US20060020120A1 - Methods for removing suspended particles from soluble protein solutions - Google Patents
Methods for removing suspended particles from soluble protein solutions Download PDFInfo
- Publication number
- US20060020120A1 US20060020120A1 US10/873,801 US87380104A US2006020120A1 US 20060020120 A1 US20060020120 A1 US 20060020120A1 US 87380104 A US87380104 A US 87380104A US 2006020120 A1 US2006020120 A1 US 2006020120A1
- Authority
- US
- United States
- Prior art keywords
- lysate
- soluble protein
- suspended particles
- protein solution
- diatomaceous earth
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 239000012460 protein solution Substances 0.000 title claims abstract description 65
- 238000000034 method Methods 0.000 title claims abstract description 58
- 239000002245 particle Substances 0.000 title claims abstract description 42
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 36
- 239000005909 Kieselgur Substances 0.000 claims abstract description 30
- 238000001914 filtration Methods 0.000 claims abstract description 30
- 239000006166 lysate Substances 0.000 claims description 90
- 108090000623 proteins and genes Proteins 0.000 claims description 64
- 235000018102 proteins Nutrition 0.000 claims description 60
- 102000004169 proteins and genes Human genes 0.000 claims description 60
- 239000002158 endotoxin Substances 0.000 claims description 19
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 claims description 12
- 239000003638 chemical reducing agent Substances 0.000 claims description 5
- 125000006239 protecting group Chemical group 0.000 claims description 5
- 125000004646 sulfenyl group Chemical group S(*)* 0.000 claims description 4
- 150000002019 disulfides Chemical class 0.000 claims description 3
- 230000003647 oxidation Effects 0.000 claims description 3
- 238000007254 oxidation reaction Methods 0.000 claims description 3
- 150000007970 thio esters Chemical class 0.000 claims description 3
- 150000003558 thiocarbamic acid derivatives Chemical class 0.000 claims description 3
- 150000003568 thioethers Chemical class 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 description 43
- 229920002873 Polyethylenimine Polymers 0.000 description 30
- 241000588724 Escherichia coli Species 0.000 description 20
- 230000001580 bacterial effect Effects 0.000 description 18
- 108020004414 DNA Proteins 0.000 description 15
- 238000005189 flocculation Methods 0.000 description 12
- 230000016615 flocculation Effects 0.000 description 12
- 230000002829 reductive effect Effects 0.000 description 10
- 239000000872 buffer Substances 0.000 description 9
- 238000005119 centrifugation Methods 0.000 description 9
- 230000008569 process Effects 0.000 description 9
- 230000009089 cytolysis Effects 0.000 description 8
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 8
- 239000011550 stock solution Substances 0.000 description 8
- 239000011324 bead Substances 0.000 description 7
- 239000013604 expression vector Substances 0.000 description 7
- 230000014509 gene expression Effects 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 230000001225 therapeutic effect Effects 0.000 description 6
- 125000003396 thiol group Chemical group [H]S* 0.000 description 6
- 101710145796 Staphylokinase Proteins 0.000 description 5
- 230000006037 cell lysis Effects 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- HAEPBEMBOAIUPN-UHFFFAOYSA-L sodium tetrathionate Chemical compound O.O.[Na+].[Na+].[O-]S(=O)(=O)SSS([O-])(=O)=O HAEPBEMBOAIUPN-UHFFFAOYSA-L 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 241000206602 Eukaryota Species 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000004744 fabric Substances 0.000 description 4
- 239000012065 filter cake Substances 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 4
- 239000012535 impurity Substances 0.000 description 4
- 238000001471 micro-filtration Methods 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 235000010265 sodium sulphite Nutrition 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000006277 sulfonation reaction Methods 0.000 description 4
- 239000004677 Nylon Substances 0.000 description 3
- 241000191967 Staphylococcus aureus Species 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 125000003275 alpha amino acid group Chemical group 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical group SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- -1 kieselguhr) Chemical compound 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 229920001778 nylon Polymers 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 238000001742 protein purification Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 241000723873 Tobacco mosaic virus Species 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000011013 endotoxin removal Methods 0.000 description 2
- 238000005187 foaming Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 239000006151 minimal media Substances 0.000 description 2
- 239000007800 oxidant agent Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000011435 rock Substances 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 238000000638 solvent extraction Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 2
- 238000003260 vortexing Methods 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- DIGQNXIGRZPYDK-WKSCXVIASA-N (2R)-6-amino-2-[[2-[[(2S)-2-[[2-[[(2R)-2-[[(2S)-2-[[(2R,3S)-2-[[2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S,3S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2R)-2-[[2-[[2-[[2-[(2-amino-1-hydroxyethylidene)amino]-3-carboxy-1-hydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1,5-dihydroxy-5-iminopentylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]hexanoic acid Chemical compound C[C@@H]([C@@H](C(=N[C@@H](CS)C(=N[C@@H](C)C(=N[C@@H](CO)C(=NCC(=N[C@@H](CCC(=N)O)C(=NC(CS)C(=N[C@H]([C@H](C)O)C(=N[C@H](CS)C(=N[C@H](CO)C(=NCC(=N[C@H](CS)C(=NCC(=N[C@H](CCCCN)C(=O)O)O)O)O)O)O)O)O)O)O)O)O)O)O)N=C([C@H](CS)N=C([C@H](CO)N=C([C@H](CO)N=C([C@H](C)N=C(CN=C([C@H](CO)N=C([C@H](CS)N=C(CN=C(C(CS)N=C(C(CC(=O)O)N=C(CN)O)O)O)O)O)O)O)O)O)O)O)O DIGQNXIGRZPYDK-WKSCXVIASA-N 0.000 description 1
