Implementation of a Quantitative Mass Spectrometry Multi-Attribute

Method (MAM) for Characterization, Quality Control Testing and Disposition of Biotherapeutics

Rich Rogers

Successful implementation of the MAM requires 3 software components

1.Automated attribute monitoring

2.Automated purity test

3.Compliant software framework

Outline

• Current attribute testing (single attribute testing)

• Multi-Attribute Method

–Workflow

–Attribute Analytics

–Mass Spec based new peak detection

• Fully compliant hardware and software package for QC release of biotherapeutics

–Meets 21 CFR part 11 requirements

• Summary

• Acknowledgements

Single attribute testing used to ensure product quality

Just. | Confidential.

1. CE-SDS

2. CEX/cIEF

3. Glycan Map

4. ID ELISA

5. HCP ELISA

6. pA ELISA

7. Color

8. Clarity

9. Osmolality

10. pH

11. SEC

12. A280

13. qPCR

14. Endotoxins

15. Bioburden

16. Bioassay

Mass Spec based Multi-Attribute Method (MAM) for Product Attribute Control (PAC) and Release

Attribute Current Method PAC and Release

Clips rCE-SDS

Multi-Attribute

Method

Charge Variants CEX-HPLC

Glycans Glycan Map

Identity Immunoassay

Process ImpuritiesHCP-ELISA,

Prot-A-ELISA

Workflow the MAM

30 minute

Digest

LC-MS/MS

Characterization

Search

AlgorithmResults

LC-MS1 only

MonitoringTargeted and

untargeted peak

detection

Compliant method

30 min Tryptic digest—Ren, D. et. al. Anal Biochem. 2009 Sep 1;392(1):12-21

MAM—Rogers, R. S. et. al. mAbs 2015 Sep 3;7(5):881-90

Targeted Attribute Analytics

Attribute Analytics using Pinpoint

Analytical Group

1. CQAs are observed by a DDA method (orbi

or QE)

2. Enter the peptides

3. Add PTMs

4. Add the isotopic distribution

Attribute Analytics Results

Comparison to traditional assays—Glycans

Comparison to traditional assays—Deamidation

0

5

10

15

20

25

30

35

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

%A

ttri

bu

te

Day of Culture

CEX (Acidic)

Deamidation

Comparison to traditional assays—Clips

0

20

40

60

80

100

120

100 99 95 90 50 0

%Cl

ips

%ABP970

Expected Level

rCE-SDS (%Clips corrected)

MAM

% unclipped mAb

Attributes that can be monitored with the MAM

• Deamidation

• Glycation

• Glycosylation

• Oxidation

• Pre-monomer

• Clips

• Unusual Glycosylation

• C-term K

• HCP and pA

• Mutations

• NGHC

• Isomerization

• Pyro-Glu

Mass Spec based new peak detection

• Absolutely essential for releasing biotherapeutics from QC

• Automated new peak detection is more sensitive than current purity tests employed by QC

• Incredibly powerful tool for detecting new critical quality attributes (revealed during stability assessment)

• Use of filters limits false positives

Sieve Workflow

Base Peak Alignment

Frame/Peak Detection

Sieve Filters

1. Only allow monoisotopic peaks

2. Peaks must have at least 2 isotopes

3. Charge states +2 through +4

4. Max Change greater than 10

We detected zero new peaks when we used SIEVE to compare duplicate digests of

the same mAb.

New Peak Detection

Overlay of control and 500 fmol spike

15 peptides spiked into an IgG2

SIEVE can detect peaks that co-elute or are below the visible base peak threshold.

SIEVE detected 13 of 15 peptides • 4 peptides were visible in the base peak chromatogram

• 6 peptides co-eluted with the IgG1 peptides

• 3 peptides below base peak threshold when visually inspected

• 2 peptides not detected did not meet the criteria for a 2nd isotope above LOD threshold

• 0 false positives

Reference

Standard

Test

Sample

Sieve is stability indicatingPinpoint: Spiked Oxidation Stability Samples

Pinpoint:

DTLM(oxid)ISR peptide

File name: Control Spike

1Spike

2Spike

3Spike

4Spike

5

% ox DTLMISR 1.45 2.32 3.37 4.21 5.49 23.72

Contro

l

Sp

ike 1

Sp

ike 2

Sp

ike 3

Sp

ike 4

Sp

ike 5

An IgG2 was spiked at different levels with an oxidation stressed

sample and analyzed using Pinpoint to quantify the oxidation

Samples were then analyzed by SIEVE

SIEVE is stability indicating

Pinpoint:

DTLM(oxid)ISR peptide

Compare Levels: 1.45% vs 3.37%

Sieve detects a new

peak in the Spike 2

sample when

compared to the

control.

File name: Control Spike

1Spike

2Spike

3Spike

4Spike

5

% ox DTLMISR 1.45 2.32 3.37 4.21 5.49 23.72

Control Trace

Spike 2 Trace

New Peak

Fully Compliant Software Package for MS based QC release of biotherapeutics

Chromeleon 7.2

21 CFR Part 11 Compliant Package

• Instrument control (UPLC and Mass Spec)

• Comprehensive compliance tools

• Automated data processing and reporting

• Hardware and Software can be validated

Chromeleon 7.2 Processing Method

Chromeleon 7.2 Attribute Report

Both samples pass oxidation specification!

Non-Targeted MS Processing

Reference

No New Peaks!

Summary

• Implementation of the MAM for process development and release of biotherapeutics requires 3

software components

– Automated attribute monitoring

– Automated purity test

–Compliant software framework

• The MAM is precise and can track attribute trends similar to conventional assays

• The MAM can directly monitor attributes (Man5, c-terminal K, NGHC, deamidation, and Iso D)

• Automated new peak detection using the MAM is more sensitive and robust than the conventional

purity assays

• Chromeleon 7.2 provides a fully compliant software package for attribute analytics and MS based

new peak detection to release biologics in a regulated environment

Acknowledgments

Just Biotherapeutics—Brittney Livingston and Randal Bass

ThermoFisher—Scott Peterman, Amol Prakash, Jennifer Sutton, Hongxia Wang, Tonya Second,

Kevin Wheeler, Mary Lopez, Zhiqi Hao, Betty Woo, Ryo Komatsuzaki, Christopher Nickel, and

Jonathan Josephs

Amgen Current and Alumni—Nancy Nightlinger, Sihong Deng, Amanda Miller, Jennifer Kerr, Yuling

Zhang, Becky Scott, Lowell Brady, Brittany Affholter, Quanzhou Luo, Wenzhou Li, Oleg Borisov,

Sabrina Benchaar, Armineh Stone, Jim Navratil, Jay Stimpson, Jim Bailey, Steve Cockrill, David

Basset, Vinny Browning III, Izydor Apostol, Gang Huang, Jette Wypych, Catherine Eakin, Bob

Bailey, and Alain Balland

Merck—Doug Richardson, Yi Wang, Huijuan Li, and David Pollard