Use of Mass Spectrometry for High Coverage Process Specific HCP Identification and Quantitation

Caprion Biosciences Inc, Montreal, CanadaChristina Bell, John Babetas, Laetitia Cortes, Vanessa Diniz Atayde, Rudolf Guilbaud, Stéphane 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

Fractionation (OPTIONAL)• Enhances 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 identification

• Relative quantitation of identified HCPs

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

Applications:PROCESS DEVELOPMENTPROCESS CHARACTERIZATION PROCESS VALIDATION

HCP QUANTITATION

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

Digestion of proteins to peptides(Trypsin enzyme)

Extensive peptide fractionation (OPTIONAL: 2 Orthogonal methods X 8 fractions each)

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

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

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

LC-MS/MS Identification & Relative Quantitation of HCP

Identification of HCP (Match detected peptides to corresponding sequences in process-specific HCP database)

Harvest In-process

step 1In-process

step 2…

In-process step N

DS

• Process blank

• UPS2 (Sigma), a proteomics dynamic range standard set comprised of 6 mixtures of 8 proteins with a dynamic range of 5 orders of magnitude (50 pmoles to 500 amoles)

• UPS2 spiked with DS

Monitoring of general MS instrument sensitivity, reproducibility and stability

Assessment of impact of DS on sensitivity

Routinely obtain LOD range 1-10 ppm

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• Vaccines

HCP IDENTIFICATION

Sensitivity of HCP Identification Using LC-MS/MS• Mock DS sample consisting of SILu™Lite SigmaMAb Universal Antibody Standard (Sigma) spiked 500:1

with UPS2 dynamic range protein standard set, injected on a high resolution Q Exactive in triplicate

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

• Two peptides from the same protein yield similar results

0

100

200

300

400

500

600

700

800

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

140

160

180

200

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 1 ppm range

• Prioritize list of HCPs • Select ≤5 surrogate

peptides per HCP

MSAIQAAWPSGTECIAKYNFHGTAEQDLPFCKGDVLTIVAVTKDPNWYKAKNKVGREGIIPANYVQKREGVKAGTKLSLMPWFHGKITREQAERLLYPPETGLFLVRESTNYPGDYTLCVSCDGKVEHYRIMYHASKLSIDEEVYFENLKMQLVEHYTSDADGLCTRLIKPKVMEGTVAAQDEFYRSGWALNMKELKLLQTIGKGEFGDVMLGDYRGNKVAVKCIKNDATA…

• Use synthetic isotope-labeled peptides to develop 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 QUANTITATION (ABSOLUTE)

➢ Mass spectrometry provides additional HCP identification andcharacterization information to complement & supplement immunoassays.

➢ Targeted mass spectrometry assays enable absolute quantitation of specificHCPs with a high level of analytical rigor, without need for antibodies.

➢ Caprion’s mass spectrometry-based platforms for HCP identification andquantitation routinely provide 1-10 ppm range sensitivity.

➢ Examples of client studies analyzed on Caprion’s platforms:

• Monitoring / identification of key clearance steps

• HCP profiling of Originator vs. Biosimilar

• Characterization of process validation run reproducibility

• Purification process optimization / resin re-use

• Characterization of HCP in clinical vs. Commercial process

• Vaccine HCP characterization

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

SUMMARY

Absolute Quantitation 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

• Experimental workflow

• A comparative analysis of in-process samples and formulated bulk DS with wide dynamic range incomposition and abundance. Data show: [A] total HCP readout in ppm (similar to ELISA) via directmeasurement of constituent HCPs, and [B] identification of individual HCPs, their relative abundance andclearance thereof.

LC-MS/MS Enables Identification and Relative Quantitation of Individual HCPs

* Top 50 of 79 HCP shown

[B] HCP Composition & Abundance

966 ppm

336 ppm

109 ppm

19 ppm

15 ppm

79 HCP*

13 HCP

6 HCP

2 HCP

2 HCP

(ppm) (Relative abundance)

[A] Total HCP

UPS2

(Protein_ID)

Spiked Level

(ppm)

No peptide

fractionation

Optional peptide

fractionation

ALBU_HUMAN 626.01 Yes Yes

CAH2_HUMAN 274.67 Yes Yes

CAH1_HUMAN 271.11 Yes Yes

LEP_HUMAN 152.43 Yes Yes

HBB_HUMAN 149.69 Yes Yes

HBA_HUMAN 142.70 Yes Yes

UBIQ_HUMAN 99.97 Yes Yes

CO5_HUMAN 80.78 Yes Yes

CATA_HUMAN 56.25 Yes Yes

SUMO1_HUMAN 36.62 Yes Yes

NQO1_HUMAN 29.00 Yes Yes

PRDX1_HUMAN 20.73 Yes Yes

PPIA_HUMAN 19.03 Yes Yes

MYG_HUMAN 16.09 Yes Yes

CYB5_HUMAN 15.12 Yes Yes

EGF_HUMAN 5.99 No Yes

SYHC_HUMAN 5.49 No Yes

KCRM_HUMAN 4.07 No Yes

NQO2_HUMAN 2.44 No Yes

RETBP_HUMAN 1.99 No No

UBC9_HUMAN 1.70 No Yes

LYSC_HUMAN 1.39 No Yes

LALBA_HUMAN 1.33 No Yes

NEDD8_HUMAN 0.86 No Yes