Bioengineered Protein A Polymer Beads for High-Affinity Antibody Purification - The authors discuss an alternative to traditional Protein A resins. - BioPharm International

ADVERTISEMENT

Bioengineered Protein A Polymer Beads for High-Affinity Antibody Purification
The authors discuss an alternative to traditional Protein A resins.


BioPharm International
Volume 24, Issue 5, pp. 36-42

Current purification of monoclonal antibodies (mAbs) as therapeutic agents relies on protein A affinity chromatography as one of its key process steps (1–4). The rapidly growing market for this class of high-value therapeutic agents makes its production process an important target for improvement. As cell lines have been engineered to produce increased titers of mAbs, the actual purification process increasingly becomes a bottleneck.


ALL FIGURES COURTESY OF THE AUTHORS
Protein A resins have greatly simplified the mAb recovery process but they are expensive, often costing millions of dollars, and their current binding capacities are insufficient to meet the needs of cell lines producing higher titers of mAbs. Currently used protein A resins are made of recombinant protein A or recombinant multiple Z–binding domains comprising derivatives of protein A that are both recombinantly produced by bacterial fermentation, purified and then chemically cross-linked to premade agarose beads. The implementation of multiple and laborious steps in the production process of protein A resin is reflected in its high retail price. The current manufacture of protein A resin inherently contributes to its limited performance. The chemical cross-linking of the protein A ligand to agarose leads to a random orientation of the ligand, and hence a loss of accessible binding sites, and a reduction of the mAb binding capacity. The protein A sepharose as porous affinity resin requires diffusive flow of the mAb solution that, in turn, increases the residence time of the mAb. Increased processing time not only impairs the economics of the production process but also facilitates enzymatic breakdown of mAbs (5).

COST-EFFECTIVE, ONE-STEP PRODUCTION OF NOVEL PROTEIN A–BASED RESINS


Figure 1: Transmission electron microscopy image of engineered E. coli showing accumulation of intracellular polyester beads (PolyBind–Z).
A new expression platform technology has been developed for the one-step production of protein A–based ligands already cross-linked to polyester beads. Escherichia coli has been successfully engineered to manufacture polyester beads that display multiple copies of the protein A–derived and antibody-binding Z domain (6). This new technology harnesses the natural polyester storage granule formation process inside bacteria for the production of ligand coated polyester beads (7) (see Figure 1). The polyester synthase (PS), which mediates polyester synthesis and bead formation, remains covalently attached to the polyester bead surface. PS was engineered to efficiently display multiple Z domains. A hybrid gene encoding the PS–Z domain fusion protein was constructed and introduced into E. coli harbouring the two genes phbA and phbB from Ralstonia eutropha. The phbA and phbB genes mediate conversion of the central metabolite acetyl–CoA to the activated polyester precursor, which is converted to polyester beads by the activity of the PS–Z domain fusion protein. This genetically engineered E. coli enabled efficient production of polyester beads displaying the PS–Z domain fusion protein at high density and functionality. These polyester beads (PolyBind–Z) were extensively analysed using gas chromatography-mass spectrometry and denaturing gel electrophoresis combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF–MS). Initial performance testing using Enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), and batch purification combined with protein quantification revealed a specific and high binding capacity for human immunoglobulin G (IgG) and various mAbs (6–8).


Figure 2: Upstream and downstream production schematic for PolyBind–Z.
Current commercial technologies for the manufacture of protein A resins require the fermentative production of protein A or its derivatives, the production of a carrier (i.e., agarose beads), and ultimately chemical cross-linking of the target protein to the carrier. This usually leads to random orientation of the protein accompanied by a decrease in specific activity/function, and requires the tedious removal of the often toxic cross-linker. Homogenous functional orientation and elimination of the need for cross-linker removal suggest further advantages of this new polyester bead-based technology. The upstream and downstream processing (see Figure 2) along with the superior functional performance of PolyBind–Z in high affinity bioseparation of antibodies will be discussed.


blog comments powered by Disqus

ADVERTISEMENT

ADVERTISEMENT

FDA Panel Unanimously Backs Secukinumab for the Treatment of Psoriasis
October 22, 2014
Amgen Sues Sanofi and Regeneron over Patent for mAb Targeting PCSK9
October 20, 2014
EMA Works to Speed Up Ebola Treatment
October 20, 2014
AbbVie/Shire Deal Officially Off
October 20, 2014
BioReliance Introduces New Predictive Assays
October 17, 2014
Author Guidelines
Source: BioPharm International,
Click here