Separation of recombinant antibodies from DNA using divalent cations

Satzer, Peter; Tscheließnigg, Anne; Sommer, Ralf J.; Jungbauer, Alois

doi:10.1002/elsc.201400080

Cited by 10 publications

(7 citation statements)

References 27 publications

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“…The insoluble polymer is then removed using depth filtration resulting in a low aggregate feed stream. Satzer and coworkers has recently shown that addition of calcium chloride to mAb conditioned medium containing phosphate will result in the formation of insoluble calcium phosphate that can effectively precipitate DNA . HCP and aggregate levels were also reported to be lower after treatment.…”

Section: Resultsmentioning

confidence: 99%

“…Satzer and coworkers has recently shown that addition of calcium chloride to mAb conditioned medium containing phosphate will result in the formation of insoluble calcium phosphate that can effectively precipitate DNA. 32 HCP and aggregate levels were also reported to be lower after treatment. As calcium chloride and phosphate buffers present no regulatory or safety hurdles, calcium phosphate precipitation was evaluated as an aggregate removal technique for scFv-IgG bispecific antibodies.…”

Section: Non-chromatographic Clarification Technologies (Calcium Phosmentioning

confidence: 97%

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An Integrated Approach to Aggregate Control for Therapeutic Bispecific Antibodies Using an Improved Three Column Mab Platform‐Like Purification Process

Andrade¹,

Arnold

Motabar³

et al. 2018

Biotechnology Progress

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Single chain variable fragment‐IgGs (scFv‐IgG) are a class of bispecific antibodies consisting of two single chain variable fragments (scFv) that are fused to an intact IgG molecule. A common trend observed for expression of scFv‐IgGs in mammalian cell culture is a higher level of aggregates (10%–30%) compared to mAbs, which results in lower purification yields in order to meet product quality targets. Furthermore, the high aggregate levels also pose robustness risks to a conventional mAb three column platform purification process which uses only the polishing steps (e.g., cation exchange chromatography [CEX]) for aggregate removal. Protein A chromatography with pH gradient elution, high performance tangential flow filtration (HP‐TFF) and calcium phosphate precipitation were evaluated at the bench scale as means of introducing orthogonal aggregate removal capabilities into other aspects of the purification process. The two most promising process variants, namely Protein A pH gradient elution followed by calcium phosphate precipitation were evaluated at pilot scale, demonstrating comparable performance. Implementing Protein A chromatography with gradient elution and/or calcium phosphate precipitation removed a sufficient portion of the aggregate burden prior to the CEX polishing step, enabling CEX to be operated robustly under conditions favoring higher monomer yield. From starting aggregate levels ranging from 15% to 23% in the condition media, levels were reduced to between 2% and 3% at the end of the CEX step. The overall yield for the optimal process was 71%. Results of this work suggest an improved three‐column mAb platform‐like purification process for purification of high aggregate scFv‐IgG bispecific antibodies is feasible. © 2018 The Authors. Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers. Biotechnol. Prog ., 35: e2720, 2019

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Section: Resultsmentioning

confidence: 99%

Section: Non-chromatographic Clarification Technologies (Calcium Phosmentioning

confidence: 97%

An Integrated Approach to Aggregate Control for Therapeutic Bispecific Antibodies Using an Improved Three Column Mab Platform‐Like Purification Process

Andrade¹,

Arnold

Motabar³

et al. 2018

Biotechnology Progress

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“…Two precipitation protocols consisting of precipitation agents CaCl 2 and caprylic acid were recently published . Addition of CaCl 2 to a cell culture supernatant (with a certain threshold PO 4 3‐ concentration) has been reported to result in formation of insoluble calcium phosphate precipitate which causes co‐precipitation of dsDNA and reduction in HCP concentration . In caprylic acid (octanoic acid, C 8 H 16 O 2 ) precipitation at low pH, the hydrophobicity of the octyl moiety causes acidic proteins in the solution to precipitate .…”