- 102100029457 Adenine phosphoribosyltransferase Human genes 0.000 description 1
- 108010024223 Adenine phosphoribosyltransferase Proteins 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 208000031104 Arterial Occlusive disease Diseases 0.000 description 1
- 241000206761 Bacillariophyta Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000701489 Cauliflower mosaic virus Species 0.000 description 1
- 101150074155 DHFR gene Proteins 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical class S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101100321817 Human parvovirus B19 (strain HV) 7.5K gene Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 108010091358 Hypoxanthine Phosphoribosyltransferase Proteins 0.000 description 1
- 102100029098 Hypoxanthine-guanine phosphoribosyltransferase Human genes 0.000 description 1
- 208000032382 Ischaemic stroke Diseases 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- 241000239218 Limulus Species 0.000 description 1
- 102000003792 Metallothionein Human genes 0.000 description 1
- 108090000157 Metallothionein Proteins 0.000 description 1
- 241000235648 Pichia Species 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 241000235070 Saccharomyces Species 0.000 description 1
- 241000272171 Scolopacidae Species 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 102000006601 Thymidine Kinase Human genes 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- 241000700618 Vaccinia virus Species 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- 206010000891 acute myocardial infarction Diseases 0.000 description 1
- 238000005377 adsorption chromatography Methods 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229940126575 aminoglycoside Drugs 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 230000000340 anti-metabolite Effects 0.000 description 1
- 229940100197 antimetabolite Drugs 0.000 description 1
- 239000002256 antimetabolite Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 208000021328 arterial occlusion Diseases 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- JJWKPURADFRFRB-UHFFFAOYSA-N carbonyl sulfide Chemical compound O=C=S JJWKPURADFRFRB-UHFFFAOYSA-N 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229910052681 coesite Inorganic materials 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 239000002826 coolant Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 229910052906 cristobalite Inorganic materials 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- VHJLVAABSRFDPM-ZXZARUISSA-N dithioerythritol Chemical compound SC[C@H](O)[C@H](O)CS VHJLVAABSRFDPM-ZXZARUISSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 108010072542 endotoxin binding proteins Proteins 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 230000020764 fibrinolysis Effects 0.000 description 1
- 239000003527 fibrinolytic agent Substances 0.000 description 1
- 239000008394 flocculating agent Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000012767 functional filler Substances 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000010423 industrial mineral Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000000622 liquid--liquid extraction Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000005272 metallurgy Methods 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- HPNSFSBZBAHARI-UHFFFAOYSA-N micophenolic acid Natural products OC1=C(CC=C(C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-UHFFFAOYSA-N 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 238000005065 mining Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- PJUIMOJAAPLTRJ-UHFFFAOYSA-N monothioglycerol Chemical compound OCC(O)CS PJUIMOJAAPLTRJ-UHFFFAOYSA-N 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- HPNSFSBZBAHARI-RUDMXATFSA-N mycophenolic acid Chemical compound OC1=C(C\C=C(/C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-RUDMXATFSA-N 0.000 description 1
- 229960000951 mycophenolic acid Drugs 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 229910000510 noble metal Inorganic materials 0.000 description 1
- 210000004287 null lymphocyte Anatomy 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 238000002515 oligonucleotide synthesis Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000011027 product recovery Methods 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 229910052682 stishovite Inorganic materials 0.000 description 1
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 description 1
- 150000003871 sulfonates Chemical class 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 229960000103 thrombolytic agent Drugs 0.000 description 1
- 230000002537 thrombolytic effect Effects 0.000 description 1
- TUQOTMZNTHZOKS-UHFFFAOYSA-N tributylphosphine Chemical compound CCCCP(CCCC)CCCC TUQOTMZNTHZOKS-UHFFFAOYSA-N 0.000 description 1
- 229910052905 tridymite Inorganic materials 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
- B01J20/10—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
- B01J20/14—Diatomaceous earth
Definitions
- the present invention relates to methods for removing suspended particles from soluble protein solutions.
- the methods of the invention are useful for removing suspended particles from secreted protein solutions and lysates, including bacterial lysates containing a heterologous protein.
- Protein purification is often a significant challenge, especially when large amounts of protein are required for therapeutic or diagnostic purposes. Procedures that simply and rapidly provide the protein of interest in pure form and high yield are very desirable, regardless of scale.
