Biomedical Engineering Reference
In-Depth Information
TABL E 3.1. Codon Usage for Most Abundant Amino Acid for
P. falciparum, H. sapiens,
and
P. pastoris
Amino
Acid
P.
falciparum
H.
sapiens
P.
pastoris
Amino
Acid
P.
falciparum
H.
sapiens
P.
pastoris
A
GCA
GC C U
L
UUA
C U G G
R
AGA
C G G
AGA
K
AAA
AA G G
N
AAU
AA C
AAU
M
AUG
AUG
AUG
D
GAU
GA C
GAU
F
UUU
UU C
UUU
C
UGU
UG C
UGU
P
CCA
CC C
CCA
Q
CAA
CA G
CAA
S
AGU
AG C
UC U
E
GAA
GA G
GAA
T
ACA
AC C C U
G
GGU
GG C
GGU
W
UGG
UGG
UGG
H
CAU
CA C
CU Y U C C
I
AUU
AU C
AUU
V
GUA
GU G U
Nucleotide that varies with respect to P. falciparum is underlined.
species in the organism [28]. Depending on the organism, each amino acid, with the
exception of methionine and tryptophan, can be encoded by two-six different synony-
mous codons. The frequencies at which these synonymous codons are used depend on
the level of protein expression and differ among organisms (Table 3.1). The Malaria
Group at EntreMed, Inc. previously reported that a mammalian codon optimized
synthetic gene encoding EBA-175 RII expressed by a DNA vaccine plasmid increased
EBA-175 RII protein expression levels in vitro and immunogenicity levels in vivo [29].
The mammalian codon optimized EBA-175 RII gene lacked the stretches of poly A's
which were responsible for the failed expression in P. pastoris. Thus, at the same time, a
parallel development effort was ongoing to evaluate whether the synthetic EBA-175 RII
gene would overcome the problem of early transcription termination events, which had
been observed in P. pastoris with the native gene, and thereby express full-length RII
protein. A similar approach was being used by two other groups (The Malaria Vaccine
Development Branch (MVDB), National Institute of Allergy and Infectious Disease,
National Institute of Health, Rockville, Maryland, and The Biomedical Primate
Research Centre, Rijswijk, The Netherlands), along with EntreMed, Inc. for the
production of P. falciparum AMA1 in P. pastoris [5, 30].
Each group independently aimed at developing an approach that would allow the
manufacturing of recombinant malarial antigens, which would maintain CQAs of the bulk
drug substances that were previously observed for the two recombinant baculovirus
proteins (EBA-175 RII [31] and AMA1 [32]). In addition, it was necessary to identify
a production process that was suitable for manufacturing a sufficient quantity of bulk
product using a robust process. The quality of the bulk drug substance was also important
with regard to its integrity, purity, functional activity, and so on (see Table 3.2 for a
complete list). In the following sections, the molecular (gene) design for recombinant
expression of these two malarial proteins in P. pastoris is presented. Limited char-
acterization of the CQAs for the EBA-175 RII bulk drug substance is presented, which
justify theQbDapproach used here that lead to its cGMPpilot-scale production. Regarding
 
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