UBC Theses and Dissertations
The CBM9 fusion tag : a new technology for inexpensive production and affinity purification of recombinant proteins Kavoosi, Mojgan
Downstream processing of proteins and other biological products has long been dominated by packed-bed chromatography (Rankin 2003). Despite the generally high cost of the technique, chromatography remains widely used because it offers extraordinarily high resolution under conditions that do not denature or alter the chemistry of the product, an imperative for therapeutic proteins since purity and activity are strict requirements. However, the inability of patients and governments to meet the rising costs of healthcare, particularly the cost of recombinant protein therapeutics, and the sharp increase in competition over the past decade for market share of recombinant-protein based treatments of major illnesses have led to intense downward pressure on the cost of goods, especially for high volume products such as monoclonal antibodies and other recombinant proteins (Morrow 2002). Industry is therefore seeking to develop more cost effective downstream processes, including cheaper and more selective forms of chromatography. Generic affinity chromatography based on affinity-tag technology has the potential to simplify downstream processing by achieving higher yields and purities than conventional modes of chromatography. However, the high cost of current affinity tag technologies, due mainly to the expense of their associated affinity chromatography media, limits their application at production scales. This thesis addresses this problem by reporting on a novel affinity chromatography platform utilizing the family 9 carbohydrate-binding module (CBM9) of xylanase 10A from T. maritima , a new affinity tag that binds to both soluble sugars and insoluble cellulose to permit the highly efficient capture and purification of CBM9-tagged fusion proteins on a very inexpensive cellulose-based affinity media. Development of this technology has required (i) design of a generic CBM9 expression vector for production of chimeric fusions containing an N-terminal CBM9, a linker region containing a suitable processing site at its C-terminus for efficient removal of the affinity tag following affinity purification, and a C-terminal target protein, (ii) development of an effective strategy to design a linker sequence to stably connect the CBM9 tag to the target protein and to permit efficient tag removal through enzyme-catalyzed cleavage, (iii) derivation and validation of a mathematical model to predict binding and elution behavior of CBM9 fusion proteins on a high-capacity cellulose column, (iv) solutions to certain technology scale-up issues, including the synthesis of a mechanically stable stationary phase, and finally, (v) validation of the performance of the technology in terms of product yield, purity and concentration factor. Two bioinformatics-based strategies were developed to successfully identify a linker with improved resistance to endogeneous proteases of the host when compared against the popular poly-glycine based linker. A simple and effective assay was developed to identify the optimal conditions for efficient tag removal post-purification. The technique, based on Luminescence Resonance Energy Transfer (LRET) prescreens a library of linkers and processing enzymes to identify a CBM9-target protein fusion with enhanced processing efficiency. A novel two-zone model (TZM) of pore diffusion is presented to describe the rate of uptake of CBM9 fusion proteins within the stationary phase of the associated affinity chromatography column and thereby provide improved predictions of product breakthrough, including elution behavior from a bacterial lysate feed. Finally, a mechanically stable cellulose-based chromatography media was synthesized to allow preparative-scale purification of recombinant proteins using CBM9. A fixed-effect two-way response surface methodology was used to optimize the concentrations of the two primary reactants, epichlorohydrin and dimethyl sulfoxide (DMSO), required to cross-link the starting material, Perloza(TM) MT100, a compressible cellulose-based chromatography resin. This resulted in a cross-linked affinity chromatography media capable of operating at an order-of-magnitude higher linear velocity than permitted by unmodified MT100. In sharp contrast to MT100, the mechanical stability and purification performance of the cross-linked media are not diminished by scale-up or repeated column use. The results of this thesis thereby provide industry with a ready-made expression vector that can be used to express any target protein as a CBM9 fusion protein and to then inexpensively purify the target recombinant protein at an overall level of performance that is either superior or comparable to current commercially available fusion-tag technologies.
Item Citations and Data