Biomedical Engineering Reference
In-Depth Information
host by overexpressing a chaperone identified as protein disulfide isomerase (PDI).
These approaches have facilitated the development of conformance parameters in which
critical process parameters and CQAs of the bulk drug substances were preset,
including parameters such as protein structure, glycosylation, functional activity,
biochemical characteristics, and so on (for a complete summary of important param-
eters, see Table 3.2). The manufacturability at pilot scale was also established such that
the production levels provided sufficient quantities of a quality bulk drug substance. The
process parameters for production were divided into four parts: molecular design of
the gene and gene product, fermentation, recovery of a secreted material, and purifica-
tion. CQAs of the bulk drug substance were evaluated throughout the production process
to understand and characterize the functional relationships between process parameters
and drug substance, which is part of a QbD approach [7, 8].
For the purpose of this chapter, three malarial proteins will be discussed with
regard to their development following a QbD approach. Two erythrocytic P. falciparum
merozoite proteins, the erythrocyte binding antigen-175 (EBA-175) and the apical
membrane antigen 1 (AMA1), will be discussed with regard to their expression in
P. pastoris and subsequent characterization of the bulk substances. A third recombinant
malarial protein, Pfs25, which is a 25 kDa transmission blocking vaccine target [11], will
be discussed briefly regarding its expression and biochemical characterization when
influenced by co-overexpression of PDI [12]. Both the blood-stage malaria proteins are
involved in merozoite invasion of erythrocytes based on in vitro growth studies and
in vivo protection studies [13, 14]. EBA-175, or more specifically a region identified
as region II (RII) (Fig. 3.1), binds sialic acid residues on its receptor glycophorin A,
in conjunction with the peptide backbone on erythrocytes [15]. Antibodies generated
against RII inhibit parasite invasion in vitro [16] and provide protection against a virulent
Figure
3.1.
Scheme of gene structure of EBA-175 showing RII including F1 and F2 domains and
the division of six other regions (upper panel). The lower panel shows the native EBA-175 RII
amino acid sequences in which the putative N-linked glycosylation sites are identified (NxS/T) by
amino acid position using the sequence identified by accession number XP_001349207. The five
aminoacid substitutions used for expressionof EBA-175RII-NGare shown ingray. Thepositionof
putative N-linked glycosylation sites are noted by asterisks. CD, cytoplasmic domain; SS, signal
sequence; TM, transmembrane domain.
