A high-throughput hydrophilic interaction liquid chromatography coupled with a charged aerosol detector method to assess trisulfides in IgG1 monoclonal antibodies using tris(2-carboxyethyl)phosphine reaction products: Tris(2-carboxyethyl)phosphine-oxide and tris(2-carboxyethyl)phosphine-sulfide

Cornell, Christopher; Karanjit, Amish; Chen, Yan; Jacobson, Fredric S.

doi:10.1016/j.chroma.2016.06.037

Cited by 6 publications

(6 citation statements)

References 18 publications

(37 reference statements)

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“…This high value of trisulfide content is in the range of several reports already published. 4,18 When using CP in process 1a, the relative amount of trisulfide linkages was drastically reduced to 9.6 and 7.2% on both days, confirming the results of Kshirsagar. 9 Results obtained by using the SSC-containing process indicate a residual mean relative trisulfide content of 3.0 and 3.1% on day 13 and day 18, respectively, pointing out to the capacity of SSC to reduce trisulfide bond formation in mAb1.…”

Section: Resultssupporting

confidence: 73%

Impact of S-sulfocysteine on fragments and trisulfide bond linkages in monoclonal antibodies

Seibel

Maier

Schnellbaecher

et al. 2017

mAbs

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The quality of recombinant proteins such as monoclonal antibodies produced using Chinese hamster ovary cell-based mammalian systems is dependent on many factors, including cell line, process and cell culture media. Due to these factors, the generated product is heterogeneous and may have chemically-induced modifications or post-translational modifications that affect antibody stability, functionality and, in some cases, patient safety. This study demonstrates that S-sulfocysteine, a cysteine derivative, can increase the antibody specific productivity in different cell lines cultivated with different processes while minimizing trisulfide linkages in generated mAbs, mainly between heavy and light chain. The supplementation of a cell culture feed with S-sulfocysteine also proved to be useful to reduce the percentage of antibody fragments generated from the monoclonal antibody. Overall, this new component used in the upstream process allows a reduction of product heterogeneity.

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Section: Resultssupporting

confidence: 73%

Impact of S-sulfocysteine on fragments and trisulfide bond linkages in monoclonal antibodies

Seibel

Maier

Schnellbaecher

et al. 2017

mAbs

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“…Taken these together, it is hypothesized that increased trisulfides caused DO F lots to require an extra of 0.2 mole of TCEP to achieve the same DAR as the DO S lots. Cornell, et al have reported an orthogonal method to quantifying trisulfides in mAbs by detection of TCEP reaction products. While this work was done on an IgG 1 , it could potentially be applied to an IgG 2 as in our case.…”

Section: Resultsmentioning

confidence: 99%

Fluctuations in dissolved oxygen concentration during a CHO cell culture process affects monoclonal antibody productivity and the sulfhydryl‐drug conjugation process

Hippach¹,

Schwartz²,

Pei³

et al. 2018

Biotechnology Progress

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During a CHO cell culture production process, important parameters are generally well controlled by a feedback mechanism (PID loop) in order to ensure consistency in both productivity and product quality. These parameters typically include pH, dissolved oxygen (DO), and temperature. While most of these parameters are very well controlled within their specific deadband, stable DO control can be challenging. Oscillations in DO concentration are not uncommon and these fluctuations can be exacerbated with an efficient mass transfer aeration strategy. In this study, where an IgG producing cell line was used, we observed increased lactate accumulation accompanied by decreased titer production in lots with fluctuations in DO concentration (DO ) when compared with lots with stable DO control (DO ). We demonstrate that DO had a greater impact on performance with respect to titer production and lactate accumulation than DO setpoint. Furthermore, we report that estimated specific oxygen uptake rates (qOURs) were lower in DO lots when compared with DO lots. We also report that purified mAb sourced from DO lots yielded lower drug-to-antibody ratio (DAR) after the sulfhydryl-targeted maleimide conjugation process when equivalent reducing agent was used. All mAb lots were within the analytical specifications for release, though a slight increase in measureable trisulfides were observed in DO mAb lots. DO control aimed to minimize fluctuations around DO setpoint was essential for us to produce consistent DAR without adjusting the conjugation process. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018.

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“…The procedure was based on previous studies. 32 Briefly, an aliquot of the Fab A reference standard (diluted to 7.5 mg/mL in 50 mM Tris buffer, pH 7.8) was treated with 2.5 mM H 2 S. After incubation for 24 h at room temperature, the sample was immediately desalted using a PD-10 column to remove excess H 2 S. After concentration and buffer exchange with PBS, the sample concentration was measured. The trisulfide enriched Fab A sample was then diluted with PBS to 2.5 mg/mL (equivalent to the clinical dosing concentration of Fab A) prior to incubation in the vitreous model.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

Predictive In Vitro Vitreous and Serum Models and Methods to Assess Thiol-Related Quality Attributes in Protein Therapeutics

Liu¹,

Chen²,

Tsui³

et al. 2020

Anal. Chem.

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In vitro models that mimic the in vivo environment can greatly facilitate and support criticality assessment of product quality attributes for therapeutic drugs to ensure product quality. An in vitro model is established to study and predict the impact of thiol-related attributes on safety or efficacy of intraocular antibody products. This model simulates the physiological redox environment of rabbit vitreous and maintains a steady-state redox potential using reduced and oxidized forms of glutathione. A similar in vitro model that mimics the thiol redox conditions of human blood has been previously established and has become a predictive tool to study intravenous (IV) therapeutic proteins. We utilized both vitreous and serum models to study the potential impact of antibody variants (trisulfides and free-thiols) on product qualities of different antibodies. The studies demonstrate that both models are effective tools to monitor changes of thiol-related attributes under physiological conditions, providing insights on these thiol-related attributes and allowing for more informed assessment of biological relevance and criticality of the attributes. Furthermore, we propose that the approach using an in vitro study for the product quality attribute assessment can be used to predict in vivo effects for future molecules during the development of biopharmaceuticals, reducing the need for live subject studies.

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A high-throughput hydrophilic interaction liquid chromatography coupled with a charged aerosol detector method to assess trisulfides in IgG1 monoclonal antibodies using tris(2-carboxyethyl)phosphine reaction products: Tris(2-carboxyethyl)phosphine-oxide and tris(2-carboxyethyl)phosphine-sulfide

Cited by 6 publications

References 18 publications

Impact of S-sulfocysteine on fragments and trisulfide bond linkages in monoclonal antibodies

Impact of S-sulfocysteine on fragments and trisulfide bond linkages in monoclonal antibodies

Fluctuations in dissolved oxygen concentration during a CHO cell culture process affects monoclonal antibody productivity and the sulfhydryl‐drug conjugation process

Predictive In Vitro Vitreous and Serum Models and Methods to Assess Thiol-Related Quality Attributes in Protein Therapeutics

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