Section: Resultsmentioning

confidence: 99%

“…In caprylic acid (octanoic acid, C 8 H 16 O 2 ) precipitation at low pH, the hydrophobicity of the octyl moiety causes acidic proteins in the solution to precipitate . Plasma product manufacturers have long utilized this method for effective precipitation of non‐immunoglobulin proteins from serum, plasma, and ascites fluid .…”

Section: Resultsmentioning

confidence: 99%

Continuous precipitation of process related impurities from clarified cell culture supernatant using a novel coiled flow inversion reactor (CFIR)

Kateja

Agarwal

Saraswat

et al. 2016

Biotechnology Journal

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Coiled Flow Inverter Reactor (CFIR) has recently been explored for facilitating continuous operation of several unit operations involved in downstream processing of biopharmaceuticals such as viral inactivation and protein refolding. The application of CFIR for continuous precipitation of clarified cell culture supernatant has been explored. The pH based precipitation is optimized in the batch mode and then in the continuous mode in CFIR using a design of experiments (DOE) study. Improved clearance of host cell DNA (52× vs. 39× in batch), improved clearance of host cell proteins (HCP) (7× vs. 6× in batch) and comparable recovery (90 vs. 91.5 % in batch) are observed along with six times higher productivity. To further demonstrate wider applicability of CFIR in performing continuous precipitation, two more case studies involving use of two different precipitation protocols (CaCl based and caprylic acid based) are also performed. In both cases, clearance of host cell DNA, HCP, and product recovery are found to be comparable or better in CFIR than in batch operations. Moreover, increase in productivity of 16 times (CaCl based) and eight times (caprylic acid based) is obtained for the two precipitation protocols, respectively. The data clearly demonstrate that CFIR can be seamlessly integrated into a continuous bioprocess train for performing continuous precipitation of clarified cell culture supernatant. To our knowledge this is the first report of such use.

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“…A starting phosphate concentration of > 4 mM has been found to promote efficient flocculation of double stranded DNA (dsDNA) [28]. The phosphate concentration of the CCCS was therefore determined and phosphate levels were found to be sufficient (4.4 ± 0.1 mM).…”

Section: Methodsmentioning

confidence: 99%

Continuous precipitation of IgG from CHO cell culture supernatant in a tubular reactor

Hammerschmidt

Hintersteiner

Lingg

et al. 2015

Biotechnology Journal

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We successfully transferred a two-stage batch precipitation-based antibody capture step to continuous mode using continuous tubular reactors. The precipitation process solely employs a cheap mineral salt (CaCl2 ) and an organic solvent (ethanol) and could replace the costly protein A capture step in the purification of recombinant antibodies from cell culture supernatant. The time from startup untill attaining steady state conditions was reached in less than 15 minutes and both reactors were operated for several hours at steady state without manual intervention, delivering antibody at a constant yield and purity. An overall yield of > 90 percent, with a host cell protein reduction from 42 777 to 9000 ppm and a DNA reduction from 359 ppm to 7 ppm, could be achieved for the antibody investigated. The precipitated antibody can be dissolved at very high concentrations (> 40 g/L) in numerous buffer systems of various pH and high and low ionic strength, thereby rendering a subsequent concentration or buffer exchange step redundant. This system enables cell culture supernatants with low or high antibody titer to be processed with constant reactor size and without changing any parameters or increasing precipitant consumption. Aggregate levels were below 1% under all conditions tested. Purification by precipitation did not affect binding to CD16a or the isoform distribution of the antibody.

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Separation of recombinant antibodies from DNA using divalent cations

Cited by 10 publications

References 27 publications

An Integrated Approach to Aggregate Control for Therapeutic Bispecific Antibodies Using an Improved Three Column Mab Platform‐Like Purification Process

An Integrated Approach to Aggregate Control for Therapeutic Bispecific Antibodies Using an Improved Three Column Mab Platform‐Like Purification Process

Continuous precipitation of process related impurities from clarified cell culture supernatant using a novel coiled flow inversion reactor (CFIR)

Continuous precipitation of IgG from CHO cell culture supernatant in a tubular reactor

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