Host Cell Protein Analysis by Mass Spectrometry. Originally presented at the 2018 Sciex Users Meeting by Michael J Nold, Ph.D., Mass Spectrometry Core Facility at KBI Biopharma.

1. a customer and science-focused
contract development & manufacturing organization
HOST CELL PROTEIN ANALYSIS
BY MASS SPECTROMETRY
Michael J Nold, Ph.D.
Director, Mass Spectrometry Core Facility

2. • Challenges with HCPs
• HCP Spectral Library Generation
• Process Clearance Monitoring
HCP– Analysis by Mass Spectrometry
Overview
2

3. HCP– Analysis by Mass Spectrometry
Challenges with HCPs
• HCPs are impurities generated when recombinant
proteins are manufactured. Recombinant proteins are
frequently expressed in host cells from various cell
lines.
• Mammalian, Microbial, Bacterial, Yeast, Plant, Insect
• Residual HCPs may affect product quality, safety and
efficacy.
• Toxicity, immune response to target, adjuvant effect, augment
ADA response, bioactivity, product stability, product potency
• Risks from a variety of factors
• Dosing and dose frequency, patient demographics, route of
administration

4. HCP– Analysis by Mass Spectrometry
Challenges with HCPs
Many Challenges:
• Variability of host cell substrates
• Large number of protein analytes
• Variability of protein population during upstream processing
• Parental cell line, cell age, cell viability, feeding strategy, etc.
• Dynamic range difference between DP and HCPs ( ≥e6 )
• Detection of HCPs that are not immunogenic à Immunoassay
may miss these
• HCP population can change during process development
• HCPs may copurify with DP – “Hitchhiker” effect

5. HCP– Analysis by Mass Spectrometry
Challenges with HCPs
Mass Spectrometry Challenges:
• Column capacity, sample loading
• DP overload on column
• Mass spectrometer sensitivity
• Dynamic range for ion detections
• False positives, negatives
• Ion statistics for identification, quantitation
• Speed – Data to results to support process development
• Does library reflect the process?
• Time to generate a new library

6. KBI HCP Reagents and Applications
PHYSICAL
CHEMICAL
ATTRIBUTE #1
PHYSICAL
CHEMICAL
ATTRIBUTE #3
PHYSICAL
CHEMICAL
ATTRIBUTE #2
6

7. HCP– MS Workflow Overview
LCMS via ACQUITY/6600 TripleTOF
Spectral Library Generation Sample Analysis
Data Processing and Library Searching
Data
Independent
Acquisition
SWATH MS
Data
Dependent
Acquisition
LC-MS/MS
CHO HCCF
Various
Separation
Methodologies
Protein Level
Additional
Separation/
Digest
Peptide
Level
Spectral
Library
1% FDR
Various
Separation
Methodologies
Protein Level
Additional
Separation/
Digest
Peptide
Level
List of
identified
proteins and
peptides
Spectral
library
matching
based on
set search
criteria and
thresholds
Sample
7

8. HCP– MS Workflow Overview
LCMS via ACQUITY/6600 TripleTOF
Spectral Library Generation Sample Analysis
Data Processing and Library Searching
Data
Independent
Acquisition
SWATH MS
Data
Dependent
Acquisition
LC-MS/MS
CHO HCCF
Various
Separation
Methodologies
Protein Level
Additional
Separation/
Digest
Peptide
Level
Spectral
Library
1% FDR
Various
Separation
Methodologies
Protein Level
Additional
Separation/
Digest
Peptide
Level
List of
identified
proteins and
peptides
Spectral
library
matching
based on
set search
criteria and
thresholds
Sample
7

9. HCP Spectral Library Generation
Sample Preparation on the Protein and Peptide Level
Process
Samples
1D Analysis - No peptide level fractionation
Process
Samples
2D Analysis - High pH RP-HPLC peptide level fractionation
Fraction 3
Fraction 2
Fraction 1
Fraction 4
Fraction 5
Peptide level fractionation of 5 of
the 8 protein fractionated samples
PHYSICAL
CHEMICAL
ATTRIBUTE #1
PHYSICAL
CHEMICAL
ATTRIBUTE #3
PHYSICAL
CHEMICAL
ATTRIBUTE #2
8

10. HCP Spectral Library Generation
Sample Load Optimization
• Load Optimization Goal:
achieve a dynamic range of
≥ 3 logs at the protein level for
each experiment.
• Utilize the ITC (ion
transmission control) Count
Conversion of the TOF MS TIC
to determine the appropriate
amount on column: optimized
load when the TOF MS TIC
apex is between 10-20%.
• ITC: dynamically adjusts
percent of the ion beam
(voltage gate) according to an
observed TIC signal to prevent
saturation.
• ITC Count Conversion plots the
ITC voltage gate response:
no signal / gate fully open = 100%
saturation / gate fully closed = 0%
TOF MS TIC – SWATH
ITC Count Conversion of the TOF MS TIC – SWATH
TOF MS TIC Apex
9

11. 1.E+04
1.E+06
1.E+08
0 10 20 30 40 50
ProteinArea
Protein Number
Log Plot of Protein Dynamic Range
HCP Spectral Library Generation
Sample Load Optimization – Depth of Database
• ADH: spiked in all samples at 300 fmol/µL**, used as a system suitability test and
the signal response for relative quantitation.
• PCM: spiked in all samples at 100 fmol/µL**, used as a retention time calibrator
allowing for retention time alignment on any chromatographic system or timescale.
** Concentration of ADH and PCM is relative to 2 µg/µL of total HCP concentration.
XIC - Yeast Alcohol Dehydrogenase (ADH)
XIC - Peptide Calibration Mix (PCM)
Graph generated using the HCPs identified in sample S3 to
conceptually show the dynamic range of identified HCPs.
10

