starts with base peak alignment of the tryptic digest

SIEVE is used to detect all of the frames (peaks)

Reference StandardTest Sample New peak detected in the spiked sample

Reference Standard

SIEVE is able to detect new peaks

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Chromeleon 7.2 SR2.0: Electronic Reporting

Main Peak

Acidic Peaks Basic Peaks

MAM provides selective and specific monitoring of biologically relevant attributes

Multi attribute method (mAb-A)Conventional purity methodCEX HPLC profile (mAb-A)

• Traditional chromatography-based purity methods often generate individual peaks that contain multiple components

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0 5 10 15 20 25 30 35 40 45 50 55 60 65 70Time (min)

0102030405060708090

17.1 26.3 27.9 29.913.0

9.9 13.5 40.022.211.0 44.025.314.1 49.832.89.4 37.2 52.116.6 59.49.0

57.7

42.85.2 37.618.236.18.7 65.814.8 61.15.8 32.4 47.239.418.5 62.033.923.6 54.0 55.6 66.6

2.06E9TIC MS amg228_crtl_110547

Acidic Peaks

BasicPeaks

Main Peak

Sample % Acidic % Main % Basic

mAb A 10 77 13

Attribute Residue Peptide Structural Region %

Deamidation N53 LC 3 LC CDR1 6.2

Oxidation

M39 H3 HC CDR1 0.2

M113 H12 HC CDR3 3.8

M253 H23 Fc 1.9

M431 H44 Fc 0.9Mannose glycan N301 H21 Fc 10.2

Clips D276/P277 H24 Fc 0.3

• MAM enables detection and quantification of specific product quality attributes.

Criteria for Evaluating a Peptide or Attribute using the Multi Attribute MethodDevelopment1. Identification of the

peptide/attribute is confirmed by MS2 fragmentation + orthogonal characterization methods (HILIC-MS for glycosylation)

2. The retention time window for the peptide/attribute is defined

3. Set appropriate filters and threshold for new peak in Sieve

Execution1. The retention time for the

peptide/attribute must be within a set retention time window (determined by characterization of the molecule)

2. The experimental mass is less than 5 ppm from the predicted mass

3. The experimental isotopic distribution fit to the theoretical must meet pre defined criteria

4. Apply filters and Threshold for new peak detection

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Comparison of Traditional Glycan Map and MAM –Excellent Agreement for Mab Fc Glycans

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EU

0.00

50.00

100.00

150.00

200.00

250.00

Minutes3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00

A2G0F

A2G1F

A2G2FA2G0A1G0 M5A1G0F

A2G1A1G1F

A2G1 M6 M7 M8

0.0%

20.0%

40.0%

60.0%

MAM

HILICGlycan map

MAM Provides ID Information– Read out of Product Specific CDR Peptides Ensures Specificity

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VH

CH1VL

CL

CH2

CH3

Glycosylation

C-Terminal LysPro Amidation

Oxidation

Oxidation

Disulfide variants

DeamidationTruncation

Isomerization Cleavage

Product Clipping: MAM vs. Reduced CE-SDS

% Clipped SpeciesSample rCE MAM*4C, T= 0 0.33 0.49

4C, T=1 Yr 0.44 0.794C, T= 2 Yrs 1.00 1.27

25C, T=26 wks 2.49 3.0840C, T=4 wks 1.87 2.39

40C, T=1 3wks 4.92 6.31

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0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

4C, T= 0 4C, T=1Yr 4C, T=2Yrs

25C,T=26wks

40C,T=4wks

40C,T=13wks

rCE

MAM*

Relative levels of Clips by MAM (H9DP + H18DP clips) and reduced CE-SDS (LMW + MMW species) are in agreement

MAM has potential to Replace Several Non-attribute Specific Assays

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Purpose Current DS Release Method Strategy

Purity - Clips rCE-SDS

Multi-Attribute Method

Purity – Charge Variants CEX-HPLC

Glycans Glycan Map

Identity Immunoassay

Process Impurities Protein-A-ELISA

VH

CH1VL

CL

CH2

CH3

Glycosylation

C-Terminal LysPro Amidation

Oxidation

Oxidation

Disulf ide variants

Deamidation

Truncation

Isomerization Cleavage

MS BasedPeptide Map

toQuantifySpecificCQAs

Multi Attribute Method has the potential to replace several Methods and Provide more Detailed Information

QUALIFICATION/VALIDATION OF MAM

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Advancing Highly Sensitive Analytical Technology

When utilizing highly sensitive methods, one has to assure that you have extensive knowledge of method capability:

• Reproducibility/Ruggedness• System Suitability• Robustness

• Sample prep conditions• Chromatography conditions• MS conditions

• Factors that Affect Results• Sensitivity, Interference, Critical method parameters

• Identification of Species• Data Interpretation• Relationship to other Methods

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Qualification Design Aligned with ICH Guidelines for Purity Methods

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Benefits of the Single Multi Attribute Method for Release:

• Automated quantification combining Orbitrap mass spectrometry technology and dedicated software for routine application in QC environment

• Science-based• Provides direct measurements of attributes of criticality captured in the QTPP • Scientifically superior to current methods (CE-SDS, CEX-HPLC, UV based

Peptide Maps). • Reduces number of release tests with the benefit of more detailed

product information

• Flexibility• Method allows for continuous input resulting from increased knowledge of the

drug attributes during process development and better understanding of their criticality from clinical experience

• Modality independent Universal Method

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PacifiChem 2015, Technical Program #242 25

Acknowledgements• Jette Wypych• Izydor Apostol• Rohini Deshpande• Sabrina Benchaar• Da Ren• Quanzhou Luo• Wenzhou Luo• Robert Hung• Helen Chung• Le Zhang• Zhongqi Zhang• Pavel Bondarenko• Suminda Hapuarachchi• Greg Flynn• Janet Cheetham• Richard Wu

• Tony Mire-Sluis• Mike Abernathy• Jason Hampson• Wendy Laderach• Armineh Stone• Rich Rogers• Nancy Nightlinger• Amanda Miller• Alain Balland• Bob Bailey• Catherine Eakin• Dean Pettit• Brent Kendrick• Amol Prakash• Scott Peterman• Jenifer Sutton• Christoph Nickel• Ryo Komatsuzaki

Q&A

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