A comparison of <scp>P</scp>rotein <scp>A</scp> chromatographic stationary phases: Performance characteristics for monoclonal antibody purification

Liu, Zhuo; Mostafa, Sigma S.; Shukla, Abhinav A.

doi:10.1002/bab.1243

Cited by 33 publications

(15 citation statements)

References 19 publications

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“…This isotherm behavior is consistent with that for IgG class antibodies in other ProA characterizations in the literature (Hahn et al, 2005;Liu et al, 2014;Perez-Almodovar and Carta, 2009;Zhang et al, 2016a). These isotherms were essentially rectangular and reached the maximum binding capacity, q m , at a protein concentration of $1 mg/mL for all three resins.…”

Section: Adsorption Isothermssupporting

confidence: 88%

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Polyclonal and monoclonal IgG binding on protein A resins—Evidence of competitive binding effects

Weinberg

Zhang²,

Crews

et al. 2017

Biotech & Bioengineering

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Protein A (ProA) chromatography is used extensively in the biopharmaceutical industry for the selective capture of both polyclonal and monoclonal antibodies (mAbs). This work provides a comparison of the adsorptive behavior of a highly heterogeneous polyclonal hIgG versus that of a mAb as well as the behavior of their mixtures on representative ProA resins. Both pH gradient elution and frontal loading experiments using human polyclonal IgG (hIgG) reveal a distribution of IgG-ProA binding strengths likely associated with multiple IgG subclasses and the heterogeneity of the variable region. pH gradient analysis of fractions collected along the breakthrough curve demonstrate a clear progression from weaker binding (higher pH eluting) to stronger binding (lower pH eluting) IgG species leaving the column suggesting the possibility of stronger binding species displacing the weaker binding ones. Displacement is directly observed by visualizing the adsorption of fluorescently labeled mAb and hIgG using confocal laser scanning microscopy (CLSM). Here, the displacement of hIgG results in a broad adsorption front compared to the sharp, "shrinking core" behavior typically observed with mAbs. Sequential CLSM adsorption experiments with a mAb and hIgG confirm that stronger or equivalent-binding hIgG species are able to displace and desorb bound mAb molecules. These phenomena are examined using a variety of ProA resins including CaptivA PriMAB, MabSelect, and MabSelect SuRe to understand the effect of different ligand properties on binding strength and competition among different IgG species. The results of these comparisons suggest that the competition kinetics are slower with ligands that have a single-point covalent attachment to the base matrix compared to a multi-point attachment. Biotechnol. Bioeng. 2017;114: 1803-1812. © 2017 Wiley Periodicals, Inc.

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Section: Adsorption Isothermssupporting

confidence: 88%

“…CaptivA PriMAB is based on 4% cross-linked agarose with an average particle diameter of 90 mm and with a recombinant version of Table I. Properties of the ProA resins used in this work (Hober et al, 2007;Jones et al, 2014;Liu et al, 2014;Repligen, 2010).…”

Section: Methodsmentioning

confidence: 99%

Polyclonal and monoclonal IgG binding on protein A resins—Evidence of competitive binding effects

Weinberg

Zhang²,

Crews

et al. 2017

Biotech & Bioengineering

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“…As shown in the figure, the two cases employing the novel elution strategies exhibit a 49% and 60% reduction in the HCPs co-eluting with the IgG fraction when compared to the conventional protein A elution method. It should be noted that the amount of HCP in the capture product shown in Figure 7 for the standard elution method is somewhat lower than typically observed in industrial practice likely because of the low IgG loadings used in the experiments performed (Liu et al, 2015). These low loadings are likely due in turn to the relatively lower peak cell density (4-6 Â 10 6 cells/mL) of the cell culture process from which the cell culture supernatant was generated (data not shown).…”

