Comparison of hydrophobic charge induction chromatography with affinity chromatography on protein A for harvest and purification of antibodies

Schwartz, Warren E.; Judd, D.L.; Wysocki, M; Guerrier, Luc; Birck-Wilson, Eszter; Boschetti, Egisto

doi:10.1016/s0021-9673(00)01013-x

Cited by 133 publications

(67 citation statements)

References 9 publications

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“…However, a method that could separate free antibody from glucoamylase-antibody fusion proteins was also sought. HCIC with MEP HyperCel was previously investigated as an alternative to protein A affinity chromatography for antibody capture and purification (20). We adapted this procedure as described in Materials and Methods and found that after loading of the column at pH 8, free antibody could be eluted within the pH range of 4.5 to 5.5, whereas glucoamylaseantibody fusion proteins did not elute until the pH was lowered further.…”

Section: Resultsmentioning

confidence: 99%

Characterization of Humanized Antibodies Secreted byAspergillus niger

Ward¹,

Lin²,

Victoria³

et al. 2004

Appl Environ Microbiol

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Two different humanized immunoglobulin G1( ) antibodies and an Fab fragment were produced by Aspergillus niger. The antibodies were secreted into the culture supernatant. Both light and heavy chains were initially synthesized as fusion proteins with native glucoamylase. After antibody assembly, cleavage by A. niger KexB protease allowed the release of free antibody. Purification by hydrophobic charge induction chromatography proved effective at removing any antibody to which glucoamylase remained attached. Glycosylation at N297 in the Fc region of the heavy chain was observed, but this site was unoccupied on approximately 50% of the heavy chains. The glycan was of the high-mannose type, with some galactose present, and the size ranged from Hex 6 GlcNAc 2 to Hex 15 GlcNAc 2 . An aglycosyl mutant form of antibody was also produced. No significant difference between the glycosylated antibody produced by Aspergillus and that produced by mammalian cell cultures was observed in tests for affinity, avidity, pharmacokinetics, or antibody-dependent cellular cytotoxicity function.

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Section: Resultsmentioning

confidence: 99%

Characterization of Humanized Antibodies Secreted byAspergillus niger

Ward¹,

Lin²,

Victoria³

et al. 2004

Appl Environ Microbiol

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“…Salt-independent antibody binding and successful elution at a somewhat higher pH range than is possible with Protein A chromatography has been demonstrated; 56,66,67 however, one critical drawback of HCIC is that it has stronger non-specific binding and can be less efficient than Protein A chromatography in reducing impurities such as host cell protein. Consequently, use of HCIC in an antibody purification process can be challenging.…”

Section: Chromatographic Processesmentioning

confidence: 99%

Recovery and purification process development for monoclonal antibody production

Liu

Winter

et al. 2010

mAbs

463

411

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“…Cation exchange chromatography removes host cell proteins, DNA, endotoxin, leached protein A and aggregates. Anion exchange chromatography is also utilized for removal of DNA, host cell proteins and endotoxin in the flow through mode, and some leached protein A purification occurs if operated in bind and elute mode (Schwartz et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

Maximizing productivity of chromatography steps for purification of monoclonal antibodies

Tugçu

Roush

Göklen

2007

Biotech & Bioengineering

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The large scale production of monoclonal antibodies presents a challenge to design efficient and cost effective downstream purification processes. We explored a two stage resin screening approach to identify the best candidates to be utilized for the platform purification of monoclonal antibodies. The study focused on commercially available affinity resins including Protein A, mimetic and mixed-mode interaction resins as well as ion exchangers used in polishing steps. An initial screening using pure proteins was followed by a final screening where selected resins were utilized for the purification of MAbs in complex mixtures. Initial screenings aimed to measure the theoretical upper limit for dynamic binding capacity (DBC) at 1% breakthrough and productivity. We confirmed that DBC of affinity, mimetic and mixed-mode resins was a strong function of the linear velocity used for loading. Productivities >27 g/(L-h), were obtained for rProtein A FF, Mabselect and Prosep rA Ultra at 2 min residence time. For the cation exchangers, we identified UNOsphere S and Fractogel SO(3) as the best candidates for our purification based on DBC. For anion exchangers operated in flowthrough mode, Q Sepharose XL and UNOsphere Q were selected from the initial screening based on DBC and resolution of IgG from BSA. Finally, a three step purification scheme was implemented using the selected affinity and ion exchangers for the purification of IgG from complex feedstocks. We found that Mabselect followed by UNOsphere Q and UNOsphere S provided the best purification scheme for our applications based on productivity.

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Comparison of hydrophobic charge induction chromatography with affinity chromatography on protein A for harvest and purification of antibodies

Cited by 133 publications

References 9 publications

Characterization of Humanized Antibodies Secreted byAspergillus niger

Characterization of Humanized Antibodies Secreted byAspergillus niger

Recovery and purification process development for monoclonal antibody production

Maximizing productivity of chromatography steps for purification of monoclonal antibodies

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