NEW PROCESS FOR DEVELOPING AND MANUFACTURING CONJUGATE VACCINES
A new technology has been developed for the production of conjugate vaccines by an in vivo conjugation process. Instead of chemically conjugating polysaccharides to proteins, the conjugate is directly synthesized
in appropriately engineered E. coli cells. Because E. coli is one of the fastest, least expensive, and highest product-to-volume systems available for the production of large molecules,
the use of E. coli is appealing for the production of vaccines. However, until recently, it has not been possible to manufacture glycoprotein
conjugates using bacterial cells.
Despite the ubiquitous presence of polysaccharides at the surface of bacterial cells, bacteria were thought to be unable to
synthesize glycoproteins, and N-linked protein glycosylation was believed to be restricted to eukaryotes. The finding of N-linked glycoproteins in the human pathogen Campylobacter jejuni disproved this theory.
Various proteins of C. jejuni have been shown to be glycosylated by a heptasaccharide. This heptasaccharide is assembled on undecaprenyl pyrophosphate (UPP),
the carrier lipid, at the cytoplasmic side of the inner membrane by the stepwise addition of nucleotide-activated monosaccharides
catalyzed by specific glycosyltransferases. The lipid-linked oligosaccharide then flip-flops (i.e., diffuses transversely)
into the periplasmic space by the flippase PglK. In the final step of N-linked protein glycosylation, the oligosaccharyltransferase PglB catalyzes the transfer of the oligosaccharide from the carrier
lipid to Asn residues within the consensus sequence Asp/Glu-Xaa-Asn-Xaa-Ser/Thr, where Xaa can be any amino acid except Pro
The gene cluster encoding this glycosylation machinery was functionally expressed in E. coli, allowing the heterologous production of Campylobacter glycoproteins in E. coli (4) and providing the first opportunity to produce N-linked glycoproteins in E. coli. In addition, the consensus amino acid sequence was introduced into different proteins that are not glycosylated in their
original organism (see Figure 2).
Figure 2: Details of an engineered glycosylation pathway in Escherichia coli. Bacterial polysaccharide antigens are synthesized
by stepwise action of glycosyltransferases at the cytoplasmic side of the membrane and polymerized after flipping. The oligosaccharyltransferase
PglB is able to transfer a different polysaccharide from the carrier lipid to Asn within the consensus sequence because of
its relaxed specificity.
The N-linked protein glycosylation biosynthetic pathway of Campylobacter has significant similarities to the polysaccharide biosynthesis pathway in bacteria (5). Because antigenic polysaccharides
of bacteria and the oligosaccharides of Campylobacter are both synthesized on the carrier lipid, undecaprenyl pyrophosphate (UPP), the two pathways were combined in E. coli. The polysaccharide-synthesizing enzymes of different pathogens were expressed in the presence of the oligosaccharyltransferase
PglB and a protein carrier (6, 7). The antigenic polysaccharides assembled on UPP are captured by PglB in the periplasm and
transferred to a protein carrier. After fermentation of E. coli, the glycoconjugate is extracted from the periplasm and purified using simple and well-known manufacturing steps similar
to those used for production and purification of recombinant proteins (see Figure 3).
Figure 3: In vivo glycosylation system for production of bioconjugates in Escherichia coli system. The bioconjugate is extracted
from the periplasm and purified by column chromatography to high purity.
ADVANTAGES OF IN VIVO RECOMBINANT TECHNOLOGY
This in vivo technology to design and produce bioconjugates offers improved versatility, efficacy, safety, speed, and cost of development,
partly resolving the challenges that the vaccine industry is currently facing. Some of the specific advantages of the technology
are as follows:
- Bioconjugation is versatile, enabling the attachment of virtually any polysaccharide to virtually any protein. This versatility
permits the development of novel conjugates that cannot be addressed with existing chemistry-based processes.
- Bioconjugates are engineered to have a specific structure optimized for efficacy. Bioconjugate vaccines can be designed to
not only generate an immune response to the polysaccharide, but also to the protein from the target organism, thereby enhancing
efficacy. No free polysaccharide is present during bioconjugate production that can inhibit the immune response.
- Bioconjugates are produced in a standard, nontoxic bacterial production system, with no risk of contamination by mammalian
infectious organisms. Moreover, bioconjugates are engineered to a reproducible structure and final product, thus minimizing
potential safety concerns. This design will lower the regulatory barriers and potentially accelerate clinical development.
- Bioconjugate process development and production are rapid and straightforward. Producing vaccine by recombinant methods in
a standard E. coli expression system and using a conserved biosynthetic pathway that may differ slightly depending on serotypes is a well-understood
and commonly used manufacturing method.
From a technical perspective, the in vivo technology has the potential to provide uniform product, easily reproducible in a low-cost expression system, with an optimized
safety and efficacy profile. These factors may decrease the regulatory barrier and the time to market and result in reduced
development and manufacturing cost.
CHALLENGES OF IN VIVO RECOMBINANT TECHNOLOGY
The in vivo technology has the potential to overcome many issues that the chemical conjugation currently face in designing and producing
conjugate vaccines. However, the following challenges are still unresolved.
- Because of the complexity of several bacterial pathogens, some vaccine candidates are still difficult to design and produce
using in vivo recombinant technology. Bacterial pathogens such as N. meningitis B or Moraxella are challenging targets because the mechanism by which the antigenic sugar is assembled and expressed on the surface is less
suitable for the in vivo glycoconjugation technology.
- The bioconjugate process is still early in development and its ultimate potential and limitations are not fully delineated.
At this point, only data from preclinical and early clinical studies on a restricted number of pathogens are available. Additional
work is required regarding process and assay development (i.e., scalability).