The effects of hitchhiker antigens co-eluting with affinity-purified research antibodies

Mechetner, Lilly; Sood, Radhika; Nguyen, Van; Gagnon, Pete; Parseghian, Missag H.

doi:10.1016/j.jchromb.2011.07.016

Cited by 17 publications

(24 citation statements)

References 20 publications

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“…Contaminant-IgG association under physiological conditions is routine with anti-chromatin antibodies because chromatin structure is so highly conserved across phyla. Monoclonal antibodies specific for chromatin from one species bind host chromatin expelled from dead host cells during cell culture production [21][22][23]. Lacking specialized purification procedures to dissociate the wrong-species antigen from the antibodies, they cause immunopotency for their authentic-species antigen to vary from lot to lot by up to a factor of 12 [23].…”

Section: Amplification Of Host Contamination By Igg-chromatin Interacmentioning

confidence: 99%

Non-immunospecific association of immunoglobulin G with chromatin during elution from protein A inflates host contamination, aggregate content, and antibody loss

Gagnon

Nian

Yang

et al. 2015

Journal of Chromatography A

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Monoclonal IgG at pH 3.5 expressed a tendency to self-associate and associate non-specifically with surfaces, including the surfaces of precipitated chromatin heteroaggregates. The tendency was elevated with protein A-eluted IgG still in elution buffer (100mM acetate, pH 3.5). Association of IgG with chromatin elements under protein A elution conditions amplified host protein contamination of the elution fraction about 15-fold, caused formation of aggregates that persisted after pH neutralization, and imposed an approximate 5% loss on IgG recovery. Neutralization released eluted IgG from its low pH associations with chromatin and caused heteroaggregate remnants to associate into large particles easily removed by microfiltration. Most effective host contaminant clearance was achieved by filtration after neutralization to pH 5.5. All chromatin-mediated liabilities were suspended by extraction of chromatin heteroaggregates in advance of protein A.

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Section: Amplification Of Host Contamination By Igg-chromatin Interacmentioning

confidence: 99%

Non-immunospecific association of immunoglobulin G with chromatin during elution from protein A inflates host contamination, aggregate content, and antibody loss

Gagnon

Nian

Yang

et al. 2015

Journal of Chromatography A

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“…Complementary to these approaches in upstream processing is the effort toward improving the downstream capture of HCPs by developing robust purification processes that are tolerant to the inherent variability in cell culture. In this regard, much attention has recently been focused on “problematic” HCPs, that is, proteins that (a) co‐elute with mAbs at the Protein A capture step (Gagnon et al, ; Gan et al, ; Hogwood, Tait, Koloteva‐Levine, Bracewell, & Smales, ; Zhang et al, ) or associate with most mAbs (Aboulaich et al, ; Gagnon et al, ; Levy, Valente, Choe, Lee, & Lenhoff, ; Mechetner, Sood, Nguyen, Gagnon, & Parseghian, ), (b) cause degradation of the product of interest through enzymatic digestion (Bee et al, ) or degradation of the excipients during storage (Chiu et al, ), or (c) present high immunogenicity risk at trace concentrations (Bailey‐Kellogg et al, ). It is important to accurately monitor the residual levels of these HCPs through the various stages of upstream and downstream processing.…”

Section: Introductionmentioning

confidence: 99%

“…This study focuses on problematic HCPs in three primary categories based on risk factors described above: (a) HCPs co‐eluting with mAbs in the capture step, herein referred to as Group I (Aboulaich et al, ; Gagnon et al, ; Levy et al, ; Mechetner et al, ; Zhang et al, ), including IgG‐associated and Protein A‐binding HCPs, (b) HCPs that cause product degradation, or Group II (Aboulaich et al, ; Bee et al, ; Chiu et al, ; Goey et al, ; Zhang et al, ), and (c) HCPs with high immunogenicity risk, or Group III (Bailey‐Kellogg et al, ; Fischer et al, ; Goey et al, ; Jawa et al, ). In typical mAb platform processes, the large majority of HCPs are removed at the product capture step with Protein A‐based adsorbents; thus, the design of the subsequent intermediate and final polishing steps is highly dependent on their ability to remove the species that co‐elute with IgG from Protein A.…”

Section: Introductionmentioning

confidence: 99%

“…Numerous studies in recent years have focused on identifying the mechanism behind co‐elution and selecting process conditions that prevent persistence of these species post‐Protein A. For a number of species, co‐elution is a result of direct association with the mAb (Aboulaich et al, ; Gagnon et al, ; Levy et al, ; Mechetner et al, ); thus, the capture of this subset of impurities without dissociation from the mAb product can result in decreased product yield, although the extent of mAb bound by these HCPs is, as of yet, debated. Novel polishing strategies that target this family of HCPs are required to lower product loss through diligent selection of wash steps or modifiers that induce dissociation if the capture of product‐bound species results in substantial product loss.…”

Section: Introductionmentioning

confidence: 99%

“…Outside of product‐associated HCPs, species that co‐elute with mAbs may result from interaction with Protein A, as it has been determined for chromatin (Gagnon et al, ). Chromatin has also been shown to cause co‐elution of “hitchhiker” HCPs (Mechetner et al, ), which associates with HCPs that bind mAbs or Protein A, thus effectively behaving as co‐eluting species.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Lavoie

Fazio

Williams

et al. 2019

Biotech & Bioengineering

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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.

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Host cell protein testing by ELISAs and the use of orthogonal methods

Zhu-Shimoni¹,

Yu²,

Nishihara³

et al. 2014

Biotech & Bioengineering

149

127

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Host cell proteins (HCPs) are among the process-related impurities monitored during recombinant protein pharmaceutical process development. The challenges of HCP detection include (1) low levels of residual HCPs present in large excess of product protein, (2) the assay must measure a large number of different protein analytes, and (3) the population of HCP species may change during process development. Suitable methods for measuring process-related impurities are needed to support process development, process validation, and control system testing. A multi-analyte enzyme-linked immunosorbent assay (ELISA) is the workhorse method for HCP testing due to its high throughput, sensitivity and selectivity. However, as the anti-HCP antibodies, the critical reagents for HCP ELISA, do not comprehensively recognize all the HCP species, it is especially important to ensure that weak and non-immunoreactive HCPs are not overlooked by the ELISA. In some cases limited amount of antibodies to HCP species or antigen excess causes dilution-dependent non-linearity with multi-product HCP ELISA. In our experience, correct interpretation of assay data can lead to isolation and identification of co-purifying HCP with the product in some cases. Moreover, even if the antibodies for a particular HCP are present in the reagent, the corresponding HCP may not be readily detected in the ELISA due to antibody/antigen binding conditions and availability of HCP epitopes. This report reviews the use of the HCP ELISA, discusses its limitations, and demonstrates the importance of orthogonal methods, including mass spectrometry, to complement the platform HCP ELISA for support of process development. In addition, risk and impact assessment for low-level HCPs is also outlined, with consideration of clinical information.

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The effects of hitchhiker antigens co-eluting with affinity-purified research antibodies

Cited by 17 publications

References 20 publications

Non-immunospecific association of immunoglobulin G with chromatin during elution from protein A inflates host contamination, aggregate content, and antibody loss

Non-immunospecific association of immunoglobulin G with chromatin during elution from protein A inflates host contamination, aggregate content, and antibody loss

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

Host cell protein testing by ELISAs and the use of orthogonal methods

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