Engineering of a two-step purification strategy for a panel of monoclonal immunoglobulin M directed against undifferentiated human embryonic stem cells

Tscheliessnig, Anne; Ong, Danny; Lee, Jeremy; Pan, Siqi; Satianegara, Gernalia; Schriebl, Kornelia; Choo, Andre; Jungbauer, Alois

doi:10.1016/j.chroma.2009.09.059

Cited by 22 publications

(11 citation statements)

References 43 publications

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“…Even for purification of recombinant proteins, use of these methods is considered problematic owing to potential losses in biological activity. However, the latest advances in upstream processes for recombinant proteins in terms of high cell density cultures and drastically improved product levels triggered the re-evaluation of these methods, particularly for purification of monoclonal antibodies [21,22] or viral proteins [23].…”

Section: Precipitation and Flocculationmentioning

confidence: 99%

Downstream processing of cell culture-derived virus particles

Wolf

Reichl

2011

Expert Review of Vaccines

121

102

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Manufacturing of cell culture-derived virus particles for vaccination and gene therapy is a rapidly growing field in the biopharmaceutical industry. The process involves a number of complex tasks and unit operations ranging from selection of host cells and virus strains for the cultivation in bioreactors to the purification and formulation of the final product. For the majority of cell culture-derived products, efforts focused on maximization of bioreactor yields, whereas design and optimization of downstream processes were often neglected. Owing to this biased focus, downstream procedures today often constitute a bottleneck in various manufacturing processes and account for the majority of the overall production costs. For efficient production methods, particularly in sight of constantly increasing economic pressure within human healthcare systems, highly productive downstream schemes have to be developed. Here, we discuss unit operations and downstream trains to purify virus particles for use as vaccines and vectors for gene therapy.

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Section: Precipitation and Flocculationmentioning

confidence: 99%

Downstream processing of cell culture-derived virus particles

Wolf

Reichl

2011

Expert Review of Vaccines

121

102

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“…5A). Beyond this critical PEG concentration, the IgG recovery decreased slightly, perhaps due to higher viscosity that kept the smaller precipitates suspended during centrifugation (Tscheliessnig et al, 2009). The purity of recovered IgG was estimated to be 70 to 90 %, regardless of PEG shape (data not shown).…”

Section: Resultsmentioning

confidence: 99%

“…In descending molecular weight, these proteins were: mAb529 monoclonal immunoglobulin M directed against undifferentiated human embryonic stem cells (IgM, 900 kDa) (Choo et al, 2008), equine spleen apoferritin (Apo, 443 kDa), sweet potato β‐amylase (β‐Amy, 200 kDa), anti‐RhD IgG (150 kDa), bovine serum albumin (BSA, 66 kDa) and bovine heart cytochrome c (Cyt c, 12.2–12.6 kDa). The IgM was purified from hybridoma mAb529 supernatant as described by Tscheliessnig et al (2009) (purity ∼80 %). The IgG was purified from the aforesaid CHO cell culture supernatant by FPLC (Amersham, Buckinghamshire, UK) using rProtein A MabSelect SuRe (GE Healthcare, Waukesha, WI) (purity ∼98 %).…”

Section: Methodsmentioning

confidence: 99%

“…The purified proteins were dissolved or buffer exchanged using PD‐10 desalting columns (GE Healthcare) into either 1× PBS (137 mM sodium chloride, 2.7 mM potassium chloride, 10 mM disodium hydrogen phosphate, 1.8 mM potassium dihydrogen phosphate) (IgM, IgG, BSA, Cyt c), or acetate buffer (0.25 M sodium acetate, 0.25 M acetic acid, 0.1 M potassium chloride) (Apo, β‐Amy). All purified protein solutions were adjusted using either hydrochloric acid or sodium hydroxide to the respective protein isoelectric points (pI) estimated to be: 6.5 for IgM (Tscheliessnig et al, 2009), 4.5 for Apo (Bulte et al, 1994), 4.6 for β‐Amy (Sigma–Aldrich product information), 8.7 for IgG (inferred from amino acid sequence using http://au.expasy.org/tools/pi_tool.html), 5.3 for BSA (Wallevik, 1973), and 10.2 for Cyt c (Sigma–Aldrich product information).…”

Section: Methodsmentioning

confidence: 99%

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Branched polyethylene glycol for protein precipitation

Sim

Tscheliessnig

et al. 2011

Biotech & Bioengineering

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The use of linear PEGs for protein precipitation raises the issues of high viscosity and limited selectivity. This paper explores PEG branching as a way to alleviate the first problem, by using 3-arm star as the model branched structure. 3-arm star PEGs of 4,000 to 9,000 Da were synthesized and characterized. The effects of PEG branching were then elucidated by comparing the branched PEG precipitants to linear versions of equivalent molecular weights, in terms of IgG recovery from CHO cell culture supernatant, precipitation selectivity, solubility of different purified proteins, and precipitation kinetics. Two distinct effects were observed: PEG branching reduced dynamic viscosity; secondly, the branched PEGs precipitated less proteins and did so more slowly. Precipitation selectivity was largely unaffected. When the branched PEGs were used at concentrations higher than their linear counterparts to give similar precipitation yields, the dynamic viscosity of the branched PEGs were noticeably lower. Interestingly, the precipitation outcome was found to be a strong function of PEG hydrodynamic radius, regardless of PEG shape and molecular weight. These observations are consistent with steric mechanisms such as volume exclusion and attractive depletion.

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“…However, these procedures typically have low capacities and are time-consuming due to inherent mass transfer limitations (Przybycien et al 2004). Furthermore, integrated precipitation and anion exchange chromatography has been reported (Tscheliessnig et al 2009). Therefore, non-chromatographic alternatives have been investigated.…”

Section: Introductionmentioning

confidence: 99%