- Removing suspended particles from soluble protein solutions is often an important practical problem in purifying proteins of therapeutic or diagnostic significance, particularly when heterologous proteins are expressed in either eukaryotic or procaryotic cells.
- Currently, several methods are used for removing suspended particles from soluble protein solutions.
- Centrifugation is a common method for removing suspended particles from soluble protein solutions.
- suspended particles may be removed from soluble protein solutions by centrifugation alone.
- soluble protein solutions particularly bacterial lysates, may be treated with a flocculating agent (e.g., polyethyleneimine (“PEI”)) which typically removes macromolecules (e.g., DNA and endotoxins) and cell debris.
- PEI polyethyleneimine
- large scale centrifugation equipment is very expensive capital equipment and is often a limiting factor in removing suspended particles from soluble protein solutions on a process scale.
- Major problems with centrifugation include low yields and air entrapment in the supernatant that can lead to substantial protein denaturation. Typical yields of protein after centrifugation are about 80%-85%.
- Aqueous two-phase partitioning is another method that has been used for removing cellular debris and suspended particles from soluble protein solutions.
- Liquid-liquid extraction relies on the incompatibility between two polymers in aqueous solution or one polymer and a salt present at high concentration. This incompatibility typically results in the formation of two separate phases of very different compositions.
- the protein molecules partition preferentially into one phase or the other, depending on their characteristics (Hayenga et al., U.S. patent application Ser. No. 09/307,549; Diamond et al., Advances in Biochem. Eng. Biotechn. 1992, 47:89-135).
- aqueous two-phase extraction is time consuming, expensive and requires large amounts of chemicals, which must be properly disposed in compliance with environmental regulations. Further, the chemicals used in extraction must be removed from the protein of interest and the two-phase distribution of protein may limit product yield. Finally, two-phase extraction lacks generality since only a limited number of proteins can be purified by this method.
- Microfiltration is another popular method for removing suspended particles from soluble protein solutions. Microfiltration uses membranes that either entrap particles on the membrane surface or within a bed of fibers found within the membrane.
- microfiltration on a process scale is a complicated operation that requires precise optimization of a number of variables such as transmembrane pressure, shear force, flow rate, concentration, pH, ionic strength, etc.
- process scale microfiltration frequently requires considerable development time.
- the present invention addresses this need by providing rapid, efficient and inexpensive methods for removing suspended particles from soluble protein solutions.
- the present invention provides soluble protein solutions, free of suspended particles in high yield, while avoiding the use of expensive capital equipment or chemicals that require expensive disposal.
- the current invention provides a method for removing suspended particles from soluble protein solutions by filtering the soluble protein solution through highly purified diatomaceous earth.
- the highly purified diatomaceous earth is CelpureTM P-1000.
- the soluble protein solution is a secreted protein solution.
- the soluble protein solution is a lysate.
- the lysate is a bacterial lysate.
- the amount of DNA and endotoxins in a bacterial lysate is reduced.
- the lysate is filtered through highly purified diatomaceous earth to remove suspended particles, which dramatically reduces lysate turbidity.
- the highly purified diatomaceous earth is packed in a filter press.
- flocculation with polyethyleneimine at between about pH 7.3 and about pH 7.7 reduces the amount of DNA and endotoxins in the lysate.
- the amount of DNA in the lysate is reduced by between about 100-fold and about 150-fold.
- the amount of endotoxins in the lysate is reduced by between about 1,000-fold and about 10,000-fold.
- the turbidity of the lysate is reduced by between about 200-fold and about 300-fold.
- the lysate is filtered through highly purified diatomaceous earth with a filter press.
- the lysate is stirred with highly purified diatomaceous earth before filtering through the filter press.
- the yield of the soluble protein solution is between about 95% and about 100% after filtration through highly purified diatomaceous earth.
- the lysate is a bacterial lysate containing a heterologous protein that was obtained by expression in bacteria
- the heterologous protein is SY161, which has the amino acid sequence shown in SEQ. ID. NO. 1.
- refractile bodies in the lysate are resolubilized.
- the bacteria is E. coli.
- the cysteine residues of the heterologous protein are blocked.
- the cysteine residues are blocked with an oxidizing agent.
- the oxidizing agent is a mixture of sodium sulfite and sodium tetrathionate. Even more preferably, about a 2:1 ratio of sodium sulfite and sodium tetrathionate are added to the heterologous protein at a pH of between about 7.8 and about 8.2.
- the blocked cysteine residues of the heterologous protein are deblocked.
- a reducing agent is used to deblock the heterologous protein. More preferably, the reducing agent is dithiothreitol.
- FIG. 1 provides the amino acid sequence (SEQ ID NO 1) of SY161.
- the present invention relates to methods for removing suspended particles from soluble protein solutions.
- the details for practicing the invention are described in the subsections below.
- Soluble protein solutions may be prepared by any art-known technique.