12. HCP Spectral Library Generation
IDA Library Data Collection and Processing
ProteinPilot™ data processing
w/ Paragon™ Algorithm.
IDA spectral library
Filter IDA spectral library
based on acceptance
criteria.
• Acceptance Criteria for Library:
• 1% FDR
• No modifications or clipped peptides
• Minimum of 2 peptide identifications per
protein
• Peptide Confidence Level: 95%
• 10 ppm mass error tolerance
• 2/2 identification replication
Acquire IDA data in duplicate for
each of 28 HCP samples/fractions.
11

13. HCP Spectral Library Generation
Contributions from 3 physiochemical preparations & fractionation
Physiochemical
Attribute 1
1850 Proteins
Physiochemical
Attribute 2
2925 Proteins
Physiochemical
Attribute 3
2137 Proteins
3802 Proteins
114
304
1047
598
976
385
378

14. • HCPs are monitored from samples
collected along a purification process,
either for process development
feedback, or monitoring a final process
for one or more batches.
• Samples are analyzed in triplicate, and
identifications will have to meet a set of
criteria at both peptide and protein
levels.
• Results include the list of identified
HCPs, and relative quantitative
information.
• As an illustration, samples were
obtained from the last four points of
sampling from one of our programs.
Protein A
Viral Inactivation
AEX* Based Polishing
(Flow Through mode)
CEX* Based Polishing
Viral Filtration
UF/DF
Clarified Harvest
*May be mixed mode
S3
S4
S5
S6
HCP Process Clearance Monitoring
Stand Alone, ELISA Support, or Process Development Support
7193 ppm
125 ppm
10 ppm
4 ppm
12

15. SWATH data file
HCP Ion
Library
Selected
Protein
Selected
Peptide
Acceptance Criteria
• 2 Peptides per protein
• 4 transitions per peptide
• FDR: 1.0%
• Peptide Confidence
Threshold > 99%
• MS/MS ppm: 20.0 ppm
• Score > 2
• 2/3 identification replication
SWATH
MS/MS
Library IDA
MS/MS
XIC – SWATH
MS/MS
SWATH Data File Processing in PeakView® SWATH™ microapp
Note: Protein selected is HCP ID 83
HCP Process Clearance Monitoring
Data Processing and Library Searching – Acceptance Criteria
13

16. 83541324
TOF MS of
sample S3
indicating the
retention times of
identified HCP ID
83, 324, and 541.
S3 S4 S5 S6
HCP 83
HCP 324
HCP 541
XICs of
identified
HCPs
Note: XICs are generated from the sum of the top four transitions per peptide.
HCP Process Clearance Monitoring
Sample Analysis
14

17. Library ID S3 S4 S5 S6
48 100.00 4.67 2.74 ND
58 100.00 37.61 4.98 ND
83 100.00 1.26 0.19 ND
301 100.00 3.77 6.03 ND
324 100.00 3.83 0.64 0.76
541 100.00 32.97 30.47 31.28
672 100.00 6.83 3.36 ND
HCP Process Clearance Monitoring
HCP Clearance in Percentage-Step 3 through Step 6
HCPs present in S5 shown
17
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
100.00
S3 S4 S5 S6
%
Timepoint
HCP's Clearance in Percentage
48 58 83 301 324 541 672

18. Library ID S3 S4 S5 S6
48 5.74E+05 2.68E+04 1.58E+04 ND
58 6.25E+05 2.35E+05 3.11E+04 ND
83 1.27E+07 1.64E+06 2.38E+04 ND
301 3.81E+05 1.43E+04 2.30E+04 ND
324 2.17E+06 8.28E+04 1.38E+04 1.65E+04
541 1.87E+05 6.16E+04 5.70E+04 5.85E+04
672 3.18E+05 2.17E+04 1.07E+04 ND
HCP Process Clearance Monitoring
HCP Clearance in Area-Step 3 through Step 6
HCPs present in S5 shown
18
1.00E+04
1.00E+05
1.00E+06
1.00E+07
S3 S4 S5 S6
ProteinArea
Timepoint
Log Plot of HCP's Clearance in Area
48 58 83 301 324 541 672

19. HCP- Quantitation
Proof of Concept – Determining Performance Expectation
Six digested proteins were spiked into a mAb digest.
Dilutions were carried out maintaining [mAb].
*MS Qual/Quant QC Mix from Sigma-Aldrich P/N MSQC1
20
Values are ppm, 30 µg of mAb was loaded on column

20. 11.6 ppm 2.32 ppm
HCP- Quantitation
Proof of Concept: Carbonic Anhydrase I, VLDALQAIK
0.464 ppm 0.0928 ppm
21

21. 62 ppm 12.4 ppm
HCP- Quantitation
Proof of Concept: NAD(P)H dehydrogenase, EGHLSPDIVAEQK
2.48 ppm 0.496 ppm
22

22. 46 ppm 9.2 ppm
HCP- Quantitation
Proof of Concept: C-reactive Protein, ESDTSYVSLK
1.84 ppm 0.368 ppm
23

23. 24 ppm 4.8 ppm
HCP- Quantitation
Proof of Concept: Catalase, NLSVEDAAR
0.96 ppm 0.192 ppm
25

24. HCP- Quantitation
Proof of Concept - Determining Performance Expectation
Range established to sub ppm levels
Green = Confirmed; Red = Not Confirmed
Values are ppm, 30 µg of mAb was loaded on column
26

25. • CHO Database in place containing 4 CHO cell lines
• 3802 Proteins
• Generation of new HCP databases within ~2-3 months
• Identifications of HCPs for process monitoring can be
made < 1 ppm, and in some cases <100 ppb
•
• Thank you SCIEX for your support!
HCP– Analysis by Mass Spectrometry
SUMMARY
27