Amy J Claydon1, Phil Widdowson1, Andrew Williamson2

1Thermo Fisher Scientific, Runcorn, UK. 2Thermo Fisher Scientific, Hemel Hempstead, UK

Reproducible chromatographic performance enables low ppm LC-MS detection of Host Cell Proteins (HCPs) in biotherapeutics

Peptides were separated by UHPLC using a ThermoScientific™ Acclaim™ VANQUISH™ C18 UHPLC column(250 x 2.1 mm, 2.2 µm) over a 90 min linear gradient of 3%- 35% B (mobile phase A: water, 0.1% formic acid, mobilephase B: acetonitrile, 0.1% formic acid) at a flow rate of0.30 mL/min and a column temperature of 60°C..UPS1 peptides were analysed by DDA (Top 10) for spectrallibrary building. Data for UPS2 and monoclonal antibodieswas acquired by DIA. All MS was performed with a ThermoScientific™ Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ mass spectrometer. Data were acquired usingThermo Scientific™ Chromeleon™ Chromatography DataSystem (CDS) Software (v.2.7.8). Proteins were identifiedusing Biognosys Spectronaut™ X software for DIA data.DIA parameters are included in Figure 1.

RESULTS

Figure 2. Base Peak Ion (BPI) chromatogram reproducibility of UPS1 digests, analysed by DDA-MS

Peptides from UPS1 protein digestion were analysed beforeand after injection of high column loading concentrations ofnivolumab peptides. Peptide separation and retention timeswere reproducible across all injections of UPS1 (Figure 2). Thisis crucial when acquiring data to produce successful spectrallibraries for protein identification from DIA data.

ABSTRACT

Proteins released from host cells during recombinantbiotherapeutic production have unknown effects on drugsafety and efficacy, therefore, levels of residual co-purifying host cell proteins (HCPs) in the final productmust be determined. Traditional immunoassayapproaches can provide only limited information onimmunologically reactive HCPs, whereas ultra-high-pressure liquid chromatography (UHPLC) - highresolution accurate mass (HRAM) mass spectrometry(MS) enables protein specific identification andquantitation.

The major challenge with HCP monitoring by UHPLC-MS analysis is the huge intrasample dynamic rangebetween the low abundance residual HCPs and theconcentrated biotherapeutic drug product. Data-independent acquisition (DIA) MS can be used toovercome this challenge, but relies on reproduciblechromatographic separations and accurate retentiontimes across multiple replicates for protein identification.

INTRODUCTIONDuring manufacture of recombinant biotherapeutics,proteins from the host cells are secreted into the cellculture. Despite multiple purification steps, some ofthese host cell proteins (HCPs) will be present in thefinal product. Residual HCPs may have detrimentaleffects on the safety and efficacy of the drug product,therefore it a regulatory requirement that levels of co-purifying HCPs must be determined prior to lot release.

The current regulatory gold-standard for assessing HCPlevels in recombinant biotherapeutics is a sandwichimmunoassay, or enzyme-linked immunosorbent assay(ELISA). However, development of suitable anti-HCPantibody reagents often demands considerable time andmoney, and ELISA-based approaches can only providelimited information, in that they offer bulk quantitation ofHCPs that are immunologically reactive to thegenerated antibodies, with no possibility of identifyingindividual proteins.

UHPLC separation combined with HRAM-MS canidentify and quantify HCPs on a protein-protein basis,enabling individual HCP monitoring throughout thepurification stages. The major challenge is to overcomethe huge intrasample dynamic range to successfullydetect low ppm levels of residual HCPs within theconcentrated biotherapeutic drug product. In contrastwith data-dependent acquisition (DDA), whichfragments only the ‘Top N’ most abundant peptides ineach Full Scan, data-independent acquisition (DIA)fragments all peptides within defined isolation widths(typically 10 - 50 Da), acquiring MSMS data for all m/zvalues within the specified mass range.

The highly complex fragmentation spectra produced byDIA requires a high-resolution mass spectrometer andspecialist software for data analysis to correctlyassociate fragment ion (MS2) data with their respectiveprecursor ions. Protein identification often relies on alibrary, created by DDA of an equivalent sample with theproteins of interest at a much higher concentration i.e.harvested culture medium or the post Protein-A fraction.Successful characterisation of the residual-HCPstherefore relies on reproducible chromatographicseparations and accurate retention times acrossmultiple replicates.

MATERIALS AND METHODSThe Universal Proteomics Standard Set (UPS1) andProteomics Dynamic Range Standard Set (UPS2)(Sigma-Aldrich) were proteolyzed (50 µL) at 75°C for 45min with Thermo Scientific™ SMART Digest™ Trypsin,Magnetic Bulk Resin,, using a Thermo Scientific™KingFisher™ Duo Prime Purification System, to a finalpeptide concentration of 5 ng/µL and 8.83 ng/µLrespectively.

The reference standard NISTmAb, (0.49 mg), spikedwith 0.02 µg UPS1, was proteolyzed as above, exceptunder non-denaturing conditions (37°C) for 3 hr (Figure1). Any undigested protein was then precipitated at 90°Cfor 10 min, and removed by centrifugation for 15 min at14,000 xg. A representative monoclonal antibody,nivolumab, was concentrated via buffer exchange with3000 MWCO Amicon® Ultra Centrifugal Filters toapproximately 100 µg/µL then proteolyzed at 75°C for45 min, with undigested protein removed as above.

CONCLUSIONS

Due to the complex nature of recombinant mAbs,non-denaturing digest conditions result in a largelyintact biotherapeutic that can be precipitated withheat-treatment and removed from the sample prior toMS analysis .

