Protein A‐Based Affinity Chromatography

Vunnum, Suresh; Vedantham, Ganesh; Hubbard, Brian

doi:10.1002/9780470444894.ch4

Cited by 18 publications

(32 citation statements)

References 37 publications

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“…[16] In many cases, the pH must be modified yet again for purification via chromatography. [17] Several pH-adjusting steps are of special interest to formulation scientists. In practice, one often makes these changes by step-by-step titrations until the target pH is reached.…”

Section: Predicting Defined Ph Shifts In Biotechnological Processesmentioning

confidence: 99%

Buffer‐free therapeutic antibody preparations provide a viable alternative to conventionally buffered solutions: From protein buffer capacity prediction to bioprocess applications

Bahrenburg

Karow

Garidel

2015

Biotechnology Journal

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Protein therapeutics, including monoclonal antibodies (mAbs), have significant buffering capacity, particularly at concentrations>50 mg/mL. This report addresses pH-related issues critical to adoption of self-buffered monoclonal antibody formulations. We evaluated solution conditions with protein concentrations ranging from 50 to 250 mg/mL. Samples were both buffer-free and conventionally buffered with citrate. Samples were non-isotonic or adjusted for isotonicity with NaCl or trehalose. Studies included accelerated temperature stability tests, shaking stability studies, and pH changes in infusion media as protein concentrate is added. We present averaged buffering slopes of capacity that can be applied to any mAb and present a general method for calculating buffering capacity of buffer-free, highly concentrated antibody liquid formulations. In temperature stability tests, neither buffer-free nor conventionally buffered solution conditions showed significant pH changes. Conventionally buffered solutions showed significantly higher opalescence than buffer-free ones. In general, buffer-free solution conditions showed less aggregation than conventionally buffered solutions. Shaking stability tests showed no differences between buffer-free and conventionally buffered solutions. "In-use" preparation experiments showed that pH in infusion bag medium can rapidly approximate that of self-buffered protein concentrate as concentrate is added. In summary, the buffer capacity of proteins can be predicted and buffer-free therapeutic antibody preparations provide a viable alternative to conventionally buffered solutions.

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Section: Predicting Defined Ph Shifts In Biotechnological Processesmentioning

confidence: 99%

Buffer‐free therapeutic antibody preparations provide a viable alternative to conventionally buffered solutions: From protein buffer capacity prediction to bioprocess applications

Bahrenburg

Karow

Garidel

2015

Biotechnology Journal

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“…In a monoclonal antibody (mAb) product purification platform, the capture step is especially convenient because it is a Protein A affinity step. 10, 15 Yet, in a modern industrial mAb purification process, the 95-98% purity of Protein A-eluted product with respect to DNA and HCP is insufficient, and a complex and expensive polishing train is still required. Complexes of DNA and DNAhistone have been reported to be a major component of cell culture fluid interfering with protein chromatography.…”

Section: Introductionmentioning

confidence: 99%

Development of adsorptive hybrid filters to enable two-step purification of biologics

Singh

Arunkumar

Michael

et al. 2016

mAbs

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Recent progress in mammalian cell culture process has resulted in significantly increased product titers, but also a substantial increase in process-and product-related impurities. Due to the diverse physicochemical properties of these impurities, there is constant need for new technologies that offer higher productivity and improved economics without sacrificing the process robustness required to meet final drug substance specifications. Here, we examined the use of new synthetic adsorptive hybrid filters (AHF) modified with the high binding capacity of quaternary amine (Emphaze TM AEX) and salt-tolerant biomimetic (Emphaze TM ST-AEX) ligands for clearance of process-related impurities like host cell protein (HCP), residual DNA, and virus. The potential to remove soluble aggregates was also examined. Our aim was to develop a mechanistic understanding of the interactions governing adsorptive removal of impurities during filtration by evaluating the effect of various filter types, feed streams, and process conditions on impurity removal. The ionic capacity of these filters was measured and correlated with their ability to remove impurities for multiple molecules. The ionic capacity of AHF significantly exceeded that of traditional adsorptive depth filters (ADF) by 40% for the Emphaze TM AEX and by 700% for the Emphaze TM ST-AEX, providing substantially higher reduction of soluble anionic impurities, including DNA, HCPs and model virus. Nevertheless, we determined that ADF with filter aid provided additional hydrophobic functionality that resulted in removal of higher molecular weight species than AHF. Implementing AHF demonstrated improved process-related impurity removal and viral clearance after Protein A chromatography and enabled a two-step purification process. The consequences of enhanced process performance are far reaching because it allows the downstream polishing train to be restructured and simplified, and chromatographic purity standards to be met with a reduced number of chromatographic steps.

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“…Antibody purification, regardless of the expression host, has been dominated by the usage of affinity chromatography [13]. Affinity chromatography has a high selectivity for effective removal of native plant proteins, subsequently reducing the number of successive unit operations required for protein purification [14].…”

Section: Introductionmentioning

confidence: 99%

“…Affinity chromatography has a high selectivity for effective removal of native plant proteins, subsequently reducing the number of successive unit operations required for protein purification [14]. Protein A chromatography is the most widely used method for antibody purification [2,13]. Protein A is a polypeptide found in the cell wall of the bacteria Staphylococcus aureus [13], which binds specifically to the Fc region of immunoglobulin G (IgG), especially the human IgG subclasses IgG 1 , IgG 2 , and IgG 4 [14].…”

Section: Introductionmentioning

confidence: 99%

“…Protein A chromatography is the most widely used method for antibody purification [2,13]. Protein A is a polypeptide found in the cell wall of the bacteria Staphylococcus aureus [13], which binds specifically to the Fc region of immunoglobulin G (IgG), especially the human IgG subclasses IgG 1 , IgG 2 , and IgG 4 [14]. Previous studies reported that direct application of tobacco extracts to protein A affinity chroma-tography resulted in strong absorption of the green components and native proteins in the tobacco extract [15], which could cause column blockage and adsorbent fouling [16].…”

Section: Introductionmentioning

confidence: 99%

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Process Development for Antibody Purification from Tobacco by Protein A Affinity Chromatography

Hey¹,

Zhang²

2011

Chem Eng & Technol

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Tobacco is possibly the most promising plant for plant‐made pharmaceuticals, e.g., antibodies, due to its high biomass yields and robust transformation technology. Protein A affinity chromatography is commonly used for antibody purification, however, direct application of tobacco extract to protein A chromatography columns may be problematic due to the nonspecific binding of native tobacco proteins (NTPs). Three different protein A resins, ProSep‐vA High Capacity, Ultra, and Ultra Plus, from Millipore were studied for antibody purification from tobacco. Experiments were run with extracts from nontransgenic tobacco and the model antibody, human immunoglobulin G (IgG). The efficiency of the wash buffers to reduce nonspecific binding of NTPs to the resins was evaluated by altering the ionic strength and pH. Considering all factors, Ultra Plus Protein A resin may be the best in antibody purification from tobacco.

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Protein A‐Based Affinity Chromatography

Cited by 18 publications

References 37 publications

Buffer‐free therapeutic antibody preparations provide a viable alternative to conventionally buffered solutions: From protein buffer capacity prediction to bioprocess applications

Buffer‐free therapeutic antibody preparations provide a viable alternative to conventionally buffered solutions: From protein buffer capacity prediction to bioprocess applications

Development of adsorptive hybrid filters to enable two-step purification of biologics

Process Development for Antibody Purification from Tobacco by Protein A Affinity Chromatography

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