Residual Host Cell Protein Promotes Polysorbate 20 Degradation in a Sulfatase Drug Product Leading to Free Fatty Acid Particles

Dixit, Nitin; Salamat‐Miller, Nazila; Salinas, Paul A.; Taylor, Katherine; Basu, Sujit K.

doi:10.1016/j.xphs.2016.02.029

Cited by 140 publications

(110 citation statements)

References 31 publications

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“…Given the structural similarities between polysorbates and triglycerides, it is hypothesized that LPL may enzymatically degrade polysorbates and consequently negatively impact mAb stability. A similar mechanism has recently been proposed to be associated with the degradation of PS-20 in a non-mAb product formulation by putative phospholipase B-like 2 (PLBL2) (Dixit et al, 2016). PLBL2 is an HCP impurity that was previously identified to have variable expression during extended culture (Valente at al., 2015).…”

Section: Introductionmentioning

confidence: 70%

“…They are thought to stabilize high-concentration mAb solutions by competing with mAbs for surface adsorption (Mahler et al, 2009) or binding to the product molecules (Lee et al, 2011). Several different routes of polysorbate degradation in formulations have previously been identified (Dixit et al, 2016; Ha et al, 2002; Khossravi et al, 2002; Kishore et al, 2011; Hall et al, 2016), with degradation leading to accelerated product degradation due to increased aggregation (Khossravi et al, 2002) or oxidation due to peroxide formation (Ha et al, 2002). …”

Section: Introductionmentioning

confidence: 99%

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Knockout of a difficult‐to‐remove CHO host cell protein, lipoprotein lipase, for improved polysorbate stability in monoclonal antibody formulations

Chiu

Valente

Levy

et al. 2016

Biotech & Bioengineering

166

154

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While the majority of host cell protein (HCP) impurities are effectively removed in typical downstream purification processes, a small population of HCPs are particularly challenging. Previous studies have identified HCPs that are challenging for a variety of reasons. Lipoprotein lipase (LPL) – a Chinese hamster ovary (CHO) HCP that functions to hydrolyze esters in triglycerides – was one of ten HCPs identified in previous studies as being susceptible to retention in downstream processing. LPL may degrade polysorbate 80 (PS-80) and polysorbate 20 (PS-20) in final product formulations due to the structural similarity between polysorbates and triglycerides. In this work, recombinant LPL was found to have enzymatic activity against PS-80 and PS-20 in a range of solution conditions that are typical of mAb formulations. LPL knockout CHO cells were created with CRISPR and TALEN technologies and resulting cell culture harvest fluid demonstrated a significantly reduced polysorbate degradation without significant impact on cell viability when compared to wild type samples.

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Section: Introductionmentioning

confidence: 70%

Section: Introductionmentioning

confidence: 99%

Knockout of a difficult‐to‐remove CHO host cell protein, lipoprotein lipase, for improved polysorbate stability in monoclonal antibody formulations

Chiu

Valente

Levy

et al. 2016

Biotech & Bioengineering

166

154

View full text Add to dashboard Cite

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“…Due to higher cell concentration and longer culture duration, the host cell proteins (HCPs) that are secreted from viable cells and released from dead cells accumulate extracellularly at a much higher level in fed‐batch cultures than they do in batch cultures, thereby potentially impairing product quality (Aboulaich et al, ; Huang et al, ). In addition, certain HCPs in culture supernatants may escape an entire purification process and remain in the final drug substances at levels that affect product stability (Dixit et al, ). Therefore, it is important to characterize the HCPs in culture supernatants to ensure optimal product quality and stability.…”

Section: Introductionmentioning

confidence: 99%

Proteomic analysis of host cell protein dynamics in the supernatant of Fc‐fusion protein‐producing CHO DG44 and DUKX‐B11 cell lines in batch and fed‐batch cultures