- soluble protein solutions may be obtained by culturing procaryotes that secrete either wild-type or heterologous proteins, lysis of procaryotes, lysis of procaryotes that express heterologous proteins, lysis of eucaryotes, lysis of eucaryotes expressing heterologous proteins, growing eucaryotes that secrete a soluble protein, dissolving commercially available proteins in solution, etc.
- Procaryotes can provide soluble protein solutions after cell lysis.
- microorganisms that secrete either wild type or heterologous proteins may be cultured to provide soluble protein solutions.
- Wild-type prokaryotic cells or those expressing heterologous proteins can be grown under a variety of conditions known to the skilled artisan. Methods of growing inocula and inoculating culturing medium are known to the skilled artisan and exemplary methods have been described in the art. Preferred media, times, temperatures and pH for culturing microorganisms are also known in the art.
- the cells are grown in a medium suitable for growth of such cells, for example, minimal media or complete (i.e., rich) media.
- Soluble protein solutions containing a heterologous protein may be advantageously produced by recombinant DNA technology using techniques well known in the art for expressing genes. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al., “Molecular Cloning,” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., Vols. 1-3: (1989), and periodic updates thereof, and Ausubel et al., eds., 1989, “Current Protocols in Molecular Biology,” Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York.
- DNA and RNA encoding any heterologous protein may be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in “Oligonucleotide Synthesis”, 1984, Gait, M. J. ed., GIRL Press, Oxford.
- a variety of host-expression vector systems may be utilized to express proteins.
- the expression systems that may be used for purposes of the invention are microorganisms such as bacteria (e.g., E. coli, B. subtilis ) transformed with recombinant bacteriophage DNA, phasmid DNA or cosmic DNA expression vectors containing a nucleotide sequence encoding the desired protein; yeast (e.g., Saccharomyces, Pichia ) transfected with recombinant yeast expression vectors containing a nucleotide sequence encoding the protein of interest; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing a nucleotide sequence encoding the protein of interest; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transfected with recombinant plasmid expression vectors (e.g., Ti plasm
- a number of selection systems may be used, such as for example, the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell, 11, 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al., 1962, Proc. Natl. Acad. Sci., USA 48, 2026), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell, 22, 817) genes can be employed in tk ⁇ , hprt ⁇ or aprt ⁇ cells, respectively.
- antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci., USA 77, 3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci., USA 78, 1527); gpt, which confers resistance to mycophenolic acid (Mulligan et al., 1981, Proc. Natl. Acad. Sci. USA 78, 2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol. 150, 1); and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene, 30, 147).
- a number of expression vectors may be selected.
- Bacteria suitable for the practice of the invention are gram positive and gram negative bacteria
- soluble protein solutions are obtained by expression of heterologous proteins in Eschericia coli (“ E. coli ”) and subsequent cell lysis.
- the protein can be expressed in a procaryotic cell using expression systems known to those of skill in the art of biotechnology. Expression systems useful for the practice of the current invention are described in U.S. Pat. Nos. 5,795,745; 5,714,346; 5,637,495; 5,496,713; 5,334,531; 4,634,677; 4,604,359; 4,601,980, all of which are incorporated herein by reference in their entirety.
- Procaryotic cells can be grown under a variety of conditions known to the skilled artisan.
- the cells are grown in a medium suitable for growth of such cells, for example, minimal media or complete (i.e., rich) media.
- Staphylokinase is a potent, uniquely fibrin-selective thrombolytic agent that has substantial therapeutic value in the treatment of acute myocardial infarction and ischemic stroke (Collen, Nature Medicine, 1998, 279).
- the staphylokinase gene has been cloned from a variety of sources including a Staphylococcus aureus strain and has been expressed in E. coli (Sako et al., Mol. Gen. Genet., 1983, 271; Behnke et al., Mol. Gen. Genet., 1987, 528; Colleen et al., Fibrinolysis 6, 203).
- a number of natural variants of the staphylokinase gene from Staphylococcus aureus have been isolated (Kim et al., Thrombosis Research, 1997, 387).
- SY161 is a staphylokinase analogue that differs at thirteen amino acids from the amino acid sequence of the native protein, while retaining significant in vivo thrombolytic activity (Collen et al., Circulation 102, 1766, 2000).
- a modification of SY161 i.e., attachment of polyethylene glycol to the lone cysteine in the protein
- a preferred soluble protein solution that may be used in practicing the current invention may be obtained by lysis of E. coli strains that express SY161.
- the current invention removes suspended particles from soluble protein solutions.
- Soluble protein solutions may be obtained from cells, cell homogenates, disrupted cells, etc. and can be prepared in a variety of ways. For example, a paste of frozen dead cells may be prepared, living cells may be frozen or living cells may be used directly in the method of the current invention.
- the method of the current invention relates to filtering suspended particles from soluble protein solutions. Suspended particles are frequently formed when cells are lysed and may include insoluble precipitates along with cell debris. Further, suspended particles are often found in solutions of soluble proteins secreted either by microorganisms or eucaryotes. Suspended particles are often difficult to remove from soluble protein solutions because of their small size.
