Download High Coverage Process Specific HCP Identification and

High Coverage Process Specific HCP Identification and Quantification
Using Mass Spectrometry
Caprion Biosciences Inc, Montreal, Canada  [email protected]
HCP QUANTIFICATION (ABSOLUTE)
Extensive Fractionation Provides Increased HCP Coverage
Regulatory trend towards increasingly deeper HCP characterization
• Regulatory framework: 42 USC 262, ICH Q6B, ICH Q8
• Current practice of using immunoassays has well-recognized gaps; little is known about individual HCPs
• Post-market commitments for development of an HCP assay with improved coverage was required for
3 of 8 BLAs approved in 2014 due to insufficient characterization of HCPs
Mass spectrometry is playing a greater role in characterization of HCPs
• Prioritize list of HCPs
• Select ≤5 signature
peptides per HCP
Method 1  2
28
11
14
2
32
2
Fractionation Method 2
CHO: 53 HCPs
Mass Spectrometry Platform Features and Applications for HCP
• Use synthetic isotope-labeled
peptides to develop LC-MRM/MS
assay conditions
Peptide detection (MS)
HCP protein sequence, trypsin digestion
E.coli: 36 HCPs
LC-MS/MS Analysis
•Enables specific detection of low
level HCP in presence of high DS
concentration
•Increased sensitivity for coverage of
low level HCPs
•DS and in-process samples
•Process-specific custom HCP
database for comprehensive HCP
identification
•Relative quantitation of identified
HCPs
Multiplexed MRM Assay
Assay qualification
•HCPs selected from previous HCP
identification studies, may include
‘problematic’ HCPs
•Assay developed using isotopelabeled standards for specific and
accurate performance
•Absolute quantitation of individual
HCPs (ppm)
•LLOQ, ULOQ, CV, recovery
24
7
24
25
19
67
92
48
Study samples
QC samples
Calibration curves
(Individual DS)
(pooled DS)
(BSA or pooled DS)
Yeast: 31 HCPs
Human: 25 HCPs
Digestion
For this example, the increased HCP coverage
at the peptide level provides increased
confidence at the protein identification level
(endogenous only)
(Low, Mid, High)
(≥7 non-zero standards)
Desalt / MRM analysis
• Use of mass spectrometry shows a decreasing number and concentration of
HCPs across the purification process. Protein (A) and spectra (B) data shown.
Monitoring of purification process
Demonstration of HCP Clearance
Compare culture media, process improvements
Evaluation of batch reproducibility and scale-up
A
B
Endogenous levels,
Precision and Accuracy
Calibration curve
(Concentration back-calculated
using calibration curve)
(Concentration back-calculated
using calibration curve)
(Peak area ratio as a function of
nominal concentration)
Quantitative Assessment of HCP Clearance
1
• Data shows successful clearance of two individual
HCPs across the purification process
• Two peptides from the same protein yield similar results
…
Digestion of proteins to peptides
(Trypsin enzyme)
Process Quality
Controls (PQC)
In-process
step N
DS
• Monoclonal Ab
• Recombinant
protein
• Fusion proteins
• Peptide drugs
Comparison of HCP content in Biosimilars vs Innovators
• Data shown from two experiments comparing Biosimilars vs Innovators using either
different manufacturing replicates (A) or lots (B).
Extensive peptide fractionation
A
 Assessment of impact of DS on sensitivity
LC-MS/MS analysis
 Routinely obtain LOD range 5-20ppm, can
be as low as 1ppm
(High resolution Q Exactive mass spectrometer)
Identification of HCP
(Match detected peptides to HCP
sequences in corresponding HCP database)
Deliverables:
1. Electronic Data Report: List of identified HCPs,
including spectral counts for relative quantification.
1
2. Written Report: Summary
study design, methods,
results discussion and conclusions.
900
200
800
180
700
600
500
400
Peptide 1
300
Peptide 2
200
B
 Caprion-specific sequences
 PQC protein mixture and
associated additives
 Common laboratory contaminants
120
100
80
Peptide 1
60
Peptide 2
0
Step 1
Step 2
Step 3
Drug
Product
Purification Step
 Client process-specific sequences
 DS sequences
 Host proteome (ex: CHO, E.coli,
human, yeast)
 Process-specific additives
140
20
0
Custom Database
160
40
100
(Two orthogonal methods)
 Monitoring of general MS instrument
sensitivity, reproducibility and stability
HCP Protein 2
HCP Protein 1
ppm (ng HCP/ mg DS)
In-process
step 2
 Highly characterized, defined mixture of ~50
human proteins spiked with and without DS.
Process blank also used.
Spike light peptides:
Demonstration of HCP Clearance During DS Purification
LC-MS/MS Identification & Relative Quantification of HCP
In-process
step 1
Spike light peptides:
Spike SIL peptides (fixed concentration)
HCP IDENTIFICATION
Harvest
No light peptide spike
ppm (ng HCP/ mg DS)
Applications for
Process Development
and Manufacturing
•
•
•
•
RT
Fragment detection (MS/MS)
Fragmentation
Absolute Quantification of HCP using LC-MRM/MS
Extensive Fractionation
1
HCP
Quantification
• Final optimized assay
monitors two fragments
(transitions) per peptide
MSAIQAAWPSGTECIAKYNFHGTAEQD
LPFCKGDVLTIVAVTKDPNWYKAKNKV
GREGIIPANYVQKREGVKAGTKLSLMP
WFHGKITREQAERLLYPPETGLFLVREST
NYPGDYTLCVSCDGKVEHYRIMYHASKL
SIDEEVYFENLKMQLVEHYTSDADGLCT
RLIKPKVMEGTVAAQDEFYRSGWALN
MKELKLLQTIGKGEFGDVMLGDYRGNK
VAVKCIKNDATA…
Fractionation Method 1
• “Immunoassay and (increasingly) mass spectrometry are highly complementary and the most
powerful methods for monitoring residual HCP levels in samples and confirming their absence in
final DSs.” - USP 1132
HCP
Identification
Targeted Multiplexed LC-MRM/MS Assay Development
• Use of two orthogonal fractionation methods typically provides 30-50% more
HCP peptide identifications and 5-20% more protein identifications
• Venn diagrams showing degree of non-overlap from use of two different
fractionation methods on different DS in various host systems (protein level)
intensity
BACKGROUND
Step 1
Step 2
Step 3
Drug
Product
Purification Step
*Sensitivity of detection routinely achieved in the 1ppm range
SUMMARY
Caprions’ Mass Spectrometry Platform has been used for the following
types of HCP client studies:
•
•
•
•
Characterization of in-process samples and DS
Demonstration of HCP clearance
Characterization of HCP from DS expressed in various host expression systems
Comparability studies of Biosimilars to Innovators