Identification of Host Cell Proteins (HCPs) in Monoclonal Antibodies At Sub-ppm Levels Using the SYNAPT XS Mass Spectrometer

In this application note we describe a highly sensitive discovery HCP assay that can identify and quantify sub-ppm levels of HCPs from a highly purified monoclonal antibody.  

Benefits

• A highly sensitive HCP discovery assay using the HDMS^E data independent acquisition (DIA) mode identifies very low levels of HCPs (down to 100 ppb) from a highly purified monoclonal antibody 
• When coupled with a literature reported sample preparation method, forty eight (48) HCPs were identified from the NIST mAb, demonstrating a considerable improvement in our ability to identify low abundance HCPs 

Residual host cell proteins (HCPs) are low-level (typically <100 ppm) process-related impurities, that might be present in protein biopharmaceuticals even after extensive purification. HCPs could produce unwanted immunogenic response in patients, reduce the efficacy or the stability of the drug or be responsible for drug degradation.¹ For these reasons, the regulatory agencies require that HCPs are identified and quantified prior to drug approval. The biopharmaceutical industry relies on ELISA assays for measuring the total HCP concentration expressed in ppm (or ng HCPs/mg biopharmaceutical). Mass spectrometry-based HCP analysis has emerged in recent years as a powerful alternative to ELISA^2-6 because it provides more extensive (proteome-wide) HCP coverage and is able to identify and measure individual HCP levels.

Sample Preparation

A highly purified mAb (NIST mAb candidate reference LRM 8670) produced in a murine cell culture was acquired from the National Institute of Standards and Technology (NIST) at a concentration of 100 mg/mL. The NIST mAb was digested using a modified protocol firstly introduced by Huang et. al from Eli Lilly & Company.³ mAb digestion was performed in a glass vial containing 100 µg of the Lys-C/Trypsin enzyme mixture (Promega, Madison, WI). A volume of 125 µL NIST mAb was mixed with 50 µL of 100 mM CaCl₂ and 625 µL of 100 mM ammonium bicarbonate. The digestion was allowed to proceed overnight (~ 16 hours) at 37 °C. The mAb sample was then denatured in the presence of 0.05% RapiGest (1 h at 60 °C), reduced in the presence of 25 mM dithiothreitol - DTT (1 h at 60 °C) and alkylated with 12 mM iodoacetamide - IAM (45 min in the dark at room temperature). Precipitation of the mAb started to occur during the alkylation step. After adding 5 µL of formic acid (FA), the digest was incubated for 30 min at 37 °C and centrifugated (10 min, 13,000 x g) to separate the precipitated mAb and the RapiGest surfactant. All digestion reagents (CaCl₂, NH₄HCO₃, DTT, IAM, and FA) were obtained from Sigma-Aldrich (St. Louis, MO). The supernatant was transferred to an autosampler vial (Waters P/N 186000327C) and five MassPREP protein digest standards were spiked at the following concentrations: 100 nM ENL (yeast enolase P/N 186002325), 40 nM ClpB (Hi3 E. coli P/N 186006012), 20 nM ADH (yeast alcohol dehydrogenase P/N 186002328), 20 nM PHO (rabbit phosphorylase b P/N 186002326), and 5 nM BSA (bovine serum albumin P/N 186002329). The amounts of each of the spiked proteins loaded on-column in 50 µL injected sample were: 5000 femtomoles ENL, 2000 fmoles ClpB, 1000 fmoles ADH, 1000 fmoles PHO and 250 fmoles BSA. LC-MS-grade solvents (acetonitrile-ACN and water) were purchased from Thermo Fisher Scientific (Waltham, MA). 

The HCP impurities contained in the NIST mAb (100 mg/mL) have been previously characterized.^2-6 Three laboratories using the same experimental setup - two dimensional microflow chromatography (high pH RP/low pH RP), coupled to a SYNAPT G2-Si Mass Spectrometer, collectively identified 35 HCPs, among which 14 HCPs were in common. This approach reached a detection limit of 1 ppm.² 

In recent years, several alternative sample preparation protocols have been introduced in order to increase the sensitivity of the LC-MS assay for identifying even lower levels of HCPs in mAb samples. These protocols either rely on the removal of the major drug species through mAb precipitation,⁴ or use low molecular-weight filters to remove the mAb and concentrate the HCPs⁵ or employ HILIC fractionation at the protein level to remove the major sample component and enrich the HCPs.⁶ Here we adapted a protocol introduced by Eli Lilly and Company⁴ to precipitate the NIST mAb, thus greatly minimizing the ion suppression and interference phenomena imposed by the more abundant NIST mAb peptides during the LC-MS analysis. Unlike the traditional digestion protocols using protein denaturation, reduction and alkylation before tryptic digestion, this approach relies on mAb resistance to trypsin digestion while in its native conformation. For this reason, HCPs non-covalently attached to the mAb are more easily digested by trypsin compared to the mAb, which stays mostly intact even during prolonged (overnight) digestion. According to this protocol, the sample is denatured, reduced, and alkylated after tryptic digestion, in order to precipitate and remove through centrifugation the major sample component. Our digestion protocol (detailed in the Experimental section) can be applied to other mAb products. 