Section: Recoveries and Hcps Bioassaysmentioning

confidence: 82%

“…Note also that the anion‐exchange group density (q R + ) and the cation‐exchange group density (q R − ) were approximately one third and one fifth, respectively, of the values expected from the amount of protein A estimated from the IgG binding capacity provided by the column manufacturer and from assuming a 1:1 binding ratio between IgG and the protein A ligand. Furthermore, since binding ratios greater than 1:1 between IgG and protein A are often observed in practice (see, e.g., Liu et al, ), this suggests that only a portion of the charged groups on protein A are accessible to the adsorbing buffering species. It can nevertheless be reasonably concluded that the good agreement between experimental results and theoretical calculations shown in Figure demonstrates that the retained pH gradients observed for the case of a protein A column are due to the same underlying mechanisms that apply to the case of ion‐exchange chromatography.…”

Section: Theorymentioning

confidence: 99%

Immunoglobulin G elution in protein A chromatography employing the method of chromatofocusing for reducing the co‐elution of impurities

Pinto

Uplekar

Moreira

et al. 2016

Biotech & Bioengineering

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Purification processes for monoclonal Immunoglobulin G (IgG) typically employ protein A chromatography as a capture step to remove most of the impurities. One major concern of the post-protein A chromatography processes is the co-elution of some of the host cell proteins (HCPs) with IgG in the capture step. In this work, a novel method for IgG elution in protein A chromatography that reduces the co-elution of HCPs is presented where a two-step pH gradient is self-formed inside a protein A chromatography column. The complexities involved in using an internally produced pH gradient in a protein A chromatography column employing adsorbed buffering species are discussed though equation-based modeling. Under the conditions employed, ELISA assays show a 60% reduction in the HCPs co-eluting with the IgG fraction when using the method as compared to conventional protein A elution without affecting the IgG yield. Evidence is also obtained which indicates that the amount of leached protein A present in free solution in the purified product is reduced by the new method. Biotechnol. Bioeng. 2017;114: 154-162. © 2016 Wiley Periodicals, Inc.

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“…Cleaning was performed after every third cycle. Cleaning reagent of 50mM NaOH, 1 M NaCl being the preferred cleaning solution by manufacturers, was used in all the three cases . Robustness of the scale down model was established by performing 40 cycles in duplicates as per the protocol defined above.…”

Section: Methodsmentioning

confidence: 99%

Protein A chromatography resin lifetime—impact of feed composition

Pathak

Rathore

Lintern

et al. 2018

Biotechnology Progress

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Adsorbent lifetime during protein A chromatography is not readily predicted or understood, representing a key challenge to be addressed for biopharmaceutical manufacturers. This article focuses on the impact of feed composition on the performance of a typical agarose-based protein A resin across a lifetime of 50 cycles. Cycling studies were performed using three different feed materials with varying levels of feed components including proteases, histones, DNA, and nonhistone proteins. Changes in the process and quality attributes were measured. The DBCs were not seen to vary between conditions although there was a reduction in particle porosity in all cases. Fluorescence spectroscopy and LC-MS/MS were used to identify the contribution and extent of fouling to the observed capacity loss. Residual protein A ligand density and deposition of foulants (HCP, residual mAb, and DNA) varied between the three feed materials. Resins cycled in feed materials containing high concentrations of HCP and histones were seen to have greater extents of capacity loss. The mode of performance loss, capacity loss, or impact on product quality was seen to vary depending on the feed material. The results indicate that feed material composition may be correlated to the rate and mode of resin aging as a basis for improved process understanding. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:412-419, 2018.

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A comparison of Protein A chromatographic stationary phases: Performance characteristics for monoclonal antibody purification

Cited by 33 publications

References 19 publications

Polyclonal and monoclonal IgG binding on protein A resins—Evidence of competitive binding effects

Polyclonal and monoclonal IgG binding on protein A resins—Evidence of competitive binding effects

Immunoglobulin G elution in protein A chromatography employing the method of chromatofocusing for reducing the co‐elution of impurities

Protein A chromatography resin lifetime—impact of feed composition

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