Name: Detecting Low-Abundant Host Cell Proteins in Drug Products Using Enrichment Beads and Limited Digestion (Video) | JoVE
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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.
<ol>
	<li>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 &#176;C for up to 3 months.</li>
	<li>Prepare...

Enrichment of Host Cell Proteins in Monoclonal Antibody Drug Products

<ol>
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H., Xiao, H., Daly, T., Li, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+host+cell+protein+analysis+in+monoclonal+antibody+products+through+molecular+weight+cutoff+enrichment.">Improved host cell protein analysis in monoclonal antibody products through molecular weight cutoff enrichment.</a> Analytical Chemistry. 92 (5), 3751-3757 (2020).</li><li>Chen, I. H., Xiao, H., Li, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+host+cell+protein+analysis+in+monoclonal+antibody+products+through+ProteoMiner.">Improved host cell protein analysis in monoclonal antibody products through ProteoMiner.</a> Analytical Biochemistry. 610, 113972 (2020).</li><li>Doneanu, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+detection+of+low-abundance+host+cell+protein+impurities+in+high-purity+monoclonal+antibodies+down+to+1+ppm+using+ion+mobility+mass+spectrometry+coupled+with+multidimensional+liquid+chromatography.">Enhanced detection of low-abundance host cell protein impurities in high-purity monoclonal antibodies down to 1 ppm using ion mobility mass spectrometry coupled with multidimensional liquid chromatography.</a> Analytical Chemistry. 87 (20), 10283-10291 (2015).</li><li>Huang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+sample+preparation+for+shotgun+proteomics+characterization+of+HCPs+in+antibodies.">A Novel sample preparation for shotgun proteomics characterization of HCPs in antibodies.</a> Analytical Chemistry. 89 (10), 5436-5444 (2017).</li><li>Johnson, R. O., Greer, T., Cejkov, M., Zheng, X., Li, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+of+FAIMS,+Protein+A+depletion,+and+native+digest+conditions+enables+deep+proteomic+profiling+of+host+cell+proteins+in+monoclonal+antibodies.">Combination of FAIMS, Protein A depletion, and native digest conditions enables deep proteomic profiling of host cell proteins in monoclonal antibodies.</a> Analytical Chemistry. 92 (15), 10478-10484 (2020).</li><li>Kreimer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+cell+protein+profiling+by+targeted+and+untargeted+analysis+of+data+independent+acquisition+mass+spectrometry+data+with+parallel+reaction+monitoring+verification.">Host cell protein profiling by targeted and untargeted analysis of data independent acquisition mass spectrometry data with parallel reaction monitoring verification.</a> Analytical Chemistry. 89 (10), 5294-5302 (2017).</li><li>Madsen, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+the+complete+characterization+of+host+cell+proteins+in+biotherapeutics+via+affinity+depletions,+LC-MS/MS,+and+multivariate+analysis.">Toward the complete characterization of host cell proteins in biotherapeutics via affinity depletions, LC-MS/MS, and multivariate analysis.</a> MAbs. 7 (6), 1128-1137 (2015).</li><li>Nie, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+sensitive+method+for+deep+profiling+of+host+cell+proteins+in+therapeutic+antibodies+by+combining+ultra-low+trypsin+concentration+digestion,+long+chromatographic+gradients,+and+boxcar+mass+spectrometry+acquisition.">Simple and sensitive method for deep profiling of host cell proteins in therapeutic antibodies by combining ultra-low trypsin concentration digestion, long chromatographic gradients, and boxcar mass spectrometry acquisition.</a> Analytical Chemistry. 93 (10), 4383-4390 (2021).</li><li>Yang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+LC-MS-Based+workflow+with+robust+0.1+ppm+sensitivity+for+identifying+residual+HCPs+in+biotherapeutic+products.">Versatile LC-MS-Based workflow with robust 0.1 ppm sensitivity for identifying residual HCPs in biotherapeutic products.</a> Analytical Chemistry. 94 (2), 723-731 (2022).</li><li>Zhang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+tracking+of+host+cell+proteins+during+monoclonal+antibody+purifications+using+mass+spectrometry.">Comprehensive tracking of host cell proteins during monoclonal antibody purifications using mass spectrometry.</a> MAbs. 6 (3), 659-670 (2014).</li><li>Zhang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Putative+phospholipase+B-Like+2+is+not+responsible+for+polysorbate+degradation+in+monoclonal+antibody+drug+products.">Putative phospholipase B-Like 2 is not responsible for polysorbate degradation in monoclonal antibody drug products.</a> Journal of Pharmaceutical Sciences. 109 (9), 2710-2718 (2020).</li><li>Zhang, J., He, J., Smith, K. 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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...

