Scale-Up of Protein Purification: Downstream Processing Issues

Milne, John Joseph

doi:10.1007/978-1-60761-913-0_5

Cited by 19 publications

(11 citation statements)

References 14 publications

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“…Thus, downstream processing has become a bottleneck in biotherapeutic production 35 . Efforts to alleviate this have mostly focused on innovations in process design to improve downstream processing capacity, speed, and economics 36,37 . It has also been suggested that cell line engineering could be used to reduce or remove problematic NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15866-w ARTICLE HCPs, lessening the need for additional purification steps 6,38 .…”

Section: Discussionmentioning

confidence: 99%

Multiplex secretome engineering enhances recombinant protein production and purity

et al. 2020

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Host cell proteins (HCPs) are process-related impurities generated during biotherapeutic protein production. HCPs can be problematic if they pose a significant metabolic demand, degrade product quality, or contaminate the final product. Here, we present an effort to create a "clean" Chinese hamster ovary (CHO) cell by disrupting multiple genes to eliminate HCPs. Using a model of CHO cell protein secretion, we predict that the elimination of unnecessary HCPs could have a non-negligible impact on protein production. We analyze the HCP content of 6-protein, 11-protein, and 14-protein knockout clones. These cell lines exhibit a substantial reduction in total HCP content (40%-70%). We also observe higher productivity and improved growth characteristics in specific clones. The reduced HCP content facilitates purification of a monoclonal antibody. Thus, substantial improvements can be made in protein titer and purity through large-scale HCP deletion, providing an avenue to increased quality and affordability of high-value biopharmaceuticals.

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Section: Discussionmentioning

confidence: 99%

Multiplex secretome engineering enhances recombinant protein production and purity

et al. 2020

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“…A detailed overview about important chromatographic and non-chromatographic issues which have to be considered during the development and also for choosing the fi xed parameters for a chromatographic step is given in the review of Milne [ 8 ].…”

Section: Methodsmentioning

confidence: 99%

“…For an overview about further important aspects for the development of a downstream procedure for protein purifi cation the reader is referred to Milne [ 8 ].…”

Section: Introductionmentioning

confidence: 99%

Sample Displacement Batch Chromatography of Proteins

Kotasińska

Richter

Kwiatkowski

et al. 2014

Methods in Molecular Biology

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In downstream processing large scale chromatography plays an important role. For its development screening experiments followed by pilot plant chromatography are mandatory steps. Here we describe fast, simple, and inexpensive methods for establishing a preparative chromatography for the separation of complex protein mixtures, based on sample displacement batch chromatography. The methods are demonstrated by anion-exchange chromatography of a human plasma protein fraction (Cohn IV-4), including the screening step and scaling up of the chromatography by a factor of 100. The results of the screening experiments and the preparative chromatography are monitored by SDS-PAGE electrophoresis. In summary we provide a protocol which should be easily adaptable for the chromatographic large scale purification of other proteins, in the laboratory as well as in industry for commercial manufacturing. For the latter these protocols cover the initial piloting steps for establishing a sample batch chromatography based on packed columns rather than batch chromatography.

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“…For clarification, crude extracts can be filtered or centrifuged to separate debris from any soluble products into the supernatant fraction. Capture and purification occurs mostly for protein products used for human therapeutic, cosmetic, or nutraceutical purposes (Milne 2017). Chromatographic operations and/or precipitation steps are commonly used to purify and concentrate proteins.…”

Section: Proteins Carbohydrates Lipids Pigmentsmentioning

confidence: 99%

Green microalgae biomolecule separations and recovery

Dixon

Wilken

2018

Bioresour. Bioprocess.

116

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Microalgae biomass has garnered significant attention as a renewable energy feedstock and alternative to petroleumbased fuels. The diverse metabolism of green microalgae species additionally provides opportunities for recovery of products for feed, food, nutraceutical, cosmetic, and biopharmaceutical industries. Recently, the concept of using microalgae as part of a biorefinery model has been adopted in place of refinery methods focused on recovering one target product. This has led to producers exploring co-production of high value and high volume products in an effort to improve process economics. With numerous potential products and applications, the biomass source or specific strain must be carefully selected to accumulate extractable levels of the target molecule(s). It is additionally imperative to understand the morphology and metabolism of the selected strain to cost-effectively manage all stages of commercial production. This review will focus specifically on microalgae in the division of Chlorophyta, or green algae and their extracellular matrices (ECM), potential for commercial products, and finally describe a holistic approach for biomolecule extraction and recovery. Additionally, cell disruption and fractionation methods for recovery of biomolecules for commercial products are highlighted along with an alternative method, aqueous enzymatic processing for multiple biomolecule extraction and recovery from green microalgae. An emphasis is placed on connecting the morphological characteristics of microalgae ECM or organelle membranes to implications on separation and purification technologies.

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Scale-Up of Protein Purification: Downstream Processing Issues

Cited by 19 publications

References 14 publications

Multiplex secretome engineering enhances recombinant protein production and purity

Multiplex secretome engineering enhances recombinant protein production and purity

Sample Displacement Batch Chromatography of Proteins

Green microalgae biomolecule separations and recovery

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