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

Forkortelser brukt i protokollen er listet opp i tilleggstabell 1.
1. Klargjøring av løsninger og buffere
MERK: De kommersielle detaljene for alle reagensene er oppført i materialfortegnelsen.
<ol><li>Forbered 0,1 M Tris-HCl, pH 8,0-oppløsning ved å tilsette 1 ml 1 M Tris-HCl, pH 8,0 i 9 ml avionisert vann i et hetteglass, og bland godt ved vortexing. Oppbevares ved 4 °C i opptil 3 måneder.</li>
	<li>Klargj...

Enrichment of Host Cell Proteins in Monoclonal Antibody Drug Products

<ol>
	<li>Aboulaich, N. <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+approach+to+monitor+clearance+of+host+cell+proteins+associated+with+monoclonal+antibodies.">A novel approach to monitor clearance of host cell proteins associated with monoclonal antibodies.</a> Biotechnology Progress. 30 (5), 1114-1124 (2014).</li><li>Goey, C. H., Alhuthali, S., Kontoravdi, C. <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+removal+from+biopharmaceutical+preparations:+Towards+the+implementation+of+quality+by+design.">Host cell protein removal from biopharmaceutical preparations: Towards the implementation of quality by design.</a> Biotechnology Advances. 36 (4), 1223-1237 (2018).</li><li>Levy, N. E., Valente, K. N., Choe, L. H., Lee, K. H., Lenhoff, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+host+cell+protein+product-associated+impurities+in+monoclonal+antibody+bioprocessing.">Identification and characterization of host cell protein product-associated impurities in monoclonal antibody bioprocessing.</a> Biotechnology and Bioengineering. 111 (5), 904-912 (2014).</li><li>Molden, R. <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+of+commercial+therapeutic+protein+drugs+as+a+benchmark+for+monoclonal+antibody-based+therapeutic+protein+development.">Host cell protein profiling of commercial therapeutic protein drugs as a benchmark for monoclonal antibody-based therapeutic protein development.</a> MAbs. 13 (1), 1955811 (2021).</li><li>Bee, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trace+levels+of+the+CHO+host+cell+protease+cathepsin+D+caused+particle+formation+in+a+monoclonal+antibody+product.">Trace levels of the CHO host cell protease cathepsin D caused particle formation in a monoclonal antibody product.</a> Biotechnology Progress. 31 (5), 1360-1369 (2015).</li><li>Bracewell, D. G., Francis, R., Smales, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+future+of+host+cell+protein+(HCP)+identification+during+process+development+and+manufacturing+linked+to+a+risk-based+management+for+their+control.">The future of host cell protein (HCP) identification during process development and manufacturing linked to a risk-based management for their control.</a> Biotechnology and Bioengineering. 112 (9), 1727-1737 (2015).</li><li>Chiu, J., 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=Knockout+of+a+difficult-to-remove+CHO+host+cell+protein,+lipoprotein+lipase,+for+improved+polysorbate+stability+in+monoclonal+antibody+formulations.">Knockout of a difficult-to-remove CHO host cell protein, lipoprotein lipase, for improved polysorbate stability in monoclonal antibody formulations.</a> Biotechnology and Bioengineering. 114 (5), 1006-1015 (2017).</li><li>Gilgunn, 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=Identification+and+tracking+of+problematic+host+cell+proteins+removed+by+a+synthetic,+highly+functionalized+nonwoven+media+in+downstream+bioprocessing+of+monoclonal+antibodies.">Identification and tracking of problematic host cell proteins removed by a synthetic, highly functionalized nonwoven media in downstream bioprocessing of monoclonal antibodies.