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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
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