Optimized Removal of Soluble Host Cell Proteins for the Recovery of met-Human Growth Hormone Inclusion Bodies from Escherichia coli Cell Lysate Using Crossflow Microfiltration

Venkiteshwaran, Adith; Heider, Patrick L.; Matosevic, Sandro; Bogsnes, Are; Staby, Arne; Sharfstein, Susan T.; Belfort, Georges

doi:10.1021/bp0700253

Cited by 17 publications

(10 citation statements)

References 39 publications

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“…Thus, although variations will exist in individual HCP expression levels from one product to another, information gained from treating these impurities as a class are likely to be broadly applicable. Given the importance of this class of contaminants, significant effort is devoted during downstream process development to achieve effective and robust clearance 16‐21…”

Section: Introductionmentioning

confidence: 99%

Host cell protein clearance during protein a chromatography: Development of an improved column wash step

Shukla

Hinckley

2008

Biotechnology Progress

203

153

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in Wiley InterScience (www.interscience.wiley.com).Host cell protein (HCP) contaminant clearance is a significant concern during downstream process development for biopharmaceuticals. Protein A chromatography as a capture step for monoclonal antibodies and Fc fusion proteins can clear a large proportion of these impurities from cell culture harvest. Nevertheless, remaining levels of this process-related impurity class do present significant constraints on the rapid development of effective and robust polishing steps. Conventionally, an intermediate pH wash is employed between column loading and elution to minimize HCP levels after Protein A chromatography. A significant mechanistic finding presented in this work is that HCP contaminants that persist following Protein A capture predominantly comprise species that associate with the product in preference to direct interaction with the chromatographic resin. This suggests that the development of improved column wash techniques to maximize HCP clearance ought to focus on disrupting protein-HCP interactions rather than Protein A-HCP interactions. A higher wash pH to preserve product -Protein A binding along with the use of additive combinations to disrupt interactions between HCPs and the product are investigated. This strategy was successfully applied to develop a broadly applicable wash condition that has the potential for eliminating the need for product specific optimization of wash conditions. A combination of 1 M urea and 10% isopropanol in the wash buffer were successfully applied as a platform wash condition for Protein A chromatography. Use of this (and other similar) wash conditions are anticipated to aid the rapid development of effective downstream processes for monoclonal antibodies and Fc fusion proteins resulting in their rapid introduction into clinical trials.

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Section: Introductionmentioning

confidence: 99%

Host cell protein clearance during protein a chromatography: Development of an improved column wash step

Shukla

Hinckley

2008

Biotechnology Progress

203

153

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“…Achieving adequate clearance of HCPs is a key aspect of the design of downstream process sequences (8)(9)(10). This has become such an integral part of downstream process development for pharmaceutically relevant proteins that papers describing novel separation techniques (11)(12)(13)(14) routinely include data on HCP clearance to demonstrate their efficacy.…”

Section: Introductionmentioning

confidence: 99%

Demonstration of Robust Host Cell Protein Clearance in Biopharmaceutical Downstream Processes

Shukla¹,

Jiang²,

Ma³

et al. 2008

Biotechnology Progress

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Residual host cell protein impurities (HCPs) are a key component of biopharmaceutical process related impurities. These impurities need to be effectively cleared through chromatographic steps in the downstream purification process to produce safe and efficacious protein biopharmaceuticals. A variety of strategies to demonstrate robust host cell protein clearance using scale-down studies are highlighted and compared. A common strategy is the "spiking" approach, which is widely employed in clearance studies for well-defined impurities. For HCPs this approach involves spiking cell culture harvest, which is rich in host cell proteins, into the load material for all chromatographic steps to assess their clearance ability. However, for studying HCP clearance, this approach suffers from the significant disadvantage that the vast majority of host cell protein impurities in a cell culture harvest sample are not relevant for a chromatographic step that is downstream of the capture step in the process. Two alternative strategies are presented here to study HCP clearance such that relevance of those species for a given chromatographic step is taken into consideration. These include a "bypass" strategy, which assumes that some of the load material for a chromatographic step bypasses that step and makes it into the load for the subsequent step. The second is a "worst-case" strategy, which utilizes information obtained from process characterization studies. This involves operating steps at a combination of their operating parameters within operating ranges that yield the poorest clearance of HCPs over that step. The eluate from the worst case run is carried forward to the next chromatographic step to assess its ability to clear HCPs. Both the bypass and worst-case approaches offer significant advantages over the spiking approach with respect to process relevance of the HCP impurity species being studied. A combination of these small-scale validation approaches with large-scale HCP clearance data from clinical manufacturing and manufacturing consistency runs is used to demonstrate robust HCP clearance for the downstream purification process of an Fc fusion protein. The demonstration of robust HCP clearance through this comprehensive strategy can potentially be used to eliminate the need for routine analytical testing or for establishing acceptance criteria for these impurities as well as to demonstrate robust operation of the entire downstream purification process.

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“…It is therefore not surprising that a large number of reports on process development have chosen HCP clearance as one of the benchmarks to demonstrate a robust and well-controlled bioprocess (Follman and Fahrner, 2004;Rathore et al, 2003;Shukla et al, 2007Venkiteshwaran et al, 2007). Furthermore, much of our knowledge regarding HCPs in biologics was obtained through process development studies.…”

Section: Impacts Of Host Cell Protein Analysis In Bioprocess Developmmentioning

confidence: 99%

Host cell proteins in biologics development: Identification, quantitation and risk assessment

Wang

Hunter

Mozier

2009

Biotech & Bioengineering

197

184

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Host cell proteins (HCPs) are those produced or encoded by the organisms and unrelated to the intended recombinant product. Some are necessary for growth, survival, and normal cellular processing whereas others may be non-essential, simply carried along as baggage. Like the recombinant product, HCPs may also be modified by the host with a number of post-translational modifications. Regardless of the utility, or lack thereof, HCPs are undesirable in the final drug substance. Though commonly present in small quantities (parts per million expressed as nanograms per milligrams of the intended recombinant protein) much effort and cost is expended by industry to remove them. The purpose of this review is to summarize what is of relevance in regards to the biology, the impact of genomics and proteomics on HCP evaluation, the regulatory expectations, analytical approaches, and various methodologies to remove HCPs with bioprocessing. Historical data, bioinformatics approaches and industrial case study examples are provided. Finally, a proposal for a risk assessment tool is provided which brings these facets together and proposes a means for manufacturers to classify and organize a control strategy leading to meaningful product specifications.

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Optimized Removal of Soluble Host Cell Proteins for the Recovery of met-Human Growth Hormone Inclusion Bodies from Escherichia coli Cell Lysate Using Crossflow Microfiltration

Cited by 17 publications

References 39 publications

Host cell protein clearance during protein a chromatography: Development of an improved column wash step

Host cell protein clearance during protein a chromatography: Development of an improved column wash step

Demonstration of Robust Host Cell Protein Clearance in Biopharmaceutical Downstream Processes

Host cell proteins in biologics development: Identification, quantitation and risk assessment

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