</a> Journal of Chromatography A. 1595, 28-38 (2019).</li><li>Graf, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+polysorbate-degrading+enzymes+in+a+monoclonal+antibody+formulation.">Identification and characterization of polysorbate-degrading enzymes in a monoclonal antibody formulation.</a> Journal of Pharmaceutical Sciences. 110 (11), 3558-3567 (2021).</li><li>Hall, T., Sandefur, S. L., Frye, C. C., Tuley, T. L., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysorbates+20+and+80+degradation+by+group+XV+lysosomal+phospholipase+A2+isomer+X1+in+monoclonal+antibody+formulations.">Polysorbates 20 and 80 degradation by group XV lysosomal phospholipase A2 isomer X1 in monoclonal antibody formulations.</a> Journal of Pharmaceutical Sciences. 105 (5), 1633-1642 (2016).</li><li>Jones, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=34;High-risk&amp;#34;+host+cell+proteins+(HCPs):+A+multi-company+collaborative+view.">34;High-risk&amp;#34; host cell proteins (HCPs): A multi-company collaborative view.</a> Biotechnology and Bioengineering. 118 (8), 2870-2885 (2021).</li><li>Li, X., 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=Identification+and+characterization+of+a+residual+host+cell+protein+hexosaminidase+B+associated+with+N-glycan+degradation+during+the+stability+study+of+a+therapeutic+recombinant+monoclonal+antibody+product.">Identification and characterization of a residual host cell protein hexosaminidase B associated with N-glycan degradation during the stability study of a therapeutic recombinant monoclonal antibody product.</a> Biotechnology Progress. 37 (3), e3128 (2021).</li><li>Zhang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+specific+causes+of+polysorbate+20+degradation+in+monoclonal+antibody+formulations+containing+multiple+lipases.">Identification of the specific causes of polysorbate 20 degradation in monoclonal antibody formulations containing multiple lipases.</a> Pharmaceutical Research. 39 (1), 75-87 (2022).</li><li>Zhang, S., 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=Degradation+of+polysorbate+20+by+Sialate+O-Acetylesterase+in+monoclonal+antibody+formulations.">Degradation of polysorbate 20 by Sialate O-Acetylesterase in monoclonal antibody formulations.</a> Journal of Pharmaceutical Sciences. 110 (12), 3866-3873 (2021).</li><li>Zhang, S., Xiao, H., Molden, R., Qiu, 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=Rapid+polysorbate+80+degradation+by+liver+carboxylesterase+in+a+monoclonal+antibody+formulated+drug+substance+at+early+stage+development.">Rapid polysorbate 80 degradation by liver carboxylesterase in a monoclonal antibody formulated drug substance at early stage development.</a> Journal of Pharmaceutical Sciences. 109 (11), 3300-3307 (2020).</li><li>Gunawan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+platform+host+cell+protein+ELISA+to+process-specific+host+cell+protein+ELISA.">Comparison of platform host cell protein ELISA to process-specific host cell protein ELISA.</a> Biotechnology and Bioengineering. 115 (2), 382-389 (2018).</li><li>Chen, I. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acids+can+induce+the+formation+of+proteinaceous+particles+in+monoclonal+antibody+formulations.">Fatty acids can induce the formation of proteinaceous particles in monoclonal antibody formulations.</a> Journal of Pharmaceutical Sciences. 111 (3), 655-662 (2022).</li><li>Uniprot1. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , (2023).</li><li>Uniprot2. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , (2023).</li></ol>

