Identification and tracking of problematic host cell proteins removed by a synthetic, highly functionalized nonwoven media in downstream bioprocessing of monoclonal antibodies

Gilgunn, Sarah; El‐Sabbahy, Hani; Albrecht, Simone; Gaikwad, Manasi; Corrigan, K.; Deakin, Lynne; Jellum, Gregory M.; Bones, Jonathan

doi:10.1016/j.chroma.2019.02.056

Cited by 48 publications

(44 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…4MP outperformed Capto Adhere with a single protein, Group I/II HCP serine protease HTRA1. While 4MP resin returned the lowest HCP binding performance, it should be noted that by both quantitative and qualitative measures, it outperforms quaternary amine ligands (Capto Q), which are currently employed on depth filtration media for clearing HCPs in harvest fluids that feature comparable salt concentration to that considered here (Gilgunn et al, 2019;Singh et al, 2017).…”

Section: Profile Of Bound Proteins By Peptide-based Resins Versus Cmentioning

confidence: 87%

Targeted capture of Chinese hamster ovary host cell proteins: Peptide ligand binding by proteomic analysis

Lavoie

Fazio

Williams

et al. 2019

Biotech & Bioengineering

View full text Add to dashboard Cite

The clearance of host cell proteins (HCPs) is of crucial importance in biomanufacturing, given their diversity in composition, structure, abundance, and occasional structural homology with the product. The current approach to HCP clearance in the manufacturing of monoclonal antibodies (mAbs) relies on product capture with Protein A followed by removal of residual HCPs in flow‐through mode using ion exchange or mixed‐mode chromatography. Recent studies have highlighted the presence of “problematic HCP” species, which are either difficult to remove (Group I), can degrade the mAb product (Group II), or trigger immunogenic reactions (Group III). To improve the clearance of these species, we developed a family of synthetic peptides that target HCPs and exhibit low binding to IgG product. In this study, these peptides were conjugated onto chromatographic resins and evaluated in terms of HCP clearance and mAb yield, using an industrial mAb‐producing CHO harvest as model supernatant. To gather detailed knowledge on the binding of individual HCPs, the unbound fractions were subjected to shotgun proteomic analysis by mass spectrometry. It was found that these peptide ligands exhibit superior HCP binding capability compared to those of the benchmark commercial resins commonly used in mAb purification. In addition, some peptide‐based resins resulted in much lower losses of product yield compared to these commercial supports. The proteomic analysis showed effective capture of many “problematic HCPs” by the peptide ligands, especially some that are weakly bound by commercial media. Collectively, these results indicate that these peptides show great promise toward the development of next‐generation adsorbents for safer and cost‐effective manufacturing of biologics.

show abstract

Section: Profile Of Bound Proteins By Peptide-based Resins Versus Cmentioning

confidence: 87%

Targeted capture of Chinese hamster ovary host cell proteins: Peptide ligand binding by proteomic analysis

Lavoie

Fazio

Williams

et al. 2019

Biotech & Bioengineering

View full text Add to dashboard Cite

show abstract

“…Numerous previous studies have demonstrated strongly reduced HCP impurity levels in the Protein A elution pool, compared to traditional depth filters (Castro-Forero, Jokondo, Voloshin, & Hester, 2015;El-Sabbahy et al, 2018;Gilgunn et al, 2019;Singh et al, 2017). One of these studies also demonstrated removal of histone proteins to below the detection limit (Gilgunn et al, 2019). Complete removal of these hydrophobic and positively charged (pI of~11.5) histones as well as reduction of the negatively charged DNA component by an anion-exchange device support that chromatin complexes and not only free DNA are eliminated.…”

Section: Various Studies Have Been Conducted To Understand What Causesmentioning

confidence: 97%

“…The bulk of these steps are aimed at purifying the target protein and removing undesired impurities such as host cell proteins (HCPs), host cell DNA (HCDNA), and viruses. HCPs are of concern as some subsets can elicit unwanted and potentially dangerous immune responses in patients, while others can affect drug efficacy or bulk protein stability (Aboulaich et al, 2014; Chiu et al, 2016; Gilgunn et al, 2019). HCP are a complex mixture of different proteins, some of which can be difficult to clear due to having similar biochemical properties as the target protein.…”

Section: Introductionmentioning

confidence: 99%

Chromatographic clarification overcomes chromatin‐mediated hitch‐hiking interactions on Protein A capture column

