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1.
J Biotechnol ; 213: 54-64, 2015 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-25747172

RESUMO

Recent studies using simple model systems have demonstrated that continuous countercurrent tangential chromatography (CCTC) has the potential to overcome many of the limitations of conventional Protein A chromatography using packed columns. The objective of this work was to optimize and implement a CCTC system for monoclonal antibody purification from clarified Chinese Hamster Ovary (CHO) cell culture fluid using a commercial Protein A resin. Several improvements were introduced to the previous CCTC system including the use of retentate pumps to maintain stable resin concentrations in the flowing slurry, the elimination of a slurry holding tank to improve productivity, and the introduction of an "after binder" to the binding step to increase antibody recovery. A kinetic binding model was developed to estimate the required residence times in the multi-stage binding step to optimize yield and productivity. Data were obtained by purifying two commercial antibodies from two different manufactures, one with low titer (∼ 0.67 g/L) and one with high titer (∼ 6.9 g/L), demonstrating the versatility of the CCTC system. Host cell protein removal, antibody yields and purities were similar to those obtained with conventional column chromatography; however, the CCTC system showed much higher productivity. These results clearly demonstrate the capabilities of continuous countercurrent tangential chromatography for the commercial purification of monoclonal antibody products.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Proteína Estafilocócica A/química , Animais , Anticorpos Monoclonais/química , Células CHO , Técnicas de Cultura de Células , Cromatografia/métodos , Cricetinae , Cricetulus
2.
Biotechnol Prog ; 31(1): 90-6, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25367682

RESUMO

Membranes used in bioprocessing applications are typically sanitized before use to insure aseptic operation. However, there is almost no information in the literature on the effects of this preuse sanitization step on the properties of the membrane. Experiments were performed with commercially available hollow fiber polysulfone (PSf) and polyethersulfone (PES) membranes with different nominal molecular weight cutoffs. Data were obtained for the membrane hydraulic permeability, dextran retention coefficients, zeta potential (surface charge), and extent of protein adsorption both before and after sanitization with 0.5 N NaOH at 45°C for 30 min. Changes in chemical composition were examined using ATR-FT-IR and XPS. Sanitization caused a large increase in the net negative charge for all membranes. There was a small reduction in hydraulic permeability and a significant increase in dextran retention for the polyethersulfone membranes, consistent with a reduction in the effective pore size. Spectroscopic analyses suggest that this change is likely due to the base-catalyzed hydrolysis of the lactam ring in polyvinylpyrrolidone (PVP) that is typically is used as a wetting/pore-forming agent in PSf and PES membranes. Preuse sanitization also appeared to have a small effect on protein adsorption, although the extent of adsorption was quite low for both the virgin and sanitized membranes. The observed changes in membrane properties could have a significant impact on the ultrafiltration performance, demonstrating the importance of standardizing the sanitization procedures even in process development and scale-down validation studies.


Assuntos
Membranas Artificiais , Polímeros/química , Hidróxido de Sódio/química , Sulfonas/química , Ultrafiltração/instrumentação , Adsorção , Biotecnologia/instrumentação , Biotecnologia/métodos , Dextranos , Permeabilidade , Porosidade , Proteínas/metabolismo
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