The downstream processes in large-scale industries for producing biotechnological products have been using diafiltration/ultrafiltration processes since the 1970s; conventional centrifugation, lyophilization, and precipitation were replaced or supplemented with industrial ultrafiltration membranes. Today, scientists have addressed two primary objectives:
(1) biological products concentration from dilute solutions (ultrafiltration); and
(2) removing small molecules by buffer exchange (diafiltartion).
Advent of the hepatitis B vaccine saw the vaccine industry undergoing a renaissance with an extensive range of new products in the 1980s. Most downstream processes used membrane processes in various forms. Vaccine antigens are extracted from three sources namely:
(1) sub-unit vaccines that contain recombinant proteins, which are expressed in prokaryotes or eukaryotes (e.g., hepatitis B surface antigen that is expressed in yeast; lipoprotein Osp A (Lyme disease vaccine) that is expressed in E.coli;
(2) toxigenic or infectious bacterial strains that produce either capsular polysaccharides or proteins as antigens, which are inactivated (e.g., antigen derived from Bordetella pertussis is used for whopping cough vaccines; capsular polysaccharides derived from Haemophilus influenza is used for Haemophilus type B vaccine); (3) live-attenuated or inactivated viral vaccines(e.g., measles, polio, hepatitis A, and mumps).
Energix B Vaccine
The SmithKline Beecham's vaccine, called Energix B, is used against hepatitis B infection. It contains an immunogen (hepatitis B surface antigen). It is first known recombinant vaccine to have received Europe's regulatory approval. Since then, the vaccine has been introduced in more than 120 countries for worldwide applications.
The surface antigen is a recombinant protein (24-kDa) that is expressed in Saccharomyces cerevisiae and accumulates in the recombinant yeast cell's vacuole during the growth stage. The antigen's expression is promotor regulated and controlled by the physiological grade of yeast cells during fermentation. The accumulation of the antigen occurs in parallel with cell growth. The fermentation scale for the growth of yeast cells is 1600 L (fed-batch process end) and upon harvest, the fermentation broth contains about 160 kilogram of yeast cell mass. Harvesting recombinant antigen from yeast cells follows centrifugation, precipitation steps, cell disruption, and clarification. The final two steps involved are the ultrafiltration and concentration steps that take place after the concentrated antigen undergoes purification (antigen) and filtration (sterile).
This process has to be repeated multiple times to extract the necessary recombinant antigen in its soluble form. The processing operations remove potential process impurities. Precipitation occurs upstream of the diafiltration/ultrafiltration; high-volumes of 3000 to 4000 L is processed. In the initial stages, the recombinant antigen transforms from monomer soluble form to spherical particulate form. These particles are observed via an electron microscopy. The mean diameter of these particles is 26 nm and the mean density is 1.2 g/cc. The spherical form of the antigen is maintained through the whole manufacturing process so that it is present in the final vaccine. The purified antigen is released in bulks through quality control tests and is adsorbed onto aluminum hydroxide to be released as final vaccine (Energix B).
An important element involved in recombinant surface antigen production is the ultrafiltration and diafiltration of crude antigen preparation that takes place at the extraction process end. The primary purposes of diafiltration/ultrafiltration are to:
(1) reduce process volumes from 4000 L to about 20 L;
(2) prepare crude antigen fraction for chromatography steps;
(3) eliminate lipid impurities and bulk proteins in the ultra-filtrate of antigen diafiltration process; and
(4) transform the preparation of soluble crude antigen into buffer system, which is compatible with chromatographic medium. The advantages lies in the fact that the recombinant protein is present in its spherical form and not in its monomeric soluble protein form (24-kDa). This enables an ultrafiltration membrane application with relatively high size exclusion of 50, 000 Da.
Sanitizing and maintaining the crossflow equipment is as important as the ultrafiltration/diafiltration step. The steps involved in maintaining the whole system include: prewash; system integrity testing; post-wash maintenance; and cycle use of cartridges.
On the whole, the diafiltration/ultrafiltration of crude product solution that is generated during Energix B (bulk) manufacturing process represent an important element in the process of manufacturing; nearly 90 percent of lipid impurities and 95 percent of protein impurities can be removed. The buffer conditions and product concentration established during diafiltration/ultrafiltration allows subsequent product solution processing.
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