Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/88—Lyases (4.)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y401/00—Carbon-carbon lyases (4.1)
  • C12Y401/01—Carboxy-lyases (4.1.1)
  • C12Y401/01015—Glutamate decarboxylase (4.1.1.15)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• GAD glutamic acid decarboxylase
• the present invention relates to a novel and improved method of producing glutamic acid decarboxylase.
• the invention relates to a novel procedure for purifying the enzyme from recombinant cells engineered to produce the enzyme or from native cells or tissues containing it.
• IDDM Insulin-dependent diabetes mellitus
• One therapeutic strategy is aimed at preventing autoimmune destruction of pancreatic beta cells via a process of "tolerisation” whereby treatment with specific autoantigens can result in immunological "re-programming” to re-establish appropriate "self from "non-self distinction.
• GAD glutamic acid decarboxylase
• GAD antibodies and GAD-reactive T-cells identifies this protein as an autoantigen and implicates its involvement in the autoimmune destruction of pancreatic beta cells - the patho-aetiological "hallmark" of IDDM.
• 89:2115-2119 used a 180 bp fragment of the rat GAD65 cDNA sequence (Eriander et al. 1991 Neuron 7:91-100 all of which are incorporated by reference) to probe a human hippocampus cDNA library.
• Non-ionic detergents Triton X-100, Triton X-l 14 and n-octylglucoside, as well as the ionic detergent: sodium dodecyl sulphate - are also specified in WO95/04137
• Tritons are not chemically-defined (thereby imposing difficulties regarding definition of a GMP-compliant process)
• Triton-solubilised proteins (including rhGAD65) are not easily dialysed or ultrafiltered. Accordingly, the inclusion of Triton X-l 14 is likely to complicate downstream processing of rhGAD65.
• Triton X-l 14-extracted insect cells expressing rhGAD65 have been found to have high viscosity - resulting in prolongation of chromatographic application times (by imposing extremely low chromatographic flow-rates). Consequently, the use of Triton X-l 14 extends run times considerably, and its inclusion is assessed by us as not enabling a cost-effective process.
• Triton X-l 14 extraction method was discovered by us to result in the co- extraction of the baculoviral glycoprotein "gp64" with rhGAD65. Furthermore, as these two proteins are not readily distinguishable (they co- migrate in SDS-PAGE) it is considered advantageous to avoid their co- purification at an early purification stage.
• Triton X-l 14 extraction method of Bordier (1981) involves 1-3 condensation steps - whereby the detergent-rich phase containing solubilised hydrophobic proteins (i.e. including rhGAD65) is separated from hydrophilic proteins in the water-rich phase (by lowering the temperature below the "cloud point" of 20°C).
• This method therefore, is not readily-scaleable, and inclusion of Triton X-l 14 condensation steps for extraction and partial purification will significantly lengthen a commercial manufacturing process.
• n-octylglucoside this is regarded as an inferior detergent due its high cost and because it readily allows microbial growth, and is not therefore conducive to a scaled-up manufacturing process.
• sodium dodecyl sulphate it is relevant to mention that this is reported (in WO95/04137) as effective at 0.1%, but completely eliminates rhGAD65 enzyme activity at 1.0%.
• x is an integer from 1 to 5 and y is an integer from 2 to 20, such as N- dodecyl-N,N-dimethyl-3-ammonio-l -propane sulphonate, surprisingly good purification results are achieved at the same time as most of the above mentioned problems are solved.
• the zwitterionic detergent is preferably used both when extracting proteins from GAD-containing cells and during subsequent purification steps, such as ion exchange chromatography.
• the use of the zwitterionic detergent/surfactant surprisingly provides an internal reference check of IEX resolution as it separates another, ca. 65kDa baculoviral chitinase protein from rhGAD 65. This chitinase protein is different from the earlier mentioned baculovims 64 kDa glycoprotein and is likely to remain a contaminant in rhGAD 65 preparations where Triton or Octylglucoside have been used.
• the present invention relates to using a zwitterionic detergent/surfactant, preferably of a type disclosed in the appended claims 4-5, in a process for purifying glutamic acid decarboxylase (GAD).
• GAD glutamic acid decarboxylase
• the invention also relates to compositions obtainable by the process, which compositions comprises GAD and a zwitterionic detergent/surfactant. These compositions are suitable as pharmaceutical for treating IDDM, cancer, etc. They can also be used as vaccine compositions against autoimmune diseases such as IDDM, rheumatoid arthritis etc. Moreover, the compositions can be used for determining the presence of antibodies against glutamic acid decarboxylase (GAD). Such assays are valuable when diagnosing autoimmune diseases.
• compositions are suitable as pharmaceutical agents for treating Stiff Man Syndrome and Graves' disease, They can also be used in vaccine compositions against Coxsackie virus, polio, mumps, parotis and rubella.
• HEPES buffer at 50 mM and pH 7.2 has been successfully used in studies of GAD from murine brain (Meeley & Martin (1983) Cellular & Molec Neurobiol. 3(l):55-68) and pig brain (Porter & Martin (1988) J. Neurochem. 51(6): 1886-91). HEPES has also been used for initial fractionation of rhGAD65 from insect cells (10 mm used by Christgau et al. 1992 J. Cell. Biol. 118(2):309- 320; all of which are incorporated by reference).
