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.
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).
ALL FIGURES COURTESY OF THE AUTHORS
COST-EFFECTIVE, ONE-STEP PRODUCTION OF NOVEL PROTEIN A–BASED RESINS
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 1: Transmission electron microscopy image of engineered E. coli showing accumulation of intracellular polyester beads
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.
Figure 2: Upstream and downstream production schematic for PolyBind–Z.