Park

Jin²,

et al. 2017

Biotech & Bioengineering

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Chinese hamster ovary (CHO) cells are the most widely used host cell lines for the commercial production of therapeutic proteins including Fc-fusion proteins. During the culture of recombinant CHO (rCHO) cells, host cell proteins (HCPs), secreted from viable cells and released from dead cells, accumulate extracellularly, potentially impairing product quality. In this study, the HCPs that accumulated extracellularly in batch and fed-batch cultures of Fc-fusion protein-producing rCHO cell lines (DG-Fc and DUKX-Fc) were identified and quantified using nanoflow liquid chromatography-tandem mass spectrometry (LC-MS/MS), followed by gene ontology and functional analysis. When the proteome database of Cricetulus griseus was used as a reference to identify the HCPs, more HCPs were identified for DG-Fc (1632 HCPs in batch culture and 1733 HCPs in fed-batch culture) than for DUKX-Fc (1114 HCPs in batch culture and 1002 HCPs in fed-batch culture). Clustering analysis of HCPs, which were classified into four clusters according to their concentration profiles during culture, showed that the concentration profiles of HCPs affecting the quality of Fc-fusion proteins correlated with changes in Fc-fusion protein quality. Taken together, the dataset of HCPs obtained in this study using the two different rCHO cell lines provides insights into the determination of appropriate target proteins to be removed during the culture and purification steps so as to ensure good Fc-fusion protein quality. Biotechnol. Bioeng. 2017;114: 2267-2278. © 2017 Wiley Periodicals, Inc.

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“…In particular, proteases789 and glycosidases1011 that accumulate in culture medium negatively affect the quality of mAbs in recombinant CHO (rCHO) cell cultures. In addition, although the concentrations of HCPs in cell culture harvests are reduced to acceptable levels after a series of purification steps, certain HCPs escape an entire purification process and remain in the final mAb drug substance at levels that affect product quality and stability1213. Therefore, it is paramount to characterize and quantify HCPs in fed-batch cultures to ensure an optimal mAb quality and perform targeted removal of HCPs during the purification steps.…”

mentioning

confidence: 99%

Proteomic Analysis of Host Cell Protein Dynamics in the Culture Supernatants of Antibody-Producing CHO Cells

Park

Jin²,

Lim³

et al. 2017

Sci Rep

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Chinese hamster ovary (CHO) cells are the most common cell line used for the production of therapeutic proteins including monoclonal antibodies (mAbs). Host cell proteins (HCPs), secreted and released from lysed cells, accumulate extracellularly during the cultures of recombinant CHO (rCHO) cells, potentially impairing product quality. In an effort to maintain good mAb quality during the cultures, HCPs accumulated extracellularly in batch and fed-batch cultures of a mAb-producing rCHO cell line were identified and quantified by nanoflow liquid chromatography-tandem mass spectrometry, followed by their gene ontology and functional analysis. Due to higher cell concentration and longer culture duration, more HCPs were identified and quantitated in fed-batch culture (2145 proteins identified and 1673 proteins quantified) than in batch culture (1934 proteins identified and 1486 proteins quantified). Clustering analysis of HCPs showed that the concentration profiles of HCPs affecting mAb quality (Lgmn, Ctsd, Gbl1, and B4galt1) correlated with changes in mAb quality attributes such as aggregation, charge variants, and N-glycosylation during the cultures. Taken together, the dataset of HCPs obtained in this study provides insights into determining the appropriate target proteins to be removed during both the cultures and purification steps for ensuring good mAb quality.

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Residual Host Cell Protein Promotes Polysorbate 20 Degradation in a Sulfatase Drug Product Leading to Free Fatty Acid Particles

Cited by 140 publications

References 31 publications

Knockout of a difficult‐to‐remove CHO host cell protein, lipoprotein lipase, for improved polysorbate stability in monoclonal antibody formulations

Knockout of a difficult‐to‐remove CHO host cell protein, lipoprotein lipase, for improved polysorbate stability in monoclonal antibody formulations

Proteomic analysis of host cell protein dynamics in the supernatant of Fc‐fusion protein‐producing CHO DG44 and DUKX‐B11 cell lines in batch and fed‐batch cultures

Proteomic Analysis of Host Cell Protein Dynamics in the Culture Supernatants of Antibody-Producing CHO Cells

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