Scale-Up of Protein Purification: Downstream Processing Issues

Milne, John Joseph

doi:10.1007/978-1-4939-6412-3_5

Cited by 17 publications

(4 citation statements)

References 24 publications

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“…Thus, downstream processing has become a bottleneck in biotherapeutic production 29 . Efforts to alleviate this have mostly focused on innovations in process design to improve downstream processing capacity, speed, and economics 30,31 . It has also been suggested that cell line engineering could be used to reduce or remove problematic HCPs, lessening the need for additional purification steps 6,32 .…”

Section: Discussionmentioning

confidence: 99%

Multiplex secretome engineering enhances recombinant protein production and purity

Kol

Ley

Wulff

et al. 2019

Preprint

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AbstractHost 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 predicted the elimination of unnecessary HCPs could have a non-negligible impact on protein production. We analyzed the total HCP content of 6-protein, 11-protein, and 14-protein knockout clones and characterized their growth in shake flasks and bioreactors. These cell lines exhibited a substantial reduction in total HCP content (40%-70%). We also observed higher productivity and improved growth characteristics, in specific clones. With the reduced HCP content, protein A and ion exchange chromatography more efficiently purified a monoclonal antibody (mAb) produced in these cells during a three-step purification process. 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

Kol

Ley

Wulff

et al. 2019

Preprint

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“…Although our maximum protein load amounts were chosen to allow for maximum resolution, one also needs to consider that our method is envisioned to be used as a secondary purification or polishing step where dynamic binding capacity is a secondary consideration. 40 This combination of IEX and linear pH gradient elution can thus be used where the lower resolution salt gradients result in insufficient separation of heterodimer from homodimers and other impurities. Although we demonstrated here the application of our method for the purification of common LC bispecifics, it can also be used for the purification of other bispecific formats as long as the impurities to be removed have a sufficiently different apparent pI.…”

Section: Discussionmentioning

confidence: 99%

Purification of common light chain IgG-like bispecific antibodies using highly linear pH gradients

Sharkey

Pudi

Moyer

et al. 2016

mAbs

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Monovalent bispecific antibodies (BsAbs) are projected to have broad clinical applications due to their ability to bind two different targets simultaneously. Although they can be produced using recombinant technologies, the correct pairing of heavy and light chains is a significant manufacturing problem. Various approaches exploit mutations or linkers to favor the formation of the desired BsAb, but a format using a single common light chain has the advantage that no other modification to the antibody is required. This strategy reduces the number of formed molecules to three (the BsAb and the two parent mAbs), but the separation of the BsAb from the two monovalent parent molecules still poses a potentially difficult purification challenge. Current methods employ ion exchange chromatography and linear salt gradients, but are only successful if the difference in the observed isoelectric points (pIs) of two parent molecules is relatively large. Here, we describe the use of highly linear pH gradients for the facile purification of common light chain BsAbs. The method is effective at separating molecules with differences in pI as little as 0.10, and differing in their sequence by only a single charged amino acid. We also demonstrate that purification resins validated for manufacturing are compatible with this approach.

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“…The complexity of the DSP is determined by the required purity of the product, which depends on the application and authorization. Performance criteria to be considered in developing an optimized purification process are speed, recovery, capacity, and resolution (Milne, 2017). A process developed in a laboratory (e.g., in 0.5 to 10 L fermenters) must be translated into a full manufacturing scale process (e.g., 20.000 to 2.000.000 L fermenters; Crater and Lievense, 2018).…”

Section: Purification Processes For Fermentation Products: What Is Fe...mentioning

confidence: 99%

Recombinant DNA in Fermentation Products is of No Regulatory Relevance

Lensch¹,

Duwenig²,

Dederer³

et al. 2022

Preprint

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A large variety of fermentation products are used in food and feed production, but also in other industries, and many of these products are produced with genetically modified microorganisms (GMMs). In food and feed production, prominent examples are amino acids, vitamins, food and feed enzymes, colorants, non-caloric sweeteners and human milk oligosaccharides. From a regulatory perspective, fermentation products are typically produced under containment. This means that premises, equipment and work processes need to be designed to prevent or at least minimize release of GMMs into the environment. The fermentation products themselves should not contain any live cells of the GMM. Over the past years, there have been concerning developments, particularly in the European Union, stipulating that also absence of recombinant DNA might be interpreted as a regulatory requirement for fermentation products produced with GMMs. In this paper, we (i) attempt to place these developments into the historical context, (ii) sketch the potential negative repercussions for the food and feed industries, (iii) elaborate on the safety of recombinant DNA, and (iv) postulate that recombinant DNA should remain an integral part of the safety assessment of fermentation products but should not be misconstrued as a criterion for regulatory classification of products of biotechnology.

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Scale-Up of Protein Purification: Downstream Processing Issues

Cited by 17 publications

References 24 publications

Multiplex secretome engineering enhances recombinant protein production and purity

Multiplex secretome engineering enhances recombinant protein production and purity

Purification of common light chain IgG-like bispecific antibodies using highly linear pH gradients

Recombinant DNA in Fermentation Products is of No Regulatory Relevance

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