Study of the mechanism of interaction of antibody (IgG) on two mixed mode sorbents.
Purification of target proteins from a crude biological mixture containing proteins, peptides and other biomolecules is the chromatographic challenge. Mixed mode chromatography offers additional selectivities to improve the overall productivity of commercial bioprocesses with novel chromatographic sorbents being introduced to overcome the problem. HEA HyperCel (n-hexyl amine) and PPA HyperCel (phenyl propyl amine) are industry scalable mixed mode chromatography sorbents where both hydrophobic and electrostatic interactions are predominant. Our study focuses on understanding the underlying mechanism of interaction of protein with the sorbent. Parameters like buffer conditions, pH and temperature were tuned to study the adsorption and desorption conditions of the protein. Dynamic binding capacity of HEA HyperCel and PPA HyperCel sorbents was studied with human IgG as a model protein. Our study shows that, in HEA the interaction of IgG to the sorbent is predominantly hydrophobic as the binding is enhanced (50-60 mg/ml of sorbent) by presence of salt in buffer and increase in temperature. Binding capacity of PPA is 50-60 mg/ml of sorbent irrespective of temperature effect and/or the presence of salt. The chromatographic experiments show that the interaction could be hydrophobic or ionic or some charge transfer mechanism depending upon the buffer conditions.
Ranjini
S Shiva
SS
Centre for Bioseparation Technology, VIT University, Vellore 632014, India.
Bimal
Devla
D
Dhivya
A P
AP
Vijayalakshmi
M A
MA
D000327:Adsorption; D002845:Chromatography / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism; D019995:Laboratory Chemicals / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D011485:Protein Binding; D013696:Temperature
D007074:Immunoglobulin G; D019995:Laboratory Chemicals; D008024:Ligands