Det finnes to versjoner av kommersielt tilgjengelige proteinberikelsesperler: en med mindre kapasitet og den andre med større kapasitet (se materialfortegnelse). Begge versjonene av anrikningsperlene inneholder ti preps i pakken. Produsentens instruksjoner antyder at hver prep fra det lille kapasitetssettet kan brukes til å berike 10 mg totalt protein. For optimal ytelse av anrikning av vertscelleprotein (HCP) fra DS, er imidlertid hver forberedelse god for fem DS-prøver. Derfor kan hvert sett brukes ...

Vertscelleproteiner (HCP) er urenheter som frigjøres fra vertsorganismens cellekultur og renses sammen med monoklonalt antistoff (mAb)1,2,3,4. Spornivåer av helsepersonell kan negativt påvirke kvaliteten på legemiddelproduktet 5,6,7,8,9,10,11,12,13,14,15, og derfor er det ønskelig med en sensitiv HCP-analysemetode for å oppdage helsepersonell i sub-ppm til ppm-nivåer.
Ortogonale metoder kan brukes til å oppdage helsepersonell i lav overflod. Enzymbundet immunosorbentanalyse (ELISA) brukes vanligvis til å kvantifisere generelle helsepersonell, og det kan også oppdage og kvantifisere individuelle helsepersonell hvis de tilsvarende antistoffene er tilgjengelige16. Produksjonen av HCP-spesifikke antistoffer er imidlertid tidkrevende og arbeidskrevende. I motsetning til dette kan væskekromatografi kombinert med massespektrometri (LC-MS) gi omfattende informasjon om individuelle helsepersonell i mAb-legemiddelprodukter og er mye brukt for HCP-identifikasjon 4,7,9,10,12,13,14,15,17,18,19,20, 21,22,23,24,25,26,27.
Flere metoder er utviklet for å oppdage helsepersonell med LC-MS/MS, inkludert begrenset fordøyelse20, filtrering17, protein A-delesjon21, immunutfelling (IP) og ProteoMiner-anrikning (PM)18. De fleste metoder tar sikte på å redusere mengden mAb og berike helsepersonell før LC-MS / MS-analyse, og dermed redusere det dynamiske området mellom mAb-peptider og HCP-peptider. Denne protokollen presenterer en proteomisk prøveanrikningsmetode som kombinerer ProteoMiner-teknologi og begrenset fordøyelse (PMLD)28. ProteoMiner-anrikningsprinsippet innebærer bruk av kommersielt tilgjengelige proteomberikelsesperler som inneholder et mangfoldig bibliotek med kombinatoriske peptidligander. Disse ligandene binder seg spesifikt til proteiner på antistoff-legemiddelprodukter, noe som muliggjør fjerning av overskytende molekyler mens de konsentrerer vertscelleproteiner med lav overflod (HCP) på deres respektive affinitetsligander. På den annen side innebærer prinsippet om begrenset fordøyelse å bruke en lav konsentrasjon av trypsin. Denne konsentrasjonen er tilstrekkelig til å fordøye helsepersonell med lav overflod, men ikke nok til å fordøye alle antistoffmedikamenter. Denne tilnærmingen muliggjør utvinning og anrikning av fordøyde HCP-peptider fra løsningen.
Sammenlignet med filtreringsmetoder er PMLD-teknikken ikke begrenset av størrelsen på de påviste helsepersonell17. Protein A-delesjonsmetoder er spesifikke for påvisning av helsepersonell assosiert med antistoffer21, mens immunutfelling er begrenset til forhåndsdefinerte helsepersonell fra en bestemt cellelinje (for eksempel den kinesiske hamsterovarien (CHO) cellelinje), hvor et anti-HCP-antistoff ble generert4. I motsetning til dette kan PMLD brukes til å oppdage helsepersonell fra alle legemiddelmoduler og vertscelleproteiner som er renset sammen med legemiddelprodukter fra forskjellige cellelinjer. I tillegg viser PMLD bedre sensitivitet sammenlignet med de nevnte metodene 17,18,20,21,24.
Denne tilnærmingen kan berike HCP-konsentrasjonen med 7000 ganger og senke deteksjonsgrensen til 0,002 ppm28. Det eksperimentelle oppsettet er illustrert i figur 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

