Biomedical Engineering Reference
In-Depth Information
Figure
3.9. Comparison of Pfs25 production by Pfs25, Pfs25-PpPDI, and Pfs25-PfPDI clones.
Clarified fermentation supernatants collected at different time points were separated on
SDS-PAGE under nonreduced conditions and Coomassie blue stain. Accumulated Pfs25 (A) and
(B) recombinant proteins are indicated by small triangles on the right. Purified Pfs25 (A) and (B)
(1
g each) are shown in the right panel as control. Overexpressed PpPDI protein was identified
(asterisk) in Pfs25-PpPDI fermentation supernatant. Reprinted with permission from Elsevier [52].
m
overexpression (Fig. 3.10), which was also confirmed by a monosaccharide composi-
tion analysis showing a fivefold reduction in mannosylation [48], provided for a
significant increase in quality as well. The benefits of overexpression of PpPDI on the
productivity of other P. pastoris-expressed malarial proteins have been observed
(Narum, unpublished and Ref. [44]).
3.4 SUMMARY
A QbD approach for molecular design has improved the quality and quantity of
recombinantly expressed malarial proteins. The capacity to produce malarial proteins
using recombinant expression systems has improved significantly with the introduction of
synthetic genes for P. pastoris. Although previous empirical information played an
important part in the early stages of the QbD approach, the targeted molecular design
of malarial genes for expression in P. pastoris has enabled the development of processes
that are suitable for manufacturing at pilot scale (Table 3.4). The P. pastoris production
clones discussed here all achieved production levels at or above 1000 vaccine doses/L
broth, which indicates their suitability for further scale-up. The use of synthetic genes not
only enables the expression of a particular protein but also permits appropriate modifica-
tions to increase the CQAs related to the quality of the bulk drug substance (Table 3.4).
Recombinant EBA-175 RII, AMA1, and Pfs25 were manufactured at pilot scale following
cGMP and the drug substances appear to mimic native malarial proteins, as indicated by
induction of antiparasite antibodies. Finally, further augmentation in both quantity and
quality appear possible by the overexpression of host chaperones such as PDI. These
measures require planned development; the result is a quality product bymolecular design.
