Enhancing Biotherapeutic Process and Product Knowledge with the Multi-Attribute Method (MAM) USP Biologics Stakeholder Forum San Francisco, January 10 th , 2020 Andrew Dawdy 1 on behalf of the Pfizer MAM Team 1,2 Biotherapeutics Pharm. Sci., Pfizer Inc, 1 St Louis, MO, USA and 2 Andover, MA, USA
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Enhancing Biotherapeutic Process and Product Knowledge with theMulti-Attribute Method (MAM)
USP Biologics Stakeholder Forum
San Francisco, January 10th, 2020
Andrew Dawdy1 on behalf of the Pfizer MAM Team1,2
Biotherapeutics Pharm. Sci., Pfizer Inc, 1St Louis, MO, USA and 2Andover, MA, USA
Outline
The benefit of MAM in Biotheraputic (BTx) Development
Development of MAM workflow for BTx Development
Implementation of MAM within Pfizer BTx Development
2
Product Quality Attributes (PQAs)
3
Quality Attribute
Efficacy(potency)
Safety /Immuno-genicity
Pharmaco-kinetics
(PK)
Criticality Assignment Matrix(CQA in red)
Severity (Harm to Patient)
Un
ce
rta
inty
(in
form
ati
on
qu
ali
ty)
10 7 5 1
10
6
4
2
Attribute understanding is clinically relevant and is foundational to a good control strategy for biotherapeutic development
ICH Q8R: CQA…should be within an appropriate limit, range or distribution to ensure the desired product quality
Analytical Testing Strategies in Biotherapeutic Development
• Release and Stability Testing
◦ Compendial Testing
◦ Content
◦ Product-related species
◦ Process-related species
◦ Potency and efficacy
• Characterization Testing
◦ Primary and Higher-Order structure
◦ Orthogonal assessments
Quality Attribute Method DS DP
Appearance Compendia √ √
Moisture, Particles, Osmolality, Reconstitution Time √
pH √ √
Protein Concentration UV √ √
Charge Isoforms iCE √ √
Heavy Chain + Light Chain CGE (reducing) √ √
Fragments CGE (reducing) √ √
Monomer CGE (nonreducing) √ √
High Molecular Mass Species HPLC SEC √ √
Peptide Profile / Identity Peptide Mapping √ √
Relative Potency Binding ELISA √ √
Glycan Fingerprint HPLC √
Endotoxin √ √
Bioburden (DS),Sterility (DP) √ √
Impurities (HCP, ProA, DNA) ELISA, qPCR √
Specification
Limits
Introduction of LC-MS-Based MAM is Revolutionizing Traditional Analytical Strategy
• Multi-Attribute Method by LC-MS
◦ The analysis of multiple product quality attributes simultaneously
◦ Automated monitoring, quantitation and detection of new peaks
• Key components of MAM
◦ Primary structure analysis, including sequence modifications
◦ Post-translational modifications
◦ Degradation mechanisms
Slide 6
MAM Consortium
• Purpose: Enable the BioPharma community to implement a robust mass spec based method for
biotherapeutic characterization and release of biotherapeutics from QC.
• Membership: >320 members from >70 companies spanning government agencies, biopharma,
mass spec vendors, software vendors, reagent vendors, and CDMOs.
HILIC – hydrophilic interaction liquid chromatography, CGE – capillary gel electrophoresis, IEX – ion exchange, iCE – imaged capillary electrophoresis
Attribute Detection & Monitoring by MAM
8
5 10 15 20 25 30 35 40 45 50 55 60 65 70
Time (min)
0
20
40
60
80
100
N-glycosylationOxidationDeamidationTerminal HeterogeneityClips and Other Modifications
Light Chain Heavy Chain
MAM is a single liquid chromatography/ mass spectrometry (LC/MS)-based method that is used to analyze multiple PQAssimultaneously. It allows for automated monitoring and quantitation of known QAs and detection of new peaks.
