Bioprocess and downstream optimization of recombinant human growth hormone in Pichia pastoris

Abstract

The methylotrophic yeast Pichia pastoris is a well-established expression host, which is often used in the production of protein pharmaceuticals. This work aimed to evaluate the effect of various concentrations of ascorbic acid in mixed feeding strategy with sorbitol/methanol on productivity of recombinant human growth hormone (r-hGH). The relevant concentration of ascorbic acid (5, 10, or 20 mmol) and 50 g/L sorbitol were added in batch-wise mode to the medium at the beginning of induction phase. The rate of methanol addition was increased stepwise during the first 12 h of production and then kept constant. Total protein and r-hGH concentrations were analyzed and the results compared with sorbitol/methanol feeding using one-way analysis of variance. Moreover, an effective clarification process using activated carbon was developed to remove process contaminants like pigments and endotoxins. Finally, a three-step chromatographic process was applied to purify the product. According to the obtained results, addition of 10 mmol ascorbic acid to sorbitol/methanol co-feeding could significantly increase cell biomass (1.7 fold), total protein (1.14 fold), and r-hGH concentration (1.43 fold). One percent activated carbon could significantly decrease pigments and endotoxins without any significant changes in r-hGH assay. The result of the study concluded that ascorbic acid in combination with sorbitol could effectively enhance the productivity of r-hGH. This study also demonstrated that activated carbon clarification is a simple method for efficient removal of endotoxin and pigment in production of recombinant protein in the yeast expression system.

Keywords: Activated carbon; Pichia pastoris; Pigment removal; ascorbic acid.

Figure 1

The effect of various ascorbic acid substrate concentrations on cell density. ****P < 0.0001; ns, non-significant, P = 0.6107.

Figure 2

The effect of different concentrations of sorbitol on total protein determined using Bradford protein assay. **P = 0.0018, ***P = 0.0007, ****P < 0.0001.

Figure 3

The effect of ascorbic acid concentration on r-hGH production. **P = 0.0029, **** P < 0.0001; ns, non-significant, P = 0.1929.

Figure 4

The effect of ascorbic acid concentration on total protein and r-hGH production and r-hGH to total protein ratio

Figure 5

(a) The effect of various concentrations of ascorbic acid on the level of protein expression, lane 1: methanol/sorbitol mixed-feed strategy, lanes 2, 3, and 4: ascorbic acid 5, 10, and 20 mmol respectively, lane 5: LMW calibration kit for SDS electrophoresis (14.4-97 kDa). (b) Densitometric analysis of fermentation run under optimal condition (lane 3).

Figure 6

The effect of activated carbon concentration on pigment removal. **P = 0.0011, ****P < 0.0001.

Figure 7

The effect of activated carbon concentration on pigment removal and r-hGH recovery

Figure 8

The effect of activated carbon treatment on endotoxin removal.

Figure 9

Shows the reverse phase high pressure liquid chromatography (RP-HPLC) chromatogram indicating the purity of recombinant human growth hormone (r-hGH) ≥ 98%.

Figure 10

(a) Size-exclusion (SEC) chromatogram of chemical reference standard (CRS) Batch 3. (b) SEC chromatogram of purified recombinant human growth hormone (r-hGH).

Figure 11

(a) Peptide mapping identification of purified recombinant human growth hormone (r-hGH). (b) Peptide mapping chromatogram of chemical reference standard (CRS) batch 3

Figure 12

(a) Reference electropherogram of charged variants distribution of somatropin chemical reference standard (CRS) batch 3, (b) Electropherogram of purified recombinant human growth hormone (r-hGH) (1 mg/mL) shows the purity of greater than 99%, I = impurity. I1, I2 (cleaved form), I3 (Gln-18 somatropin), I4 (deamidated forms).