Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-02-08
2002-08-06
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069300, C435S235100, C435S254200, C435S254210, C435S254220, C435S254230, C435S320100, C435S477000, C530S350000, C536S023100, C536S023720
Reexamination Certificate
active
06428984
ABSTRACT:
The present invention relates to a method for recovering recombinant HBsAg from HBsAg expressing yeast cells.
The destruction of the cell wall of a microorganism—cell disruption—is the first step in recovering intracellular, biologically active proteins. On a production scale, mechanical disruption methods, such as wet milling in ball mills, have turned out to be suited in recent years.
The high-speed agitator ball mills of a closed constructional type have developed from the conventional ball mills used in pigment processing because of the continuously increasing demands made on comminution and dispersion. They consist of a hollow cylinder rotating about a vertical or horizontal axis, which is filled with mixed oxide beads of glass or zirconium up to a filling degree of 80%. The rotational speed can here not be increased in any desired way for increasing impact/pressure crushing because the centrifugal forces compensate the grinding effect. Furthermore, at increased speeds or filling degrees thermal problems arise that might damage the product to be recovered. A continuous grinding material/grinding body separation is carried out with a sieve cartridge, a rotating sieve gap or with a coaxial annular gap integrated into the bearing housing. The grinding material is cooled via a double jacket around the grinding container; the rotating shaft can also be cooled in part. The disruption principle of wet milling consists in transmitting motional energy from the agitator to the grinding body. This is predominantly done by adhesion forces in combination with displacement forces that are due to the assembly and type of agitator elements. The grinding chamber is activated by this and by cohesive forces. The acceleration of the grinding body in radial direction effects the formation of a laminar flow. Depending on the absolute speed and the size of the grinding bodies moved, the differential speed profiles between the grinding body layers effect high shear forces which apart from collision results are mainly responsible for the destruction of the cell wall of the microorganism.
Cells walls can also be broken mechanically by high pressure homogenizers. Such a device consists essentially of a high-pressure piston pump and a homogenizing unit. A homogenization valve will open when the set pressure is reached, the cell suspension being then pressed through the valve unit at a very high speed. The cell suspension is thus heated by about 2.5° C. per 100 bar. After having left the disruption valve unit the cell suspension is cooled by means of a heat exchanger. Thus, in contrast to wet milling, the disruption material is here heated for a short period of time. While passing through the homogenizing unit the cell suspension is subjected to very high turbulence, cavitation and shear forces. The disruption principle of a high pressure homogenizer consists in the sudden reduction of the energy density in the cell suspension within an extremely short period of time, i.e. the high pressure difference and the rapid pressure drop can be regarded as the main factor for the degree of disruption.
Both mechanical disruption methods are used for different microorganisms. The selection of the respective method depends on the type of the microorganism, in particular on its structure. For instance, both cell disintegration methods are tested in Pittroff, M. et al., DECHEMA-Biotechnol. Conf.; (1990), 4, Pt.B, 1055-60 for various microorganisms, such as
Saccharomyces cerevisiae, Micrococcus luteus
and
Escherichia coli,
and the conclusion could be drawn that morphological differences in the microorganism affect the disintegration performance of the two methods.
For instance, Pittroff, M. et al., DECHEMA-Biotechnol. Conf.; (1992, 5, Pt.B, 687-91, and Pittroff, M. et al, Chem. Ing. Techn.; (1992), 64, 10, 950-53 point out that the high-pressure disruption method is preferably carried out for specific microorganisms, such as rod-shaped bacteria. By contrast, the ball milling method is preferably used for cocci having a round structure. The same disintegration degree was observed for both methods in the disruption of yeast cells of the species
Saccgarintces cerevisiae.
Furthermore, Luther, H. et al., Acta—Biotechnol.; (1992), 12, 4, 281-91, describe a further comparison between ball mills and high pressure homogenizers for purifying proteins from
Saccgarintces cerevisiae
or
Escherichia coli.
It is confirmed that both yeast and bacterial cells can be disrupted using ball mills or high pressure homogenizers. It has been found to be a disadvantage of high pressure homogenizers that slime-forming microorganisms or mechanical contamination very easily lead to occlusions. It is pointed out with respect to yeast cells that the high pressure homogenizer destroys fewer cells. Although this has not been mentioned explicitly, a lower yield from the high pressure homogenizer has to be assumed.
Schütte, H., Biol. Recombinant-Microorg. Anim. Cells; (1991) Oholo 34 Meet., 293-305 also confirm that high pressure homogenizers effect an efficient disruption of yeast and bacterial cells, but are not very suitable for mycelial organisms.
Saccgarintces cerevisiae
disruption using a high pressure homogenizer was studied in Baldwin, C., Biotechnol. Tech; (1990), 96, 4, 329-34. It was found that the breakage of cells by means of a high pressure homogenizer generally gave low disruption yields (40% in 5 passes). A total disruption of yeast cells in the high pressure homogenizer could only be observed if an enzyme treatment with zymolase from
Oerskovia xanthineolytica
had been performed previously.
As becomes further apparent from the extensive studies that have so far been carried out, the type of the protein to be purified is of decisive importance to the selection of the respective disruption method. Of particular interest is here the cell disruption of hepatitis B surface antigen (HBsAg)-expressing cells.
Choo, K. B. et al., Biochem. Biophys. Res. Commun.; (1985), 131, 1, 160-66 study the use of ball mills for the disruption of
Saccgarintces cerevisiae
cells for the recovery of HBsAg. However, only a low amount of HBsAg could be extracted in particulate form.
Fenton D. M. et al., Abstr. Ann. Med. Am. Soc. Microbiol.; (1984), 84 Meet. 193 describe the release of recombinant HBsAg from
Saccgarintces cerevisiae
by means of cell disruption in a high pressure homogenizer. It is pointed out in this document that the solubilization of HBsAg in an active, i.e. antigenic, form on a large scale poses problems. Sufficient cell breakage and satisfactory protein release were only observed in a high pressure homogenizer after ten passes, and a maximum HBsAg release could only be observed after 15 passes. The authors drew the conclusion that the release of antigenically active HBsAg requires the disruption of subcellular structures.
Hence, up to now it has not been possible with the two disruption methods to provide suitable systems for the optimum production of HBsAg by cell disruption.
It is therefore the object of the present invention to develop a method for recovering a high yield of recombinant HBsAg from recombinant microorganisms.
According to the invention this object is achieved by a method for recovering recombinant HBsAg, wherein recombinant methylotrophic yeast cells which are capable of expressing HBsAg are disrupted using a high pressure homogenizer, and HBsAg is recovered from the cell debris obtained.
Surprisingly enough, it has been found that in the cell disruption of HBsAg-expressing
Hansenula polymorpha
cells in a high pressure homogenizer a considerably higher product yield per g of cell dry weight could be achieved than with the conventional methods, in particular the cell disruption of
Saccharomyces cerevisiae
by means of a high pressure homogenizer or glass bead mills. The method of the invention is thus a considerable improvement over the formerly known methods used for recovering HBsAg from microorganisms.
In the method of the invention, recombinant HBsAg is recovered from recombinant methylotrophic yeas
Pointek Michael
Weniger Michael
Carlson Karen Cochrane
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Kam Chih-Min
Rhein Biotech Gesellschaft fur Neue Biotechnologische Porzesse u
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