- suspended particles are removed from a soluble protein solution obtained by secretion. In another embodiment, suspended particles are removed from a soluble protein solution obtained by cell lysis.
- bacterial cells are lysed to form soluble protein solutions.
- E. coli cells that express a heterologous protein, such as SY161 are lysed.
- E. coli cells that express wild type protein are lysed.
- a number of methods well-known in the art may be used to lyse bacterial cells such as bead mills, osmotic shock, freeze fracture and enzymatic treatment.
- a high pressure homogenizer such as a microfluidizer, is used to lyse bacterial cells.
- Lysis of bacterial cells releases substantial amounts of DNA and endotoxins into the lysate.
- the amount of DNA and endotoxins in the bacterial lysate are reduced prior to removing suspended particles.
- Many methods for removing DNA and endotoxins from bacterial lysates are known to those of skill in the art. These methods, include but are not limited to, ammonium sulfate precipitation, anion exchange chromatography or filtration.
- DNA and endotoxins in a bacterial lysate are reduced by flocculation.
- flocculation is performed with polyethyleneimine at between about pH 7.3 and about pH 7.7.
- Other flocculation methods are known to those of skill in the art.
- flocculation reduces the amount of DNA in the lysate by between about 100-fold to about 150-fold and the amount of endotoxins by between about 1,000-fold and about 10,000-fold, as measured by conventional DNA threshold methods and Limulus Amoebocyte Lysate (LAL) methods, respectively.
- LAL Limulus Amoebocyte Lysate
- Diatomaceous earth i.e., kieselguhr
- diatomite is a light colored, porous sedimentary rock composed of the silaceous shells of diatoms, which are unicellar aquatic plants of microscopic size.
- diatomite is commonly called diatomite.
- Diatomaceous earth has been used in a number of different situations, including but not limited to, separation, adsorption, support and functional filler applications (Breese, (1994) “ Industrial Minerals and Rocks,” 6 th ed., Littleton, Colo.: Society for Mining, Metallurgy and Exploration, pp. 397-412).
- Diatomaceous earth is known in the art as a filtration aid and has been used, for example, in the processing of oils, beverages, solvents and chemicals on an industrial scale.
- Diatomaceous earth may be obtained in a variety of different grades and purity.
- highly purified diatomaceous earth is used to practice the current invention.
- Methods for preparing highly purified diatomaceous earth have been described in the art (Shiuh et al, U.S. Pat. No. 5,656,568, which is herein incorporated by reference).
- CelpureTM most preferably, CelpureTM P-1000
- a grade of highly purified diatomaceous earth is used to practice the current invention (Advanced Minerals, Inc., 130 Castilian Drive, Santa Barbara, Calif., 93117).
- Highly purified diatomaceous earth such as CelpureTM has the following general characteristics: extremely high purity, low density, low soluble impurity content, low total impurity content, and high brightness (Shiuh et al., U.S. Pat. No. 5,656,568).
- the highly purified diatomaceous earth (i.e., CelpureTM) used to practice the invention should have been leached in appropriate media (e.g., by acid treatment) to remove soluble impurities, have a total SiO 2 content of at least about 95% and a silica specific volume of greater than about 3.4 (Shiuh et al., U.S. Pat. No. 5,656,568).
- the bead size of the highly purified diatomaceous earth used to practice the current invention is typically determined by the volume of the soluble protein solution. While small bead sizes may provide reasonable protein recovery, the amount of back pressure generated is unacceptable when large volumes of soluble protein are filtered. Generally, larger beads provide a better filter cake and lower back pressure and are preferred for at least these reasons.
- the lysate is stirred with highly purified diatomaceous earth before filtration through a filter press.
- the yield of soluble protein solution after filtration through highly purified diatomaceous earth is between about 95% and about 100%, as measured by quantitative reverse phase HPLC.
- cysteine residues in the soluble protein solution may be desirable.
- Cysteine protection may prevent protein intermolecular or intramolecular disulfide bond formation and/or undesirable sulfhydryl oxidation.
- the soluble protein solution is treated with a sulfhydryl protecting group, which may be selected from the many reagents that have been described in the art (see e.g., Greene et al, “Protective Groups in Organic Synthesis”, Chapter 6, John Wiley & Sons).
- Appropriate sulfhydryl protecting groups include, but are not limited to, disulfides, sulfenyl compounds, thiocarbamates, thiocarbonates, thioesters, thioethers, etc.
- the sulfhydryl groups of cysteine residues of the soluble protein solution are blocked by oxidation to a disulfide or sulfenyl group.
- sulfonation with sodium sulfate and sodium tetrathionate is used to block the sulfhydryl groups.
- Other methods for forming sulfonates are known to the skilled artisan.
- about a 2:1 ratio of sodium sulfite and sodium tetrathionate are added to the soluble protein solution, which is adjusted to a pH of between about 7.8 and about 8.2.
