Proteomic profiling of recombinant Escherichia coli in high-cell-density fermentations for improved production of an antibody fragment biopharmaceutical

Abstract

By using two-dimensional polyacrylamide gel electrophoresis, a proteomic analysis over time was conducted with high-cell-density, industrial, phosphate-limited Escherichia coli fermentations at the 10-liter scale. During production, a recombinant, humanized antibody fragment was secreted and assembled in a soluble form in the periplasm. E. coli protein changes associated with culture conditions were distinguished from protein changes associated with heterologous protein expression. Protein spots were monitored quantitatively and qualitatively. Differentially expressed proteins were quantitatively assessed by using a t-test method with a 1% false discovery rate as a significance criterion. As determined by this criterion, 81 protein spots changed significantly between 14 and 72 h (final time) of the control fermentations (vector only). Qualitative (on-off) comparisons indicated that 20 more protein spots were present only at 14 or 72 h in the control fermentations. These changes reflected physiological responses to the culture conditions. In control and production fermentations at 72 h, 25 protein spots were significantly differentially expressed. In addition, 19 protein spots were present only in control or production fermentations at this time. The quantitative and qualitative changes were attributable to overexpression of recombinant protein. The physiological changes observed during the fermentations included the up-regulation of phosphate starvation proteins and the down-regulation of ribosomal proteins and nucleotide biosynthesis proteins. Synthesis of the stress protein phage shock protein A (PspA) was strongly correlated with synthesis of a recombinant product. This suggested that manipulation of PspA levels might improve the soluble recombinant protein yield in the periplasm for this bioprocess. Indeed, controlled coexpression of PspA during production led to a moderate, but statistically significant, improvement in the yield.

FIG. 1.

Process for manufacture of anti-CD18 F(ab′)₂-LZ. (a) Idealized fermentation profiles for the process. The arrows indicate sampling times. (b) Growth curves for control and production runs. Symbols: □ and ▪, replicate control fermentations of 59A7 harboring pBR322; ▵ and ▴, replicate production fermentations of 59A7 harboring pS1130. (c) Total amounts of light chain (LC) and heavy chain (HC) expressed over time in a production fermentation. The results were derived from a single reversed-phase HPLC assay, but the trends and values are representative of the results of duplicate assays. Symbols: ▪, recombinant light chain; ▴, heavy chain.

FIG. 2.

Two-dimensional protein profiles for end-of-run (72-h) samples for control (A) and production (B) gels. Qualitative (on-off) protein spot changes are indicated by circles. Green crosses indicate protein spots detected in production fermentations but not in control fermentations. Blue triangles indicate protein spots detected in control fermentations but not in production fermentations. Red plus signs indicate protein spots that were up-regulated in production fermentations compared to control fermentations, and red circles indicate protein spots that were up-regulated in control fermentations compared to production fermentations. Prominent protein spots that were up-regulated in the production gel and which were identified as anti-CD18 light chain (anti-CD18 LC) and PspA are labeled. IEF, isoelectric focusing; SDS, sodium dodecyl sulfate.

FIG. 3.

Zoomed-in views of 2-D gels for control and production fermentations at 14 and 72 h. (a) Recombinant antibody expression in production fermentations and corresponding regions of the gels for control fermentations. Dominant and minor light chain (LC) isoforms and heavy chain (HC) are indicated. The heavy chain was not highly soluble in the 2-D gel system, so it migrated as a smear. (b) Phosphate starvation proteins UgpB and PstS, which were quantified, and PhnD, which was not easily quantified in the gels.

FIG. 4.

Protein quantity dynamics over the course of the fermentations. Time course graphs are grouped into the following categories (reading from left to right): anti-CD18 light chain (LC) isoforms (a), phosphate starvation proteins (b), ribosomal proteins (c), nucleotide biosynthesis proteins (d), nucleotide degradation proteins (e), oxidative damage defense proteins (f), and stress proteins (g). The red circles are data for samples from the production fermentations. The blue triangles are data for samples from the control fermentations. The data are the geometric mean spot quantities for the four gels corresponding to each sample point. The error bars were generated as described in Materials and Methods. The protein quantities represent the sum of isoforms in many cases.

FIG. 5.

Western blot analysis conducted with anti-PspA antisera to show differential PspA induction from pPspA. IPTG was added at different levels in replicate fermentations at 29 h. Samples for immunoblot analysis were taken 6 h postinduction.

FIG. 6.

Effects of manipulating PspA expression with various levels of IPTG prior to product induction on recombinant antibody yield. IPTG was added at 29 h. The bars indicate averages of three independent fermentations for addition of no IPTG or 0.02 mM IPTG, and the error bars indicate one standard deviation. Soluble, assembled F(ab′)₂-LZ was measured by protein G affinity chromatography.