Pfizer Platform MAM Workflow
9
Low-Artifact Digestion
(e.g. Trypsin)
High resolution, Accurate mass
LC-MS/MS(CID/HCD/ETD)
PTM / HotspotSearch
Generate site-specific
catalog of PQAs
LC/MS Only
New peak detection
Targeted quantitation of
PQA catalog End State: A routine MS-based assay to
monitor expected and unexpected attributes via
comparative sample analysis
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
SS
C
S S
S S
NH2
NH2 NH2
NH2
COOH COOH
COOH COOH
C
Characterization
Routine Monitoring
Automatic ID and Quantitation of PQAs using:
1. Chromatographic retention time
2. HR/AM = high resolution (e.g. 140,000) /
accurate mass (<5 ppm)
3. Isotopes =12,13C; 14,15N; 16,18O; 32,33,34S
BioPharma Finder Software
Chromeleon Chromatography Data System Software Chromeleon Chromatography Data
System Software
Thermo Scientific™ Vanquish™ UHPLC System
Thermo Scientific™ Orbitrap Fusion™ Tribrid™ Mass Spectrometer
hardware at two cross-site analytical laboratories
QE+ QE+E+ E+Lab 1 Lab 2
0
5
10
15
20
25
Met Ox MAM Met Ox MAM Met Ox MAM Met Ox MAM Met Ox MAM Met Ox MAM Met Ox MAM Met Ox MAM Met Ox MAM Met Ox MAM Met Ox MAM Met Ox MAM Met Ox MAM Met Ox MAM Met Ox MAM Met Ox MAM Met Ox MAM Met Ox MAM Met Ox MAM Met Ox MAM Met Ox MAM Met Ox MAM Met Ox MAM Met Ox MAM Met Ox MAM Met Ox MAM Met Ox MAM Met Ox MAM Met Ox MAM Met Ox MAM
• Many PQAs were measured with precision over time using a mAb standard• Data provides a basis for establishment of system suitability and assay acceptance criteria
CV 7.7%
CV 9.5%
CV 4.4%
CV 18.2%
CV 3.7%
CV 14.1%
CV 0.9% CV 6.8%
CV 6.9%
CV 8.8% CV 12.8% CV 5.7%
Lab 1Lab 2
• iCE provides global charge variant information, but no specific information on identification or location of attributes
Drug Substance Batches Subjected to 40° C Stress for 18 Weeks
0
10
20
30
40
50
60
70
0 w
k
3 w
k
6 w
k
9 w
k
12 w
k
18 w
k
0 w
k
3 w
k
6 w
k
9 w
k
12 w
k
18 w
k
Process 1 Process 2%
Ab
un
da
nc
e
Acidic Species by iCE
Basic
Main0 wk
18 wk
iCE ProfileExamples of
Charge VariantsAsn deamidation
Sialylated N-GlycansN-terminal pyro Glu
C-terminal LysC-terminal amidated Pro
Lys glycation
Charge Variants: Global to Local
Acidic
Drug Substance Batches Subjected to 40° C Stress for 18 WeeksAcidic Species by MAM
0
5
10
15
20
25
30
35
40
0 w
k
3 w
k
6 w
k
9 w
k
12 w
k
18 w
k
0 w
k
3 w
k
6 w
k
9 w
k
12 w
k
18 w
k
Process 1 Process 2%
Ab
un
da
nc
e
• Increase in acidic species by iCE trends with increase observed by MAM• MAM provides ID and site-specificity for each acidic PQA
Asn 2XXG2F + NeuAc
Asn 3XXDeamidation
Acidic Basic
Main0 wk
18 wk
iCE ProfileExamples of
Charge VariantsAsn deamidation
Sialylated N-GlycansN-terminal pyro Glu
C-terminal LysC-terminal amidated Pro
Lys glycation
Asn 2XXG2F + NeuAc
Asn 3XXDeamidation
Charge Variants: Global to Local
• Traditional methionine oxidation assay (LC-UV) targets one specific site in the Fc-region• MAM quantitates multiple sites and types of oxidation in a single assay (LC-MS)
Drug Substance Batches Subjectedto 40° C Stress for 18 Weeks
1. Non-targeted analysis detects unidentified peak increasing upon thermal stress
2. Peak identified as a D/P clip within Fc region
0
1
2
3
4
0 6 12
Time (Weeks)
% A
bu
nd
an
ce
0
1
2
3
4
0 6 12
Time (Weeks)
% A
bu
nd
an
ce
0
1
2
3
4
0 6 12
Time (Weeks)%
Ab
un
da
nc
e
0
1
2
3
4
0 6 12
Time (Weeks)
% A
bu
nd
an
ce
rCGEMAM
rCGEMAM
rCGEMAM
rCGEMAM
Batch 1 Batch 2
Batch 3 Batch 4
Non-Targeted Peak Detection
5xIncrease
3. D/P clip monitoring by MAM correlates well with clipping data from traditional rCGE assay.
18 wk at 40C
0 wk at 40C
4 DS Batches Subjected to Thermal Stress (40° C)
Future Steps: Sample Prep Automation
Automation results are highly comparable to
the manual approach of sample preparation
Pfizer Confidential | 24
Hamilton Microlab STAR
IMCS SizeX 100 Tips
0 2 4 6 8 10 12
Cumulative Days for One Sample
Future Steps: Replacement of Traditional Assays
MAM has the capability to gather attribute information 5X faster than
running the traditional repertoire of assays
Pfizer Confidential | 25
MAM
Traditional
Assays
Time estimate includes sample receipt,
preparation, incubation times, instrument
setup, instrument analysis, data collection,
interpretation and archival
• Pfizer has created a functional MAM platform and the resulting data are agreeing well with the results from traditional assays
• MAM provides great benefit for biotherapeutic development by enhancing analytical process and product knowledge and efficiencies from early- through late-stage
• Upon analysis of a sufficient aggretate of side-by-side data, Pfizer plans to further increase analytical efficiencies by replacing select traditional assays with MAM
• Pfizer is moving forward with compliance and risk assessments in order to enable implementation of MAM into QC laboratories within the next 5 years