Vertscelleproteiner (HCP) er urenheter som kan påvirke terapeutiske proteiner negativt, selv i små mengder. For å evaluere den potensielle risikoen forbundet med narkotikaprodukter, er det utviklet metoder for å identifisere helsepersonell med lav overflod. En avgjørende tilnærming for å utvikle en sensitiv HCP-deteksjonsmetode innebærer å berike helsepersonell samtidig som monoklonale antistoffer (mAbs) fjernes før analyse, ved bruk av væskekromatografi-massespektrometri (LC-MS).
Denne protokollen gir detaljerte instruksjoner for berikelse av vertscelleproteiner ved bruk av kommersielt tilgjengelige proteomanrikningsperler. Disse perlene inneholder et mangfoldig bibliotek av heksapeptidligander med spesifikke affiniteter for forskjellige proteiner. Protokollen inneholder også begrenset fordøyelse og påfølgende peptiddeteksjon ved bruk av nano LC-MS / MS. Ved å bruke disse teknikkene kan helsepersonell med lav overflod berikes over 7000 ganger, noe som resulterer i en imponerende deteksjonsgrense så lav som 0,002 ppm. Denne protokollen gjør det mulig å oppdage 850 helsepersonell med høy grad av sikkerhet ved hjelp av en NIST mAb. Videre er den designet for å være brukervennlig og inkluderer en videodemonstrasjon for å hjelpe til med implementeringen. Ved å følge disse trinnene kan forskere effektivt berike og oppdage helsepersonell, noe som øker følsomheten og nøyaktigheten av risikovurdering for legemidler.

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.

Author Spotlight: Detecting Low-Abundant Host Cell Proteins in Drug Products Using Enrichment Beads and Limited Digestion 

Vertscelleproteinanalyse ved hjelp av anrikningsperler kombinert med begrenset fordøyelse

Denne protokollen presenterte en arbeidsflyt for prøvepreparering, kalt proteinberikelse kombinert med begrenset fordøyelse (PMLD), for analyse av vertscelleproteiner (HCP) i en monoklonalt antistoff (mAb) prøve. Figur 1 illustrerer den trinnvise prosedyren for PMLD. Forskerne sammenlignet resultatene av HCP-analyse ved hjelp av direkte fordøyelse (vist i topppanelet i figur 2) og PMLD (vist i bunnpanelet i figur 2). TIC-profile...

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

En protokoll presenteres for berikelse av vertscelleproteiner (HCP) fra legemiddelprodukter (DP) og påvisning av peptider ved bruk av proteomanrikningsperler. Metoden er demonstrert ved hjelp av et egenprodusert monoklonalt antistoff (mAb) legemiddelstoff (DS), som er et godt karakterisert referansemateriale for å evaluere og sammenligne ulike metoder med hensyn til ytelse.

Host cell proteins (HCPs) are impurities that can adversely affect therapeutic proteins, even in small quantities. To evaluate the potential risks ...

Vi har utviklet en svært sensitiv metode for å oppdage de med lav rikelig vertscelleprotein fra legemiddelproduktet ved hjelp av proteomanrikningsperlene. Denne tilnærmingen hjelper oss å finne årsaken og risikoen forbundet med legemiddelproduktet. Det er flere måter å forbedre deteksjonsgrensen for HCP-analyse, den ikke-målrettede metoden, inkludert, men ikke begrenset til, protein A-uttømming, begrenset fordøyelse, molekylvektavskjæring, immunutfelling.Den målrettede måten å oppdage vertscelleproteinet på, inkludert væskekromatografi, overvåking av flere reaksjoner og parallell reaksjonsovervåking av ikke-væskekromatografi. Den største utfordringen forbundet med disse HCP-analysene er det signifikante dynamiske området mellom antistoffmedikamenter og vertscelleproteiner. Så de fleste metoder tar sikte på å redusere mengden antistoff og berike HCP før LCMS-analyse for å redusere det dynamiske området mellom antistoffpeptider og HCP-peptider.Så ProteoMiner-teknologien og den begrensede fordøyelsesprotokollen forbedrer deteksjonsgrensen for HCP-analyse betydelig sammenlignet med andre metoder. Det er ikke-partisk sammenlignet med immunutfelling og kan brukes på forskjellige medikamentmoduler sammenlignet med protein A-uttømmingsmetode. Denne protokollen er også enkel og grei.Så ProteoMiner-teknologi og begrenset fordøyelsesprotokoll hjelper oss med å identifisere flere helsepersonell som nedbryter polysorbat og fragmentantistoffmedikamentet. I kombinasjon med nano LCMPRM-metoden kan anrikningsmetoden også gi kvantitative resultater for å finne den biologiske relevante konsentrasjonen av problematiske vertscelleproteiner.