Host Cell Protein Analysis using Enrichment Beads Coupled with Limited Digestion

Summary

A protocol is presented for enriching host cell proteins (HCPs) from drug products (DP) and detecting peptides using proteome enrichment beads. The method is demonstrated using an in-house manufactured monoclonal antibody (mAb) drug substance (DS), which is a well-characterized reference material for evaluating and comparing different methods in terms of performance.

Abstract

Host cell proteins (HCPs) are impurities that can adversely affect therapeutic proteins, even in small quantities. To evaluate the potential risks associated with drug products, methods have been developed to identify low-abundance HCPs. A crucial approach for developing a sensitive HCP detection method involves enriching HCPs while simultaneously removing monoclonal antibodies (mAbs) before analysis, utilizing liquid chromatography-mass spectrometry (LC-MS).

This protocol offers detailed instructions for enriching host cell proteins using commercially available proteome enrichment beads. These beads contain a diverse library of hexapeptide ligands with specific affinities for different proteins. The protocol also incorporates limited digestion and subsequent peptide detection using nano LC-MS/MS. By employing these techniques, HCPs with low abundance can be enriched over 7000-fold, resulting in an impressive detection limit as low as 0.002 ppm. Significantly, this protocol enables the detection of 850 HCPs with a high level of confidence using a NIST mAb. Moreover, it is designed to be user-friendly and includes a video demonstration to assist with its implementation. By following these steps, researchers can effectively enrich and detect HCPs, enhancing the sensitivity and accuracy of risk assessment for drug products.

Introduction

Host cell proteins (HCPs) are impurities that are released from the cell culture of the host organism and co-purified with monoclonal antibody (mAb)^1,2,3,4. Trace levels of HCPs can negatively impact the quality of the drug product^{5,6,7,8,9,10,11,12,}

Protocol

Abbreviations used in the protocol are listed in Supplementary Table 1.

1. Preparation of solutions and buffers

NOTE: The commercial details of all the reagents are listed in the Table of Materials.

1. Prepare 0.1 M Tris-HCl, pH 8.0 solution by adding 1 mL of 1 M Tris-HCl, pH 8.0 into 9 mL of deionized water in a glass vial, and mix well by vortexing. Store at 4 °C for up to 3 months.
2. Prepare.......

Discussion

There are two versions of commercially available protein enrichment beads: one with a smaller capacity and the other with a larger capacity (see Table of Materials). Both versions of the enrichment beads contain ten preps in the package. The manufacturer's instructions suggest that each prep from the small capacity kit can be used to enrich 10 mg of total protein. However, for optimal performance of host cell protein (HCP) enrichment from DS, each prep is good for five DS samples. Therefore, each kit can.......

Materials

Name	Company	Catalog Number	Comments
16 G, Metal Hub Needle, 2 in, point style 3	Hamilton	91016
Acclaim PepMap 100 C18 trap column (20 cm × 0.075 mm)	Thermo Fisher	164535
Acetonitrile	Fisher-Scientific	A955
Acetonitrile with 0.1% Formic Acid (v/v), Optima LC/MS Grade	Fisher-Scientific	LS120-4
Amicon Ultra-0.5 Centrifugal Filter Unit	Millipore Sigma	UFC5010
C18 analytical column (0.075 mm × 1.7 μm × 30 cm, 100 Å)	CoAnn Technologies	HEB07503001718I
Centrifuge 5424	Eppendorf	5405000646
Dithiothreitol (DTT)	Thermo Fisher	A39255
Frit for SPE cartridges, 9.5 mm, 3 mL, 100/pk	Agilent	12131020
GL-Tip GC	GL Sciences Inc	7820-11201
in-house mAb	Regeneron		concentration 200 mg/mL
Iodoacetamide (30 x 9.3 mg)	Thermo Fisher	A39271
Isopropanol	Fisher-Scientific	149320025
L-Histidine	Sigma Aldrich	H6034
L-Histidine monohydrochloride monohydrate	Sigma Aldrich	53370
Methanol	Fisher-Scientific	A456-4
Milli-Q	Millpore	30035
NanoDrop 2000	Thermo Scientific	ND-2000
Orbitrap Exploris 480	Thermo Fisher	BRE725539
Protein LoBind Tube 0.5 mL	Eppendorf (VWR)	22431064
Protein LoBind Tube 2.0 mL	Eppendorf (VWR)	22431102
Proteome Discoverer software 2.4	Thermo Scientific
ProteoMiner Protein Enrichment Large-Capacity Kit	Bio-Rad	1633007
ProteoMiner Protein Enrichment Small-Capacity Kit	Bio-Rad	1633006
Sodium deoxycholate (SDC)	Sigma Aldrich	D6750
Sodium lauroyl sarcosinate (SLS)	Sigma Aldrich	L5777
SpeedVac	Labconco	7970010
Thermomixer R	Eppendorf	22670107
Trifluoracetic acid (TFA)	Fisher-Scientific	28904
Trypsin (Sequencing Grade Modified)  (5 x 20 ug)	Promega	V5111
Tube Revolver Rotator	Thermo Fisher	88881001
UltiMate 3000 RSLC nano system	Thermo Fisher	ULTIM3000RSLCNANO
UltraPure 1 M Tris-HCl pH 8.0	Thermo Fisher	15568-025
Vortex Genie 2	VWR	102091-234
Water with 0.1% Formic Acid (v/v), Optima LC/MS Grade	Fisher-Scientific	LS118-4