Analysis of host-cell proteins in biotherapeutic proteins by comprehensive online two-dimensional liquid chromatography/mass spectrometry

Doneanu, Catalin E.; Xenopoulos, Alex; Fadgen, Keith E.; Murphy, James Peter; Skilton, St John; Prentice, Holly; Stapels, Martha; Chen, Weibin

doi:10.4161/mabs.4.1.18748

Cited by 152 publications

(162 citation statements)

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“…Even after sophisticated purification steps, low levels of HCPs may still remain in a final purified biotherapeutic,29 although it is important to note that the ratio of DnaK : scFv used in these experiments (1 : 1000) was higher than would be expected in most biotherapeutic preparations used clinically. Accepted limits of HCPs are generally 1–100 parts per million 29. However, 1 part per million HCPs could result in 0·1 μg HCP in a 100‐mg dose (biotherapeutics given to patients can reach doses well above 100 mg30, 31).…”

Section: Discussionmentioning

confidence: 96%

Influence of Escherichia coli chaperone DnaK on protein immunogenicity

et al. 2016

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SummaryThe production of anti‐drug antibodies can impact significantly upon the safety and efficacy of biotherapeutics. It is known that various factors, including aggregation and the presence of process‐related impurities, can modify and augment the immunogenic potential of proteins. The purpose of the investigations reported here was to characterize in mice the influence of aggregation and host cell protein impurities on the immunogenicity of a humanized single‐chain antibody variable fragment (scFv), and mouse albumin. Host cell protein impurities within an scFv preparation purified from Escherichia coli displayed adjuvant‐like activity for responses to the scFv in BALB/c strain mice. The 70 000 MW E. coli chaperone protein DnaK was identified as a key contaminant of scFv by mass spectrometric analysis. Preparations of scFv lacking detectable DnaK were spiked with recombinant E. coli DnaK to mimic the process‐related impurity. Mice were immunized with monomeric and aggregated preparations, with and without 0·1% DnaK by mass. Aggregation alone enhanced IgM and IgG2a antibody responses, but had no significant effect on total IgG or IgG1 responses. The addition of DnaK further enhanced IgG and IgG2a antibody responses, but only in the presence of aggregated protein. DnaK was shown to be associated with the aggregated scFv by Western blot analysis. Experiments with mouse albumin showed an overall increase in immunogenicity with protein aggregation alone, and the presence of DnaK increased the vigour of the IgG2a antibody response further. Collectively these data reveal that DnaK has the potential to modify and enhance immunogenicity when associated with aggregated protein.

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Section: Discussionmentioning

confidence: 96%

Influence of Escherichia coli chaperone DnaK on protein immunogenicity

et al. 2016

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“…[4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] In this methodology, proteins are first digested enzymatically. The resulting peptides are then separated (usually by liquid chromatography), and subsequently detected by mass/ charge via a mass spectrometer.…”

Section: Introductionmentioning

confidence: 99%

“…[4][5][6]11,14 Since HCPs are several orders of magnitude lower in concentration compared to the protein therapeutic, 2-dimensional LC separation helps increase the sensitivity of HCP detection. HCP quantitation is then achieved typically by spiking in one or more protein standards at known concentrations.…”

Section: Introductionmentioning

confidence: 99%

“…HCP quantitation is then achieved typically by spiking in one or more protein standards at known concentrations. [4][5][6]11,20 While these methodologies have substantially increased the depth of HCP characterization, often no proteins (or very few) are detected in final, commercial-grade drug products. Another problem is that the protein standards used will likely have different response factors in the mass spectrometer compared to that of host cell impurities, thus confounding quantitation of specific HCP.…”

Section: Introductionmentioning

confidence: 99%

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Toward the complete characterization of host cell proteins in biotherapeutics via affinity depletions, LC-MS/MS, and multivariate analysis

Farutin²,

Carbeau³

et al. 2015

mAbs

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“…As part of an effort to help users understand the conditions under which 2D-LC provides more resolving power than 1D-LC separations for peptide analyses, the Heinisch group recently demonstrated that the peak capacity of 2D-LC separations is superior to that of 1D-LC separations for analysis times longer than about five minutes [18]. In the area of Host Cell Protein (HCP) analysis, Donenau and coworkers have demonstrated the use of heartcutting 2D-LC [19,20] and offline comprehensive 2D-LC [21] to detect trace-level peptide markers of HCPs. This is very important to quality assurance for biotherapeutic mAbs.…”

Section: Peptide Level Analysismentioning

confidence: 99%

The growing role of two-dimensional LC in the biopharmaceutical industry

Wang¹,

Buckenmaier²,

Stoll³

2017

J Appl Bioanal

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IntroductionOver the past two decades, three major trends have been observed in the bioanalysis world.• First, in liquid chromatography (LC) two-dimensional liquid chromatography (2D-LC) has emerged as one of the most active areas of technology advancement [1][2][3]. In 2D-LC, a conventional separation is carried out in the first dimension and aliquots of the effluent are collected and injected to a second-dimension column that has very different separation selectivity compared to the first-dimension column. Therefore, much higher peak capacity, and thus resolving power, can be achieved in 2D-LC compared to 1D-LC. This increased resolving power can be used to increase the likelihood of separating a complex mixture, or decrease the time required to fully separate simpler mixtures. In addition, 2D-LC allows the coupling of two completely different separation modes (e.g. reversed-phase and ionic exchange) in one method. This makes it possible to measure multiple attributes of a target analyte in one method instead of two separate methods. Although 2D-LC research has been going on for more than three decades, the speed of innovation and commercialization has accelerated in the last ten years due to efforts at both universities and instrument companies.• Second, Mass Spectrometry (MS) has become an indispensable tool in bioanalysis [4]. The ability of new MS instruments to more accurately characterize large molecules keeps improving. However, several challenges still remain. MS works best with volatile buffers of limited concentration. The ionization suppression effect still occurs when compounds with very different concentrations (e.g. several orders of magnitude) co-elute in chromatographic separations. These challenges to MS make LC separation a critical part of any bioanalysis workflow.• Finally, in the application area, biopharmaceutical research has become the fastest growing area in the pharmaceutical industry. In particular, monoclonal antibodies (mAbs) are currently the most important class of biotherapeutic molecules. As of 2016, seven of the top-ten-selling drugs were biologics, and six of these were mAb related [5]. In particular, the number one drug Humira (adalimumab) had an annual sales of $15.7 Billion dollars in 2016. Due to the large size of these antibodies at about 150 kDa molecular weight, analyzing them is very challenging. It often takes a suite of analytical tools to fully characterize the molecule and ensure good quality control of drug products involving these molecules. These three trends are developing at the same time and at high speed. It is our opinion that the combination of 2D-LC with MS is emerging as an exciting and essential tool for efficient, high quality biopharmaceutical analysis. In this article, we will discuss examples that demonstrate the power of 2D-LC-MS in this application area.

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Analysis of host-cell proteins in biotherapeutic proteins by comprehensive online two-dimensional liquid chromatography/mass spectrometry

Abstract: (2012) Analysis of host-cell proteins in biotherapeutic proteins by comprehensive online two-dimensional liquid chromatography/mass spectrometry, mAbs, 4:1, 24-44,

Cited by 152 publications

References 41 publications

Influence of Escherichia coli chaperone DnaK on protein immunogenicity

Influence of Escherichia coli chaperone DnaK on protein immunogenicity

Toward the complete characterization of host cell proteins in biotherapeutics via affinity depletions, LC-MS/MS, and multivariate analysis

The growing role of two-dimensional LC in the biopharmaceutical industry

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