Abstract
Protein adsorption on solid state media is important for the industrial affinity chromatography of biotherapeutics and for preparing materials for self-interaction chromatography where fundamental protein solution thermodynamic properties are measured. The adsorption of three model proteins (lysozyme, catalase and BSA) and two antibodies (a monoclonal and a polyclonal antibody) have been investigated on commercial affinity chromatography media with different surface functionalities (Formyl, Tresyl and Amino). Both the extent of protein immobilised (mg protein/ml media) and the reaction kinetics are reported for a range of reaction conditions, including pH, differing buffers as well as the presence of secondary reactants (glutaraldehyde, sodium cyanoborohydride, EDC and NHS). Compared to the reaction conditions recommended by manufacturers as well as those reported in previous published work, significant increases in the extent of protein immobilisation and reaction kinetics are reported here. The addition of glutaraldehyde or sodium cyanoborohydride was found to be especially effective even when not directly needed for the adsorption to happen. For mAb and pIgG, immobilisation levels of 50 and 31 mg of protein/ml of resin respectively were achieved, which are 100% or more than previously reported. Enhanced levels were achieved for lysozyme of 120 mg/ml with very rapid reaction kinetics (< 1 h) with sodium cyanoborohydride. It can be concluded that specific chromatography resins with Tresyl activated support offered enhanced levels of protein immobilisation due to their ability to react to form amine or thio-ether linkages with proteins. Additionally, glutaraldehyde can result in higher immobilisation levels whilst it can also accelerate immobilisation reaction kinetics.
Highlights
One of the fastest growing sectors in the pharmaceutical industry is the production of protein based therapeutics that have been shown to be successful in treatments of cancer, heart disease, diabetes, autoimmune diseases and infectious diseases (Pavlou and Reichert 2004)
This paper considers two model proteins as well as a therapeutic monoclonal antibody and a polyclonal antibody
The protein remaining after immobilisation has a higher concentration of aggregates, which approximates in total to the amount of aggregated protein left in the original solution after the amount bound on the resin is considered
Summary
One of the fastest growing sectors in the pharmaceutical industry is the production of protein based therapeutics that have been shown to be successful in treatments of cancer, heart disease, diabetes, autoimmune diseases and infectious diseases (Pavlou and Reichert 2004). Antibody therapeutics are part of an important class within the biopharmaceutical industry, especially, since monoclonal antibodies have been used for many therapeutic conditions (Shukla and Kandula 2009). Despite their success in treatments and the growing numbers of protein based pharmaceuticals, the manufacture of these proteins in a cost-effective and reliable way remains a challenge (Hernandez 2015; Shukla and Gottschalk 2013). The ability to predict, restrict or minimise aggregation would still be crucial for a successful manufacturing or formulation process (Vázquez-Rey and Lang 2011)
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