• DTT Reducing agents are frequently used for stabilisation during extraction and purification of proteins, and use of DTT has been recommended for protein stabilisation during detergent extiaction (Hjelmeland and Chrambach 1984 Methods Enzymol. 114:305-318).
• a high concentration of DTT 50 mM
• Zwittergen 3-12 for purification ofthe human interferon "Betaseron” (Russel-Harde et al. 1995 J. Interferon & Cytokine Res. 15:31-37, all of which are incorporated by reference).
• a reducing agent is included to minimise inter- and intra-molecular disulphide bridge formation by maintaining thiol groups (cyteine residues) in their reduced state.
• thiol groups cyteine residues
• PBP Pyridoxal-5 -phosphate
• This stabilising agent has also used (at 0.5 mM) for extraction of GAD from human brain (Blinderman et al. (1978) Eur. J. Biochem. 86: 143-152); for rat brain (used at 0,2 mM by Wu et al. (1985) Methods Enzymology 113:3-10); and for bovine brain (used at 0,2 mM by Wu (1982) Proc. Natl. Acad. Sci. USA 79:4270-4, all of which are incorporated by reference).
• Use of 0.2-1.0 mM PLP is also mentioned in WO95/04137.
• protease inhibitors are often mandatory in the extraction and purification of recombinant proteins from eukaryotic expression systems.
• certain of these e.g. APMSF
• APMSF e.g. APMSF
• the separate addition of individual protease inhibitors prior to cell extraction was unattractive during the design of our process, and has been avoided by us by the addition of single tablets of a pre-made "cocktail" of complete protease inhibitors.
• Fig. 1 shows a Western blot disclosing the presence absence of GAD in a cell homogenate and in the supernatant as well as in the pellet after the cell homogenate has been ultracentrifugated.
• the cell homogenate has been prepared according to the present invention.
• Fig 2 discloses the result of an ion exchange chromatography purification step ac- cording to a preferred embodiment of the present invention
• Fig 3 illustrates the degree of purification after ion exchange chromatography by showing an electrophoresis gel in which lane 1 corresponds to unpurified protein extract and lanes 2-6 correspond to different dilutions of the purified rhGAD65 enzyme;
• Fig 4 discloses rhGAD65 -specific immunoreactivity by Western blotting.
• Recombinant cells expressing GAD were prepared as disclosed in Christgau et al.,
• Zwittergent 3-12 N-Dodecyl-N,N-dimethyl-3-ammonio-l-propansulphonate
• HEPES 119.15 g was dissolved in 350 ml sterile distilled water. pH was adjusted to 7.2. The volume was adjusted to 500 ml and the buffer was passed through a sterile filter, 0.2 microns.
• Ion-exchange chromatography using Source Q 15 was used for "capture” and first purification of rhGAD65 from extracts of GAD-infected Sf9 cells. Briefly, after extract loading and column washing (10 bed volumes), a gradient to 0.3 M NaCI (over 10 bed volumes) was started, which caused elution of rhGAD65 as a component of the first of two major UV-absorbing peaks at 0J5 M salt. All fractions were collected, aliquots from all fractions removed for analysis to locate eluate fractions with rhGAD65, and all fractions/aliquots were frozen/stored at - 70°C. The aliquots alone (that had been removed from their respective fractions at the time of collection) were then thawed and analysed.
• fractions were selected to maximise GAD concenhation (i.e. those with maximal staining intensity of GAD65 band in SDS-PAGE, Western immunoblot, and enzyme activity) while simultaneously minimising protein contaminants. These contaminants were defined as bands visualised on SDS-PAGE gels (e.g. see Fig. 5.5) that do not bind GAD65 antibodies on Western analysis (e.g. see Fig. 5.6). Selected fractions were then thawed, bulked, and immediately refrozen (and stored at -70°C) to provide IEX- purified rhGAD65.
• Zwittergent 3-12 N-Dodecyl-N,N-dimethyl-3-ammonio-l -propane sulphonate
• Zwittergent 3-12 1.5 g pH was adjusted to 7.2 with cone. Hcl. Adjust the volume to 1000 ml.
• Buffer B (was as Buffer A (above) containing 0.3 M NaCI. Condutivity was ca: 20 mS/cm)
• the buffer was prepared as Buffer A. NaCI was added before adjusting pH and vo- lume.
• the buffer was prepared as Buffer A. 3 "Complete" tablets was added for every 125 ml of buffer. Sample preparation
• the FPLC system included the simultaneous and continuous measurement and display of the following run parameters :
• the IEX system used was FPLC (Pharmacia, SE), and the chromatography was largely carried out according to the manufacturer's recommendations.
• Fig. 2 discloses the salt gradient part ofthe obtained chromatogram.
• the GAD enzyme activity of the eluted fractions were determined according to Blindermann et al (1978)., Eur. J. Biochem. 86: 143-152. The results are both presented in the following table, and in Fig. 2.
• the activity peak in Fig. 2 is situated about two fractions after the main peak. This is due to the amount of eluate between the OD 280 detector and the fraction collector. Hence, the main peak and the activity peak are closely correlated.
• Fig. 3 discloses an SDS-PAGE gel in which lane 1 corresponds to unpurified protein extract and lanes 2-7 correspond to different dilutions of fraction 61 showing the highest degree of purity.
• Fig. 4 discloses the result of Western blotting of fractions 55-97. rhGAD65 is only present in fractions 59-63.

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• 1998
  • 1998-04-21 EP EP98920768A patent/EP0977835A1/de not_active Withdrawn
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