Application of a Mass Spectrometry Based Multi-Attribute ... · ≤ 10 CFU/10 mL. 0: MAM is a...
Transcript of Application of a Mass Spectrometry Based Multi-Attribute ... · ≤ 10 CFU/10 mL. 0: MAM is a...
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
5
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
6
• 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
7
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
9
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
11
Attribute Quantitation
Instrument Control
Chromeleon 7.2 SR 2.0
13
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
13
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
14
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
15
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
16
Comparison of Traditional Glycan Map and MAM –Excellent Agreement for Mab Fc Glycans
17
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
18
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
19
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
20
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
21
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
22
Qualification Design Aligned with ICH Guidelines for Purity Methods
23
----
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
24
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
26