Velde¹,

Saller

Eyer³

et al. 2020

Biotech & Bioengineering

View full text Add to dashboard Cite

Protein A capture chromatography is a critical unit operation in the clearance of host cell protein (HCP) impurities in monoclonal antibody (mAb) purification processes. Though one of the most effective purification steps, variable levels of protein impurities are often observed in the eluate. Coelution of HCP impurities is suggested to be strongly affected by the presence of chromatin complexes (Gagnon et al., 2014; Koehler et al., 2019). We investigated the effect of removal of DNA complex and HCP reduction pre-Protein A on the HCP clearance performance of the Protein A capture step itself. We found that only reduction of DNA in the Protein A load consistently lowered HCP in the Protein A eluate. Reduction of HCP in the Protein A load stream did not produce a significant increase in the chromatography HCP clearance performance. These results are consistent across three different biosimilar therapeutic mAbs expressed by the same Chinese hamster ovary (CHO) cell line (i.e., CHO BC® of Polpharma Biologics). This result demonstrates that optimization of the mAb purification process utilizing Protein A as the primary capture step depends primarily on being able to effectively clear DNA and associated complexes early in the process, rather than trying to incorporate HCP reduction at the harvest cell culture fluid.

show abstract

“…ELISA analysis of these samples showed that the total HCP concentrations were greatly reduced from 510,762 ppm in OM, to 1,081 ppm in PrA, 557 ppm in CEX, and finally only 7 ppm in the AEX. In the SWATH-MS analysis, total 1,900 proteins were quantified (at 20% CV cutoff and 1% peptide FDR in OM) from one microgram of each DSP sample, including previously reported difficult-to-remove and problematic HCPs 43 – 45 (Online-only Table 1 ). Heat map analysis of all the identified proteins showed that majority of the impurities were removed after protein A affinity chromatography (Fig.…”

Section: Technical Validationmentioning

confidence: 99%

A comprehensive CHO SWATH-MS spectral library for robust quantitative profiling of 10,000 proteins

et al. 2020

View full text Add to dashboard Cite

Sequential window acquisition of all theoretical fragment-ion spectra (SWATH) is a data-independent acquisition (DIA) strategy that requires a specific spectral library to generate unbiased and consistent quantitative data matrices of all peptides. SWATH-MS is a promising approach for in-depth proteomic profiling of Chinese hamster Ovary (CHO) cell lines, improving mechanistic understanding of process optimization, and real-time monitoring of process parameters in biologics R&D and manufacturing. However, no spectral library for CHO cells is publicly available. Here we present a comprehensive CHO global spectral library to measure the abundance of more than 10,000 proteins consisting of 199,102 identified peptides from a CHO-K1 cell proteome. The robustness, accuracy and consistency of the spectral library were validated for high confidence in protein identification and reproducible quantification in different CHO-derived cell lines, instrumental setups and downstream processing samples. The availability of a comprehensive SWATH CHO global spectral library will facilitate detailed characterization of upstream and downstream processes, as well as quality by design (QbD) in biomanufacturing. The data have been deposited to ProteomeXchange (PXD016047). Background & Summary Chinese hamster ovary (CHO) cells are widely studied in biomedical research and have been used for the production of nearly 70% recombinant therapeutic proteins, including the blockbuster monoclonal antibodies (mAbs) such as adalimumab (Humira), bevacizumab (Avastin), and trastuzumab (Herceptin) 1-4. As with other biopharmaceuticals, the production of recombinant mAbs using CHO cells is strictly regulated by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) 5. The quality by design (QbD) approach is emphasized in order to ensure all aspects of CHO mAb product development and manufacturing are evaluated and consistent 6-9. Hence, in order to enhance the mAb product quality while minimizing potential adverse effects, a thorough understanding of the recombinant mAbs, the early-stage product development process as well as the production and purification process is critical. Mass spectrometry (MS)-based proteomics techniques have been applied to facilitate the QbD approach for optimal bioprocess design, and enhanced quality and yield of biotherapeutic products 10-13. For example, the data-dependent acquisition (DDA) shotgun proteomics has been demonstrated to complement the traditional immunoassays (western blotting and ELISA) to monitor and analyze known and/or previously unseen host cell proteins (HCPs) in the drug manufacturing processes 14-16. Nevertheless, due to the stochastic nature of peptide sampling by DDA-MS technique, multi-dimensional separation or fractionation steps and lengthy chromatographic gradients are often required to increase the proteome coverage over a large dynamic range of protein concentrations. Additionally, the advanced MS methodologies usually require well-trained professionals to operate t...

show abstract

Identification and tracking of problematic host cell proteins removed by a synthetic, highly functionalized nonwoven media in downstream bioprocessing of monoclonal antibodies

Cited by 48 publications

References 40 publications

Targeted capture of Chinese hamster ovary host cell proteins: Peptide ligand binding by proteomic analysis

Targeted capture of Chinese hamster ovary host cell proteins: Peptide ligand binding by proteomic analysis

Chromatographic clarification overcomes chromatin‐mediated hitch‐hiking interactions on Protein A capture column

A comprehensive CHO SWATH-MS spectral library for robust quantitative profiling of 10,000 proteins

Contact Info

Product

Resources

About