Downstream Processing

Ohmura et al. described the application of packed-bed adsorption (PBA) for the purification of rHSA
requiring filtration and chromatography steps. Alternatively, using expanded-bed adsorption (EBA) the
product can be captured directly from the cell containing fermentation broth. Sumi et al. reported significant
improvements by a 50% reduction of the downstream time and a 45% increase of the overall yield compared
with the results using the conventional purification method. Some cost comparisons of EBA and PBA
processes have been published, e.g. by Curbelo et al. for bovine serum albumin from P. pastoris broth and by
Amersham Biosciences for monoclonal antibody.

In this case study, we compare the use of EBA and PBA as alternative downstream routes in the production of
rHSA (EBA process, PBA process). Figure 11.2 shows the flow diagrams of the recovery section for the EBA
and PBA process. The application of the EBA process allows the removal of at least three downstream
processing steps (microfiltration and two ultrafiltrations) that is expected to lead to a better purification yield
and a reduction in the purification time. The process flow diagrams of the bioreaction section and the
downstream section following the EBA step and the PBA step, respectively, are identical in both cases (as
shown in Figure 11.1).

EBA Process. Under the acidic conditions necessary for the EBA that uses strongly cationic adsorbent
particles, rHSA would be rapidly degraded by proteases contained in the culture medium. Therefore, the
proteases are inactivated by heating the fermentation broth to 68 ◦C for 30 minutes in the presence of 10mM
sodium caprylate as stabilizer and 10 mM cysteine and 100 mM aminoguanidine hydrochloride to suppress
the coloration caused by heating. The heat treatment is done in the bioreactor (P-3), saving an additional tank.
To represent the rHSA denaturation due to the heat treatment, a reaction operation is used with a reaction
extend of 4% converting rHSA into waste proteins.

The heated solution has an electric conductivity of about 20 mS/cm. An optimum binding of rHSA to the
strongly cationic adsorbent particles is at an electrical conductivity of 8–12 mS/cm. For a better adsorption,
the solution is diluted (1:1) in the vessel P-8 using acetate buffer (50 mM) and distilled water. The pH value is
adjusted to 4.5 using acetic acid.

The diluted solution containing the cells is then loaded upwardly into the EBA column (P-9), which has been
equilibrated with an acetate buffer. The target rHSA binds to the adsorbent particles, while impurities are
discarded. The equilibration buffer is used for washing. rHSA is recovered by feeding downwardly a
phosphate buffer (100 mM, pH 9).

For decolorization, the rHSA solution is then heat-treated again in P-10 at 60 °C for 1 hour in the presence of
10 mM cysteine, 5 mM sodium caprylate, and 100 mM aminoguanidine hydrochloride at pH 7.5. The solution
is then adjusted to pH 6.8 using phosphoric acid and the salt concentration is reduced to 200 mM by adding
water. The adjusted solution is loaded onto the hydrophobic interaction chromatography (HIC) column (P-
11), where impurities are retained.

In next step, the salt concentration of the solution is again reduced to about 100 mM (P-12), and loaded onto
the anion exchanger (P-13). rHSA flows through the column, while coloring matters and trace impurities are
removed. In the diafiltration step (P-14) the phosphate buffer (pH 6.8) is replaced by an acetate buffer (pH
4.5), which is required for the chelate resin treatment (P-16). The chelate resin treatment again retains
coloring matter. Pyrogens are removed by ultrafiltration (P-17, molecular weight cut-off: 100 kDa). In a
further ultrafiltration step (P-18), the solution is concentrated and then freeze-dried (P-21).

PBA Process. In the PBA process, the cell biomass is separated from the fermentation broth by microfiltration
(P-23). The solution is then further concentrated by ultrafiltration (P-24). The proteases in the solution are
inactivated by heat in P-9 at 60 ◦C for 1 hour (also for decolorization). The heat-treated solution is adjusted to
pH 4.5 using acetic acid. Polymerized high-molecular-weight contaminants are removed by ultrafiltration (P-
25). The rHSA solution is then loaded onto the PBA column (P-26), where the product is retained. Acetate
buffer is used for washing and for equilibration of the column. Similar to the EBA process, rHSA is recovered
by feeding in a phosphate buffer. The further purification steps (P-10 and subsequent units) are identical to
the EBA process.