- the soluble protein solution is a bacterial lysate containing a heterologous protein
- the sulfhydryl groups of cysteine-residues are protected after cell lysis and before flocculation.
- the cysteine protecting group should also be readily removable. Many methods for converting disulfides, sulfenyl compounds, thiocarbamates, thiocarbonates, thioesters, thioethers, etc. to the free thiol have been described in the art (see e.g., Greene et al., “Protective Groups in Organic Synthesis”, Chapter 6, John Wiley & Sons). In an exemplary embodiment, when the cysteine residues in the soluble protein solution have been protected by sulfonation, they are deblocked with a reducing agent.
- reducing agents include, but are not limited to, sodium borohydride, mercaptans (e.g., 2-mercaptoethanol, methythioglycoloate, 3-mercapto-1,2-propanediol, 3-mercaptoproprionic acid, dithioerythritol and dithiothreitol), tri-n-butyl phosphine, hydrogen in the presence of noble metal catalysts and alkali in liquid ammonia.
- dithiothreitol is used to deprotect the cysteine residues of the soluble protein solution when a sulfonate has been used as the protecting group.
- the cysteine protecting group may be removed after lysate flocculation (e.g., when the soluble protein solution is a bacterial lysate containing a heterologous protein) or any other subsequent purification step.
- heterologous protein may be precipitated within the bacterial cell as refractile bodies.
- cell lysis will provide a lysate that contains substantial amounts of refractile bodies.
- these refractile bodies are resolubilized and the resulting heterologous protein restored to active form, prior to removing suspended particles from the lysate. Otherwise, large quantities of the heterologous protein will be removed during filtration, which greatly reduces the overall yield of the process.
- Methods for resolubilizing refractile bodies and restoring the resulting heterologous protein to active form are known to the skilled artisan (see, e.g., Jones et al., U.S. Pat. No. 4,512,922).
- refractile bodies are resolubilized and restored to active form prior to lysate flocculation.
- Soluble protein solutions may be further processed, for example, in order to provide a soluble protein solution of a higher level of purity.
- the level of purity required will depend on the potential use of the protein. For example, therapeutic uses will typically require extensive further purification following application of the method of the current invention.
- Any protein purification methods known to the skilled artisan may be used for further purification. Such techniques have been extensively described in “New Protein Techniques: Methods in Molecular Biology,” Walker, J. M., ed., Humana Press, Clifton, N.J., 1988; and Protein Purification: Principles and Practice, 3rd. Ed., Scopes, R. K., Springer-Verlag, New York, N.Y., 1987. In general, techniques including, but not limited to, ammonium sulfate precipitation, centrifugation, ion exchange chromatography, affinity chromatography, gel filtration, reverse-phase chromatography (and the HPLC or FPLC forms thereof), and adsorption chromatography may be used to further purify a soluble protein solution.
- SY161 may be produced in E. coli strain TG1 transformed with plasmid pMc5-SY161-S3C. This clone represents 13 mutations from the original Staphylokinase gene subcloned from Staphylococcus aureus.
- the E. coli cells expressing SY161 were harvested by centrifugation and stored at ⁇ 70° C. prior to use.
- the frozen cell paste was broken into pieces and suspended in about 7.0 volumes (weight/volume) of lysate buffer (50 mM sodium phosphate, pH 9.5 containing 5 mM EDTA) using an overhead mixer set at between about 500 RPM to about 1000 RPM. Mixing was continued until the cell paste was completely suspended in the lysate buffer.
- a microfluidizer unit was assembled by connecting the required air pressure lines, coolant lines and hoses. The microfluidizer was then purged with lysate buffer and the pressure was adjusted to between about 13,000 psi to about 14,000 psi.
- the suspended cell paste was transferred to a pressure vessel, which was then sealed and adjusted to a pressure of about 30 psi.
- a stainless steel in-line filter was then connected to the bottom of the pressure vessel to prevent large cell clumps from clogging the microfluidizer.
- a feed line was attached to the pressure vessel containing the suspended cell paste. The homogenizer was turned on, the feed valve was opened and the pressure of the system was allowed to equilibrate until it was between about 13,000 psi to about 14,000 psi.
- the once-lysed cell suspension was collected in a clean tank and the system was rinsed with appropriate quantities of lysate buffer. The above procedure was then repeated to provide a twice-lysed cell suspension containing SY161.
- the lysate prepared in Section 5.1 was stirred until well suspended. If necessary, the pH of the lysate was adjusted to about 8.0 ⁇ 0.2 with either dilute acid or base.
- the target lysate volume may be calculated by multiplying the weight of the cell paste used in the procedure above by 10. Lysate buffer was added with stirring until the desired volume was reached.
- the amount of sulfitolysis stock solution that was added to the lysate may be readily approximated by multiplying the lysate volume by 0.05 (the stock solution was a mixture of 200 mg/ml sodium sulfite and 100 mg/ml sodium tetrathionate).
