High Coverage Process Specific HCP Identification and Quantification Using Mass Spectrometry

Caprion Biosciences Inc, Montreal, Canada Laura McIntosh, John Babetas, Laetitia Cortes, Rudolf Guilbaud , Stephane Parent, Michael Schirm, Lorella Di Donato

BACKGROUND

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

• “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

Mass Spectrometry Platform Features and Applications for HCP

HCP

Identification

Extensive Fractionation •Enables specific detection of low level HCP in presence of high DS concentration

•Increased sensitivity for coverage of low level HCPs

LC-MS/MS Analysis •DS and in-process samples

•Process-specific custom HCP database for comprehensive HCP identification

•Relative quantitation of identified HCPs

HCP Quantification

Multiplexed MRM Assay •HCPs selected from previous HCP identification studies, ‘problematic’ HCPs

•Assay developed using isotope-labeled standards for specific and accurate performance

Assay qualification •Absolute quantitation of individual HCPs (ppm)

•LLOQ, ULOQ, CV, recovery

• Monitoring of purification process • Demonstration of HCP Clearance • Compare culture media, process improvements • Evaluation of batch reproducibility and scale-up

Applications for Process Development and Manufacturing

Digestion of proteins to peptides (Trypsin enzyme)

Extensive peptide fractionation (Two orthogonal methods)

LC-MS/MS analysis (High resolution Q Exactive mass spectrometer)

1. Electronic Data Report: List of identified HCPs, including spectral counts for relative quantification.

2. Written Report: Summary study design, methods, results discussion and conclusions.

LC-MS/MS Identification & Relative Quantification of HCP

Identification of HCP (Match detected peptides to HCP sequences in

corresponding HCP database)

Harvest In-process

step 1 In-process

step 2 …

In-process step N

DS

Highly characterized, defined mixture of ~50 human proteins spiked with and without DS. Process blank also used.

Monitoring of general MS instrument sensitivity, reproducibility and stability

Assessment of impact of DS on sensitivity

Routinely obtain LOD range 1-10ppm

Deliverables:

Process Quality Controls (PQC)

Custom Database

Client process-specific sequences

DS sequences

Host proteome (ex: CHO, E.coli, human, yeast)

Process-specific additives

Caprion-specific sequences

PQC protein mixture and associated additives

Common laboratory contaminants

• Monoclonal Ab • Recombinant protein • Fusion proteins • Peptide drugs

HCP IDENTIFICATION

Extensive Fractionation Provides Increased HCP Coverage

CHO: 53 HCPs E.coli: 36 HCPs

Yeast: 31 HCPs Human: 25 HCPs

• Degree of HCP coverage shown for different DS in various host systems (protein level) using two orthogonal fractionation methods; this approach typically provides 30-50% more peptide and 5-20% more protein identifications

Fractionation Method 1

Fractionation Method 2

Method 1 2

For this example, the increased HCP coverage at the peptide level provides increased confidence at the protein identification level

32 2 2 11 14 28

7 24 24 92 48 67 19 25

Demonstration of HCP Clearance During DS Purification • Use of mass spectrometry shows a decreasing number and concentration of HCPs across the purification

process. Protein (A) and spectra (B) data shown.

A B

Comparison of HCP content in Biosimilars vs Innovators • Data shown from two experiments comparing Biosimilars vs Innovators using either different replicates

(A) or lots (B).

A B

• Data shows successful clearance of two individual HCPs across the purification process

• Two peptides from the same protein yield similar results

0

100

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900

Step 1 Step 2 Step 3 DrugProduct

ppm

(ng

HCP

/ m

g D

S)

Purification Step

HCP Protein 1

Peptide 1

Peptide 2

0

20

40

60

80

100

120

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Step 1 Step 2 Step 3 DrugProduct

ppm

(ng

HCP

/ m

g D

S)

Purification Step

HCP Protein 2

Peptide 1

Peptide 2

Quantitative Assessment of HCP Clearance

*Sensitivity of detection routinely achieved in the 1ppm range

• Prioritize list of HCPs • Select ≤5 surrogate

peptides per HCP

MSAIQAAWPSGTECIAKYNFHGTAEQDLPFCKGDVLTIVAVTKDPNWYKAKNKVGREGIIPANYVQKREGVKAGTKLSLMPWFHGKITREQAERLLYPPETGLFLVRESTNYPGDYTLCVSCDGKVEHYRIMYHASKLSIDEEVYFENLKMQLVEHYTSDADGLCTRLIKPKVMEGTVAAQDEFYRSGWALNMKELKLLQTIGKGEFGDVMLGDYRGNKVAVKCIKNDATA…

• Use synthetic isotope-labeled peptides to develop LC-MRM/MS assay conditions

Targeted Multiplexed LC-MRM/MS Assay Development

inte

nsi

ty

RT

• Final optimized assay monitors 2 fragments (transitions) per peptide

HCP protein sequence, trypsin digestion

Peptide detection (MS) Fragmentation Fragment detection (MS/MS)

HCP QUANTIFICATION (ABSOLUTE)

Caprion’s mass spectrometry-based platforms routinely achieve sensitivity in

the 1-10ppm range for both HCP identification and absolute quantitation

Caprion’s 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 different host expression systems

• Comparability studies of Biosimilars to Innovators

• Evaluation of manufacturing process robustness

For more information, please send inquiries to: info@caprion.com

SUMMARY

Absolute Quantification of HCP using LC-MRM/MS

No light peptide spike (endogenous only)

Spike light peptides: (Low, Mid, High)

Desalt / MRM analysis

Endogenous levels (Concentration back-calculated using

calibration curve)

Spike light peptides: (≥7 non-zero standards)

Calibration curve (Peak area ratio as a function of nominal

concentration)

Digestion

Study samples (Individual DS)

Precision and Accuracy (Concentration back-calculated using

calibration curve)

Calibration curves (BSA or pooled DS)

Spike SIL peptides (fixed concentration)

QC samples (pooled DS)

• Experimental workflow