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 product5,6,7,8,9,10,11,12,13,14,15, and therefore, a sensitive HCP analysis method is desired to detect HCPs in sub-ppm to ppm levels.
Orthogonal methods can be applied to detect HCPs in low abundance. Enzyme-linked immunosorbent assay (ELISA) is generally used to quantitate overall HCPs, and it can also detect and quantitate individual HCPs if the corresponding antibodies are available16. However, the production of HCP-specific antibodies is time-consuming and labor-intensive. In contrast, liquid chromatography coupled with mass spectrometry (LC-MS) can provide comprehensive information about individual HCPs in mAb drug products and is widely applied for HCP identification4,7,9,10,12,13,14,15,17,18,19,20,21,22,23,24,25,26,27.
Several methods have been developed to detect HCPs with LC-MS/MS, including limited digestion20, filtration17, Protein A deletion21, immunoprecipitation (IP), and ProteoMiner enrichment (PM)18. Most methods aim to reduce the amount of mAb and enrich HCPs prior to LC-MS/MS analysis, thereby decreasing the dynamic range between mAb peptides and HCP peptides. This protocol presents a proteomic sample enrichment method that combines ProteoMiner technology and limited digestion (PMLD)28. The ProteoMiner enrichment principle involves using commercially available proteome enrichment beads containing a diverse library of combinatorial peptide ligands. These ligands specifically bind to proteins on antibody-drug products, allowing for the removal of excess molecules while concentrating low-abundance host cell proteins (HCPs) on their respective affinity ligands. On the other hand, the principle of limited digestion involves using a low concentration of trypsin. This concentration is sufficient to digest low-abundance HCPs but not enough to digest all antibody drug products. This approach enables the recovery and enrichment of digested HCP peptides from the solution.
Compared to filtration methods, the PMLD technique is not limited by the size of the detected HCPs17. Protein A deletion methods are specific to detecting HCPs associated with antibodies21, while immunoprecipitation is restricted to predefined HCPs from a particular cell line (such as the Chinese Hamster Ovary (CHO) cell line), where an anti-HCP antibody was generated4. In contrast, PMLD can be applied to detect HCPs from any drug modules and host cell proteins co-purified with drug products from various cell lines. Additionally, PMLD exhibits better sensitivity compared to the mentioned methods17,18,20,21,24.
This approach can enrich the HCP concentration by 7000-fold and lower the detection limit to 0.002 ppm28. The experimental setup is illustrated in Figure 1.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>16 G, Metal Hub Needle, 2 in, point style 3</td>
			<td>Hamilton</td>
			<td>91016</td>
			<td></td>
		</tr>
		<tr>
			<td>Acclaim PepMap 100 C18 trap column (20 cm &#215; 0.075 mm)</td>
			<td>Thermo Fisher</td>
			<td>164535</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Fisher-Scientific</td>
			<td>A955</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile with 0.1% Formic Acid (v/v), Optima LC/MS Grade&#160;</td>
			<td>Fisher-Scientific</td>
			<td>LS120-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-0.5 Centrifugal Filter Unit</td>
			<td>Millipore Sigma</td>
			<td>UFC5010</td>
			<td></td>
		</tr>
		<tr>
			<td>C18 analytical column (0.075 mm &#215; 1.7 &#956;m &#215; 30 cm, 100 &#197;)</td>
			<td>CoAnn Technologies</td>
			<td>HEB07503001718I</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5424</td>
			<td>Eppendorf</td>
			<td>5405000646</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)&#160;</td>
			<td>Thermo Fisher</td>
			<td>A39255</td>
			<td></td>
		</tr>
		<tr>
			<td>Frit for SPE cartridges, 9.5 mm, 3 mL, 100/pk</td>
			<td>Agilent</td>
			<td>12131020</td>
			<td></td>
		</tr>
		<tr>
			<td>GL-Tip GC</td>
