Separation of immunoglobulin G precipitate from contaminating proteins using microfiltration

Neal, Greg; Francis, Richard; Shamlou, P. Ayazi; Keshavarz-Moore, E

doi:10.1042/ba20030129

Cited by 12 publications

(12 citation statements)

References 22 publications

(28 reference statements)

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“…Since the S30 extract prepared under these heavily diluted conditions exhibited no loss of production efficiency, we attempted to use the TFF system to dialyze a large volume of S30 extract. In other words, the dialysis step for solvent exchange and removal of low-molecular chemicals was replaced with diafiltration (55,56). An ultrafiltration membrane (filtration area, 0.11 m 2 ; MWCO, 10 kDa) was applied to a TFF system, and the pump and valves were configured in a manner similar to that during cell harvesting, in fact, valves 2 and 3 were fully opened.…”

Section: Resultsmentioning

confidence: 99%

A reproducible and scalable procedure for preparing bacterial extracts for cell-free protein synthesis

Katsura

Matsuda

Tomabechi

et al. 2017

The Journal of Biochemistry

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Cell-free protein synthesis is a useful method for preparing proteins for functional or structural analyses. However, batch-to-batch variability with regard to protein synthesis activity remains a problem for large-scale production of cell extract in the laboratory. To address this issue, we have developed a novel procedure for large-scale preparation of bacterial cell extract with high protein synthesis activity. The developed procedure comprises cell cultivation using a fermentor, harvesting and washing of cells by tangential flow filtration, cell disruption with high-pressure homogenizer and continuous diafiltration. By optimizing and combining these methods, ∼100 ml of the cell extract was prepared from 150 g of Escherichia coli cells. The protein synthesis activities, defined as the yield of protein per unit of absorbance at 260 nm of the cell extract, were shown to be reproducible, and the average activity of several batches was twice that obtained using a previously reported method. In addition, combinatorial use of the high-pressure homogenizer and diafiltration increased the scalability, indicating that the cell concentration at disruption varies from 0.04 to 1 g/ml. Furthermore, addition of Gam protein and examinations of the N-terminal sequence rendered the extract prepared here useful for rapid screening with linear DNA templates.

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Section: Resultsmentioning

confidence: 99%

A reproducible and scalable procedure for preparing bacterial extracts for cell-free protein synthesis

Katsura

Matsuda

Tomabechi

et al. 2017

The Journal of Biochemistry

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“…Titre improvement Up to 19 One month to develop the Biacore assay; 6 months to write a validation protocol, undertake all the experiments and sign off the assay; 2 months to undertake trials on small sheep flocks and then scale up the immunisation protocol. Furthermore, based on an assumed 8% increase in titre per month, up to a maximum 80% rise, up to 10 further months would be needed Packed and expanded bed IgG capture 6 Purchasing, validating and qualifying the column and the subsequent ultrafilter as well as installing the associated instrumentation, process control equipment and electrical supply and then revalidating the remainder of the process downstream of where the centrifuge was originally Removing the ion exchange step 5 Development of the extra affinity wash step and revalidation of the affinity step and the steps downstream Fig.…”

Section: Tasks Involvedmentioning

confidence: 99%

“…The above method was applied to an industrial scale process operated by Protherics U.K. Limited (Blaenwaun, Ffostrasol, Llandysul, Wales, UK), which makes polyclonal F AB (CroFab TM ) for the treatment of rattlesnake envenomation [17][18][19]. A variety of changes are possible to boost annual product mass and reduce batch times and costs.…”

Section: Industrial Case Studymentioning

confidence: 99%

A decision-support model for evaluating changes in biopharmaceutical manufacturing processes

Chhatre

Francis²,

O'Donovan³

et al. 2006

Bioprocess Biosyst Eng

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A simulation is described that evaluates the impacts of altering bio-manufacturing processes. Modifications designed to improve production levels, times and costs were assessed, including increasing feed volumes/titres, replacing initial downstream stages with packed or expanded bed affinity steps and removing ion exchange steps. Options were evaluated for manufactured product mass, COG, batch times and development costs and timescales. Metrics were combined using multi-attribute-decision-making techniques generating a single assessment metric for each option. The utility of this approach was illustrated by application to an FDA-approved process manufacturing rattlesnake anti-venom (Protherics U.K.). Currently, ovine serum containing anti-venom IgG is purified by precipitation/centrifugation, prior to antibody proteolysis by papain. An ion exchanger removes F(C), before affinity chromatography yields the final anti-venom. An expanded bed affinity column operating with an 80% higher IgG titre, 66% higher feed volume and without the ion exchanger delivered the best multi-attribute-decision-making value, potentially providing the most desirable alternative.

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“…Companies have to bear significant risks of failure from competitive, regulatory, economic and clinical factors (3)(4)(5)(6)(7). Against this backdrop, a number of techniques now exist that will serve to accelerate and enhance R&D, including genomics and high throughput screening (8) and the application of scale-down methodologies (9)(10)(11)(12). Computer simulations have demonstrated a similar potential, enabling rapid and inexpensive exploration of different process options (13) and facilitating comparison of alternatives (14).…”

Section: Introductionmentioning

confidence: 96%

The Integrated Simulation and Assessment of the Impacts of Process Change in Biotherapeutic Antibody Production

Chhatre

Jones²,

Francis³

et al. 2006

Biotechnol. Prog.

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Growing commercial pressures in the pharmaceutical industry are establishing a need for robust computer simulations of whole bioprocesses to allow rapid prediction of the effects of changes made to manufacturing operations. This paper presents an integrated process simulation that models the cGMP manufacture of the FDA-approved biotherapeutic CroFab, an IgG fragment used to treat rattlesnake envenomation (Protherics U.K. Limited, Blaenwaun, Ffostrasol, Llandysul, Wales, U.K.). Initially, the product is isolated from ovine serum by precipitation and centrifugation, before enzymatic digestion of the IgG to produce FAB and FC fragments. These are purified by ion exchange and affinity chromatography to remove the FC and non-specific FAB fragments from the final venom-specific FAB product. The model was constructed in a discrete event simulation environment and used to determine the potential impact of a series of changes to the process, such as increasing the step efficiencies or volumes of chromatographic matrices, upon product yields and process times. The study indicated that the overall FAB yield was particularly sensitive to changes in the digestive and affinity chromatographic step efficiencies, which have a predicted 30% greater impact on process FAB yield than do the precipitation or centrifugation stages. The study showed that increasing the volume of affinity matrix has a negligible impact upon total process time. Although results such as these would require experimental verification within the physical constraints of the process and the facility, the model predictions are still useful in allowing rapid "what-if" scenario analysis of the likely impacts of process changes within such an integrated production process.

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Separation of immunoglobulin G precipitate from contaminating proteins using microfiltration

Cited by 12 publications

References 22 publications

A reproducible and scalable procedure for preparing bacterial extracts for cell-free protein synthesis

A reproducible and scalable procedure for preparing bacterial extracts for cell-free protein synthesis

A decision-support model for evaluating changes in biopharmaceutical manufacturing processes

The Integrated Simulation and Assessment of the Impacts of Process Change in Biotherapeutic Antibody Production

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