Recombinant vaccine protein purification

FIGURE 10 Optimization of elution salt step for a recombinant vaccine protein purification. A step elution ladder with increments of 100 m M salt for 5 min durations, from 0 to I /VI was used MA, Sartobind Q-15 (anionic 100 cm2). Load conditions double load, 15 m/VI Tris, pH 8.1 flow rate 5 mL/ min. Most of the vaccine protein elutes in the 200 m/VI salt step. FT indicates the flow through peaks. (Courtesy of R. McMaster, Aventis Pasteur.)... 

FIGURE I I Optimization of pH elution steps for recombinant vaccine protein purification. Incremental pH step gradients 5.0, 5.5, 6.0, 6.5, and 8.0 are shown. Membrane 100 cm2 C (Sartobind Cl00) Load conditions 15 m/VI citrate, pH 4.75, flow rate 7 mL/min. The product elutes at pH shift of 5.5. (Courtesy of R. McMaster, Aventis Pasteur.)... 

A. Purification of Proteins on MA Case Study of Recombinant Vaccine Protein... 

Figure 5.6 Positive-ion electrospray spectrum obtained from the major component in the LC-MS analysis of a purified recombinant 62 kDa protein using a Cig microbore 50 X 1 mm column and a flow rate of 50 p.lmin . The starting buffer (buffer A ) was 0.1% TEA in water, while the gradient buffer (buffer B ) consisted of 0.1% TEA in acetonitrile-water (9 1 vol/vol). The running conditions consisted of 0% B for 5 min, followed by a linear gradient of 100% B for 55 min. Reprinted from J. Chromatogr., B, 685, McAtee, C. P., Zhang, Y., Yarbough, P. O., Fuerst, T. R., Stone, K. L., Samander, S. and Williams, K. R., Purification and characterization of a recombinant hepatitis E protein vaccine candidate by liquid chromatography-mass spectrometry , 91-104, Copyright (1996), with permission from Elsevier Science.

Recombinant proteins with unique properties can potentially generate new markets and penetrate into existing markets if they can be supplied on a large scale. An ideal system would produce the safest biologically active material at the lowest cost, and would be used in combination with an inexpensive and simple purification process. So far, there have been several examples of the high-yield production of recombinant proteins in transgenic crop plants, mainly in the area of molecular medicines such as antibodies, enzymes and vaccines [45, 48-50]. Modern agricultural practices offer... 

Figure 3.9. Generalized overview of the industrial-scale manufacture of recombinant E2 classical swine fever-based vaccine, using insect cell culture production systems. Clean (uninfected) cells are initially cultured in 500-1000 litre bioreactors for several days, followed by viral addition. Upon product recovery, viral inactivating agents such as /i-propiolactone or 2-bromoethyl-iminebromide are added in order to destroy any free viral particles in the product stream. No chromatographic purification is generally undertaken as the product is substantially pure the cell culture media is protein-free and the recombinant product is the only protein exported in any quantity by the producer cells. Excipients added can include liquid paraffin and polysorbate 80 (required to generate an emulsion). Thiomersal may also be added as a preservative. The final product generally displays a shelf-life of 18 months when stored refrigerated...

Display on bacteria can have various applications, such as the generation of recombinant bacterial vaccines, the screening of peptide libraries, and the use of cells as a source of immobilized proteins for purification or for enzymatic processes [2]. 

It is currently unclear whether, based on the current state of the art, a patent following the above-mentioned example could be extended to recombinant derivatives of the native protein. One might argue that, once the native protein is known and accessible, it needs no inventiveness to sequence the amino acids for parts of this protein, synthesize the corresponding DNAs, use these as probes to identify and isolate the entire coding sequence of the protein, which is then inserted into a suitable expression system to produce the protein in any desired form and quantity. Experience, however, teaches that it still requires some non-obvious steps and usually more than a limited degree of experimentation (often even a stroke of luck) to get there and to achieve the desired utility with recombinant polypeptides. For a vaccine it may be necessary to find and express the important epitopes in an appropriate (still unknown) way and to develop adequate purification and further processing protocols (with unpredictable technical... 

Inorganic membranes have been mentioned in other usage in the biotechnology and pharmaceutical industries. For example, ziiconia and alumina-based ceramic membranes have been incorporated in the following operations [Cueille and Ferreira, 1989] purification and concentration of antibiotics, vitamins, amino-acids, organic acids, enzymes, biopolymers and biopeptides for the fermentation steps in the more conventional applications human blood derivatives, vaccines, recombinant proteins, cells culture and monoclonal antibiotics in newer applications and pyrogen lemoval for ultrapure water. 

Reprinted from McAtee et. at. "Purification and Characterization of a Recombinant Hepatitis E Protein Vaccine Candidate by Liquid Chromatography-Mass Spectrometry" with kind permission from Elsevier Science-NL, Sara Burgerhartstraat 25, 1055 KV Amsterdam, The Netherlands. 

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