			<td>GL Sciences Inc&#160;&#160;</td>
			<td>7820-11201</td>
			<td></td>
		</tr>
		<tr>
			<td>in-house mAb</td>
			<td>Regeneron</td>
			<td></td>
			<td>concentration 200 mg/mL</td>
		</tr>
		<tr>
			<td>Iodoacetamide (30 x 9.3 mg)</td>
			<td>Thermo Fisher</td>
			<td>A39271</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher-Scientific</td>
			<td>149320025</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Histidine</td>
			<td>Sigma Aldrich</td>
			<td>H6034</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Histidine monohydrochloride monohydrate</td>
			<td>Sigma Aldrich</td>
			<td>53370</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher-Scientific</td>
			<td>A456-4&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q</td>
			<td>Millpore</td>
			<td>30035</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000</td>
			<td>Thermo Scientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbitrap Exploris 480</td>
			<td>Thermo Fisher</td>
			<td>BRE725539</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind Tube 0.5 mL</td>
			<td>Eppendorf (VWR)</td>
			<td>22431064</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind Tube 2.0 mL</td>
			<td>Eppendorf (VWR)</td>
			<td>22431102</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteome Discoverer software 2.4</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ProteoMiner Protein Enrichment Large-Capacity Kit</td>
			<td>Bio-Rad</td>
			<td>1633007</td>
			<td></td>
		</tr>
		<tr>
			<td>ProteoMiner Protein Enrichment Small-Capacity Kit</td>
			<td>Bio-Rad</td>
			<td>1633006</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate (SDC)</td>
			<td>Sigma Aldrich</td>
			<td>D6750</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium lauroyl sarcosinate (SLS)&#160;</td>
			<td>Sigma Aldrich</td>
			<td>L5777</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeedVac</td>
			<td>Labconco</td>
			<td>7970010</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermomixer R</td>
			<td>Eppendorf</td>
			<td>22670107</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoracetic acid (TFA)</td>
			<td>Fisher-Scientific</td>
			<td>28904</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin (Sequencing Grade Modified)&#160; (5 x 20 ug)</td>
			<td>Promega</td>
			<td>V5111</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube Revolver Rotator</td>
			<td>Thermo Fisher</td>
			<td>88881001</td>
			<td></td>
		</tr>
		<tr>
			<td>UltiMate 3000 RSLC nano system</td>
			<td>Thermo Fisher</td>
			<td>ULTIM3000RSLCNANO</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure 1 M Tris-HCl pH 8.0</td>
			<td>Thermo Fisher</td>
			<td>15568-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Genie 2</td>
			<td>VWR</td>
			<td>102091-234</td>
			<td></td>
		</tr>
		<tr>
			<td>Water with 0.1% Formic Acid (v/v), Optima LC/MS Grade&#160;</td>
			<td>Fisher-Scientific</td>
			<td>LS118-4&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

host cell protein analysis using enrichment beads coupled with limited digestion

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.

Host Cell Protein Analysis using Enrichment Beads Coupled with Limited Digestion

This protocol presented a sample preparation workflow, termed protein enrichment coupled with limited digestion (PMLD), for the analysis of host cell proteins (HCPs) in a monoclonal antibody (mAb) sample. Figure 1 illustrates the step-by-step procedure of PMLD. The researchers compared the results of HCP analysis using direct digestion (shown in the top panel of Figure 2) and PMLD (shown in the bottom panel of Figure 2). The Total I...

Watch this Scientific Journal Video about Detecting Low-Abundant Host Cell Proteins in Drug Products Using Enrichment Beads and Limited Digestion at JoVE.com

Detecting Low-Abundant Host Cell Proteins in Drug Products Using Enrichment Beads and Limited Digestion (Video) | JoVE

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.