- the appropriate amount of sulfitolysis stock solution was added to the lysate, which was mixed for about 4.0 hours at room temperature until sulfonation of SY161 was complete.
- a 10% phosphoric acid was slowly added with mixing to the sulfonated lysate prepared in Section 5.2 until the lysate reached a pH of about 7.5 ⁇ 0.2.
- a 5% (w/w) polyethyleneimine (“PEI”) stock solution was prepared by diluting 50% PEI to 5% and adjusting the pH to about 7.5 ⁇ 0.2 with hydrochloric acid.
- the volume of PEI stock solution used for flocculation may be estimated by dividing the volume of sulfonated lysate by 25. The appropriate amount of PEI stock solution was added to the lysate to provide a final PEI concentration of about 0.2%.
- the flow rate of PEI addition was a critical parameter and may be calculated by multiplying the volume of pH adjusted sulfonated lysate by 0.8, which provided an appropriate flow rate in milliliter per minute. If PEI was added at too rapid of a rate the product protein was co-flocculated, which significantly reduced the yield of the process. The calculated volume of 5% PEI was added to the sulfonated lysate at the flow rate calculated from the formula provided above. The sulfonated lysate was gently stirred during PEI addition, although vortexing or foaming was avoided.
- the amount of highly purified diatomaceous earth (e.g., CelpureTM P-1000) added to the lysate prepared in Section 5.3 may be estimated by multiplying the volume of the lysate in liters by 0.06, which provided a CelpureTM P-1000 concentration of about 6%.
- Table 1 provides the relationship among the bead size, percentage of activity and product recovery. Small beads such as CelpureTM P-65 provided reasonable recovery but generated higher back-pressure, which is unacceptable in large scale. Generally, larger beads provided a better filter cake and preferred for this reason.
- the calculated amount of CelpureTM P-1000 was added to the lysate with mixing. Importantly, mixing should be done at the lowest rate necessary to provide a suspension of CelpureTM P-1000.
- the filter press was prepared as follows. Fresh filter pads (preferably, filter cloth septums from Nylon filter cloth S/46412-4-CHS made by Komline-Sanderson) were installed and a filter press (preferably, a Begerow BECO-ASF 4000 filter press) was rinsed and equilibrated. It should be noted that Nylon filter cloth was critical to filtration of the lysate through highly purified diatomaceous earth. The hold-up volume of the filter press may be estimated at this time. The amount of CelpureTM P-1000 pre-coat used in the filter press may be calculated by multiplying the filtration surface area, in square meters, by 1000 (each filter sheet is 0.14 m 2 ).
- the required amount of CelpureTM P-1000 was suspended in approximately 50 liters of filtration buffer (i.e., 50 mM sodium phosphate, pH 7.5) and the filter press was pre-coated by circulating the CelpureTM P-1000 suspension through the filter press until the suspension became clear. Lysate filtration commenced immediately at a flow rate of between about 5 and about 10 liters per minute. The filtrate and filter cake wash were collected and any liquid remaining in the filter press was removed by flushing with compressed air. The filter cake retained cell debris such as suspended particles and flocculated material. The turbidity of the lysate was reduced from about 1800 Nephelometric Turbidity Units (“NTU”) to less than about 10 NTU. The yield of SY161 was between about 95% and about 100%. The clear solution was directly used in further applications.
- NTU Nephelometric Turbidity Units
- E. coli null cells E. coli TGI, pMc5-8 ( ⁇ clone) for expression of SY161 were harvested by centrifugation and stored at ⁇ 70° C. prior to use.
- the frozen cell paste was suspended in about 7.0 volumes (weight/volume) of lysate buffer (50 mM sodium phosphate buffer, pH 9.5, containing 5 mM EDTA).
- the frozen cell paste was stirred for about 0.5 hour with a Silverson Lab Mixer Emulsifier (Model L4R) at about 3,000 rpm to resuspend the cells.
- a microfluidizer (Model 110Y) was connected to compressed air and the cooling chamber was filled with ice.
- the homogenizer was purged with lysate buffer and the pressure was adjusted to between about 13,000 psi to about 14,000 psi.
- the suspended cells were fed into a homogenizer and lysed under the operational pressure of between about 13,000 psi to about 14,000 psi.
- the once-lysed cell suspension was collected in a clean container and the system was rinsed with appropriate quantities of lysate buffer. The above procedure was then repeated to provide a twice-lysed cell suspension containing E. coli host cell proteins.
- phosphoric acid was slowly added to the lysate prepared in Section 7.1 with mixing, until the lysate pH reached a pH of about 7.5 ⁇ 0.2.
- a 5% (w/w) PEI stock solution was prepared by diluting 50% PEI to 5% and adjusting the pH to 7.5 ⁇ 0.2 with hydrochloric 35 acid. The appropriate amount of PEI stock solution was then added to provide a final PEI concentration of about 0.2%.
