Biomedical Engineering Reference
In-Depth Information
indicate the use of combination therapies, potentially making drug delivery more
expensive. Due to insecticide resistance by the mosquito and drug resistance by the
parasite, it is generally accepted that the development of a malaria vaccine is critical to
future efforts for the control (or possible elimination) of malaria.
The malaria parasite has a complex life cycle, which involves release of the parasite
(sporozoite) while probing for a blood vessel into the individual's skin before a blood
meal. The parasite travels to and develops within a liver hepatocyte, where it eventually
ruptures and releases parasites (merozoites) that cyclically invade and develop within
erythrocytes. Subsets of the parasitized erythrocytes develop into male and female
parasites (gametocytes) that are subsequently taken up by a mosquito during a blood
meal. Following this blood meal, fertilization occurs in the midgut of the mosquito and
the parasite then migrates through the mosquito midgut to replicate beneath the midgut
wall. The complexity of the malaria parasite life cycle, in which each stage is immuno-
logically distinct, has led to diverse approaches for vaccine development. The diverse
approaches include various stage-specific vaccine targets: sporozoite and liver stages or a
“pre-erythrocytic” vaccine; blood-stage or an “erythrocytic” vaccine, which would
include a pregnancy vaccine; and a transmission blocking vaccine. These stage-specific
vaccines would in principle provide sterile immunity, disease control, or reducedmalaria
transmission to humans. In conjunction with numerous stage-specific parasite targets,
there are numerous vaccine delivery systems being studied, including DNA vaccines,
attenuated sporozoite vaccines, viral-vectored vaccines, virus-like particle vaccines,
recombinant protein vaccines, or combinations of these different delivery systems (for a
summary of the above, see Ref. [3]). To date, the only malaria vaccine that has shown
some clinical efficacy is RTS/S, a pre-erythrocytic virus-like particle vaccine [4].
In the mid-1990s, the development of recombinant protein vaccines for malaria was
limited, in principle, to recombinant expression in Escherichia coli or baculovirus.
E. coli production necessitated the development of refold procedures for provision of
relevant protein structure while baculovirus production was limited by scalability of
fermentation and multiplicity of viral infection rates. Therefore, we (members of the
Malaria Group at EntreMed, Inc.) as well as others [5, 6] were interested to develop the
methylotrophic yeast Pichia pastoris, a eukaryotic expression system, for scalable
production of correctly folded recombinant malarial proteins from P. falciparum for
human clinical trials. However, based on initial experimentation, it became apparent that
a systematic approach would be needed to achieve this result.
Quality by Design (QbD) is a planned approach to pharmaceutical development that
may or may not use prior knowledge for planning purposes [7, 8]. QbD is currently being
applied to development of new pharmaceutical products and in particular to the
development of therapeutic monoclonal antibodies [9, 10]. In this chapter, the progres-
sion from preclinical development through pilot-scale production of several malarial
proteins following a QbD approach for development of a pharmaceutical drug substance
will be discussed. In the case studies described here, the following will be presented:
(1) use of a QbD approach to design a synthetic gene and its gene product (drug
substance) with specified critical quality attributes (CQAs) for production using the
P. pastoris expression system; (2) a comparison of codon usage in gene design for
P. pastoris expression of a malarial protein; and (3) impact of modifying the expression