Protein digestion, using the SMART Digest trypsin kit,with magnetic resin, in combination with theKingFisher Duo Prime purification system, produceshighly reproducible peptide digests, under bothdenaturing and non-denaturing conditions.

The automated approach also simplifies theprocedure compared to standard in-solution proteindigestion protocols.

To bring any low abundant HCPs into the detectablerange of the mass spectrometer, and thus enabledetection of individual HCPs at the low ppm level inthe final drug product, a column overloading amountof biotherapeutic must be injected. Even under thesechallenging conditions, the Acclaim VANQUISH C18UHPLC column continued to deliver high efficiencyand excellent separation.

Combining the DIA data analysis algorithms inSpectronaut X and its integrated search engine,Pulsar, with HRAM Orbitrap data enables high proteincoverage and comprehensive data mining foridentification of low abundance proteins within abiotherapeutic drug product, using both a denaturingand non-denaturing digestion approach.

Identification of HCPs in a biotherapeutic drugproduct requires a suitable spectral library to begenerated. This requires samples of the drug productbefore the final polishing steps, such as the harvestcell culture fluid (HCCF) or post-Protein A fraction.Optimization of false discovery rates (FDR) statisticswill also be essential.

REFERENCES1. USP 41 Published General Chapter <1132>

Residual Host Cell Protein Measurement inBiopharmaceuticals. May 2018

2. Huang, et al. A Novel Sample Preparation forShotgun Proteomics Characterization of HCPs inAntibodies. Analytical Chemistry, 2017, 89(10),5436-5444

3. Houel et al. Improving the Dynamic Range of HostCell Proteins Analysis Using an HRAM OrbitrapMass Spectrometer. ASMS Conference Poster,2018

4. Füssl et al. Comprehensive Characterisation of theHeterogeneity of Adalimumab via Charge VariantAnalysis Hyphenated On-line to Native HighResolution Orbitrap Mass Spectrometry. mAbs,2019, 11(1), 116-128

ACKNOWLEDGEMENTSWe would like to thank our customers and colleaguesin the Bio/Pharma and Product Marketing teams forsupport. We would also like to thank to the supportteam at Biognosys for their help with the Spectronautsoftware

TRADEMARKS/LICENSING© 2019 Thermo Fisher Scientific Inc. All rightsreserved. All trademarks are the property of ThermoFisher Scientific and its subsidiaries unless otherwisespecified. Spectronaut is a trademark of Biognosys AG.This information is presented as an example of thecapabilities of Thermo Fisher Scientific products. It isnot intended to encourage use of these products in anymanners that might infringe the intellectual propertyrights of others. Specifications, terms and pricing aresubject to change. Not all products are available in allcountries. Please consult your local salesrepresentatives for details.

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Figure 1. Overview schematic of HCP analysis protocol

Table 1. Retention Time RSD for nivolumab peptides across the BPI chromatogram from Chromeleon CDS

Data were acquired over two days, including multiple replicatesof UPS1 peptide separations, interspersed with replicates ofmonoclonal antibody (mAb) peptide separations that wereinjected at high column loading concentrations; 20 µL injectionscontaining equivalent of up to 150 µg mAb peptides. To ensureretention time reproducibility, any blank injections (40 min total)within the sequence maintained the same wash and re-equilibration times and chromatographic conditions as theanalytical separations (120 min total).

Despite the very high peptide load, the Acclaim VANQUISHC18 UHPLC column (250 x 2.1 mm, 2.2 µm), maintained itschromatographic performance, with excellent retention timereproducibility, across multiple replicate injections of digestedmAb (Figure 3, Table 1).

Figure 3. Mirrored BPI chromatograms of the first (upper) and last (lower) UHPLC separations of 150 µg nivolumab loaded on column, analysed by DIA-MS

NISTmAb, spiked with low ppm levels of UPS1, was analysedby DIA and the data searched against the same spectral library(Figure 5). For positive identification, each protein must beidentified with at least two unique peptides, or with the samepeptide in more than one replicate.

Figure 5. UPS1 proteins identified in NISTmAb (green bars) with respective calculated ppm (ng UPS1 protein: mg NISTmAb)

Peaks across the BPI chromatograms were integrated usingChromeleon CDS algorithms Cobra™ and SmartPeaks™ toassess their retention times and calculate the relative standarddeviation (RSD). For all integrated peptides, RSD was <0.5%,despite the very high peptide load (Table 1).

UPS2 was analysed in triplicate and data searched against thespectral library created in Spectronaut by Pulsar (Figure 4). Alow peptide load, to challenge the limit of detection (LoD) of themass spectrometer was injected. The most abundant proteinsin the UPS2 mix were loaded at 0.4 pmol/protein, withabundance decreasing in 10 fold increments to a lower limit of0.04 fmol/protein. Individual protein amount in ng wascalculated using the given molecular weights.

Figure 4. Protein coverage and associated ng loading on column of UPS2 proteins identified from DIA data

Protein AccessionAverage

Coverage#

peptidesppm

2.60% 1 2.264.30% 2 2.26

SYHC_HUMAN 3.90% 2 1.98

30% 8 1.5612.30% 4 1.566.60% 2 1.472.10% 1 1.47

GSTA1_HUMAN 6.80% 2 0.87

PRDX1_HUMAN 9.60% 2 0.75

21.60% 3 0.699.70% 2 0.699.80% 1 0.589.80% 1 0.588.80% 1 0.558.80% 1 0.5536.70% 4 0.52

17% 2 0.526.80% 1 0.506.80% 1 0.5011.90% 1 0.2511.90% 1 0.25

ALBU_HUMAN

TAU_HUMAN

MYG_HUMAN

PPIA_HUMAN

KCRM_HUMAN

IFNG_HUMAN

FABPH_HUMAN

SYUG_HUMAN

LEP_HUMAN