The digested sample was analyzed in triplicate using both MS^E and HDMS^E acquisition modes and the datasets were processed in Progenesis QI for Proteomics v4.2 for HCP identification and quantification. Only the HCPs identified in all three LC-MS replicates with 3 or more peptides per protein were reported. The MS^E acquisition was able to identify only 5 of the most abundant HCPs, reaching a detection limit of about 10 ppm (data not presented). In contrast with MS^E, the HDMS^E acquisition was able to identify significantly more HCPs - forty eight (48), improving the detection limit by 100 fold and reaching a detection level of 100 ppb. A detailed list of all the HCPs identified in all three replicates across 3 orders of magnitude is presented in Table 1. Compared to MS^E, the sensitivity of HDMS^E is greatly enhanced because in this acquisition mode the coeluting peptide precursors are separated by very fast (15-20 millisecond) ion mobility separations before fragmentation. As a result, the SYNAPT XS Mass Spectrometer provides cleaner MS/MS fragmentation spectra enabling many more HCP identifications. In addition, the collision energy (CE) used for peptide fragmentation is further optimized in the HDMS^E mode, by synchronizing the applied CE on the transfer cell with the IMS drift time of peptide precursors.

The list of HCPs identified in the HDMS^E experiment was compared against the HCP identification reported in two previous publications^4,6 and the results are summarized in the Venn diagram shown in Figure 2. All three datasets contain a subset of 14 HCPs which was initially reported by our group.² In addition, each of the three datasets contains a unique subset of HCPs, identified only by a single laboratory: in our case 20 HCPs were only identified by the HDMS^E acquisition on SYNAPT XS. Fourteen out of these 20 HCPs (highlighted in yellow in Table 1) were measured to have concentrations in the sub-ppm range (100–1000 ppb) demonstrating the excellent sensitivity of this assay. Clearly, while it is easier to identify the higher abundance HCPs contained in the NIST mAb sample, it appears that each different sample preparation procedure renders some unique very low level HCPs. This observation suggests the need to try different sample preparation methods in order to acquire a comprehensive HCP profile for this challenging mAb sample. 

• A highly sensitive HCP discovery assay using HDMS^E data independent acquisition can identify very low levels of HCPs (sub-ppm) from a highly purified monoclonal antibody 
• Forty eight (48) HCPs were identified from the NIST mAb, considerably improving our previous results reporting only 14 proteins identifications 

1. Hogwood BD, Smales MC. Measurement and Control of Host Cell Proteins (HCPs) in CHO Cell Bioprocesses, Curr Opin Biotechnol, 2014, 30, 153–160. 
2. Doneanu CE, Anderson M, Williams BJ, Lauber MA, Chakraborty A, Chen W. 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, Anal Chem, 2015, 87, 10283–10291. 
3. Weibin C, Doneanu CE, Lauber MA, Koza S, Prakash K, Stapels M, Fountain KJ. Improved Identification and Quantification of Host Cell Proteins (HCPs) in Biotherapeutics Using Liquid Chromatography-Mass Spectrometry, book chapter in Technologies for Therapeutic Monoclonal Antibody Characterization, Vol 3, ACS Symposium Series, 2015, 357–393.
4. Huang L, Wang N, Mitchell CE, Brownlee T, Maple SR, De Felippis MR. A Novel Sample Preparation for Shotgun Proteomics Characterization of HCPs in Antibodies, Anal Chem, 2017, 89, 5436–5444. 
5. Chen IH, Xiao H, Daly T, Ning L. Improved Host Cell Protein Analysis in Monoclonal Antibody Products through Molecular Weight Cutoff Enrichment, Anal Chem, 2020, 92, 3751–3757. 
6. Wang Q, Slaney TR, Wu W, Ludwig R, Tao L, Leone A. Enhancing Host-Cell Protein Detection in Protein Therapeutics Using HILIC Enrichment and Proteomic, Anal Chem, 2020, 92, 10327–10335.