- the flow rate of PEI addition was calculated by multiplying the volume of the lysate by 0.8, which provided an appropriate flow rate in milliliter per minute. If PEI was added too rapidly, the E. coli proteins can be co-flocculated which would significantly reduce the yield of the process. The lysate was gently stirred during the PEI addition, although vortexing or foaming was avoided.
- the amount of highly purified diatomaceous earth (e.g., CelpureTM P-1000) added to the flocculated lysate was estimated by multiplying the volume of the flocculated lysate in liters by 0.09, which provides a CelpureTM P-1000 concentration of 9%.
- the calculated amount of CelpureTM P-1000 (about 450 g of CelpureTM P-1000 was added to about 5L of lysate) was added to the lysate with mixing.
- a Komline-Sanderson Avery Filter Press, Model 177 Laboratory Filter Press and Nylon filter cloth were used in the filtration process.
- the system hold-up volume was about 1.8 L.
- the lysate-DE mixture was recycled by pumping the mixture through the filter press with a Sandpiper pump, (model PB 1 ⁇ 2-A) and maintaining the air pressure between about 25 psi and 40 psi until the filtrate was cleared.
- the filtrate was then directed with an outlet tube to a clean container.
- the hold-up liquid was removed by connecting the filter press to compressed air.
- An SDS-PAGE gel indicated that no protein species were lost during flocculation and filtration. However, most DNA and endotoxin were removed from the lysate.
- the turbidity reading of the lysate was 1875 NTU, while the amount of endotoxins was greater than about 3,000,000 EU/ml (see line 1, Table 2) prior to addition of PEI and filtration in Experiment 1. After filtration, the amount of endotoxins (i.e., 30 EU) and suspended particles (i.e., 7 NTU) were greatly reduced (compare line 1 to line 2, Table 2) in Experiment 1.
- the turbidity reading of the lysate was 2625 NTU, while the amount of endotoxins were greater than about 3,000,000 EU/ml (see line 3, Table 3) prior to filtration in Experiment 2 (no PEI added). After filtration, the amount of endotoxins (i.e., 3,000,000 EU) and suspended particles (i.e., 130 NTU) were reduced (compare line 3 to line 4, Table 2) but were significantly greater than when PEI was added to the lysate in Experiment 1 (compare line 2 and line 4, Table 2). Thus, PEI addition may increase the removal of suspended particles by about 10-20-fold and reduce the amount of endotoxin in the filtrate by about 10,000-fold.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Water Supply & Treatment (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Engineering & Computer Science (AREA)
- Geochemistry & Mineralogy (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Inorganic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention provides soluble protein solutions, free of suspended particles in high yield. More particularly, the current invention provides a method for removing suspended particles from soluble protein solutions by filtering the soluble protein solution through highly purified diatomaceous earth.
Description
- This application claims the benefit under 35 U.S.C. § 119 (e) of U.S. Provisional Application No. 60/241,967, filed Oct. 19, 2000, which is incorporated herein by reference in its entirety.
- The present invention relates to methods for removing suspended particles from soluble protein solutions. In particular, the methods of the invention are useful for removing suspended particles from secreted protein solutions and lysates, including bacterial lysates containing a heterologous protein.
- Proteins play critical roles in functions such as metabolism, gene expression, signal transduction, cellular and extracellular structures, which are essential to the survival and/or reproduction of any living organism. Many proteins may be used in therapeutic and/or diagnostic applications, particularly when available in pure form. Contaminants often prevent realization of therapeutic and/or diagnostic goals and may endanger the health of a patient.
- Protein purification is often a significant challenge, especially when large amounts of protein are required for therapeutic or diagnostic purposes. Procedures that simply and rapidly provide the protein of interest in pure form and high yield are very desirable, regardless of scale.
- Removing suspended particles from soluble protein solutions is often an important practical problem in purifying proteins of therapeutic or diagnostic significance, particularly when heterologous proteins are expressed in either eukaryotic or procaryotic cells. Currently, several methods are used for removing suspended particles from soluble protein solutions.
- Centrifugation is a common method for removing suspended particles from soluble protein solutions. In some situations, suspended particles may be removed from soluble protein solutions by centrifugation alone. In other instances, prior to centrifugation, soluble protein solutions, particularly bacterial lysates, may be treated with a flocculating agent (e.g., polyethyleneimine (“PEI”)) which typically removes macromolecules (e.g., DNA and endotoxins) and cell debris. However, large scale centrifugation equipment is very expensive capital equipment and is often a limiting factor in removing suspended particles from soluble protein solutions on a process scale. Major problems with centrifugation include low yields and air entrapment in the supernatant that can lead to substantial protein denaturation. Typical yields of protein after centrifugation are about 80%-85%.
- Aqueous two-phase partitioning is another method that has been used for removing cellular debris and suspended particles from soluble protein solutions. Liquid-liquid extraction relies on the incompatibility between two polymers in aqueous solution or one polymer and a salt present at high concentration. This incompatibility typically results in the formation of two separate phases of very different compositions. The protein molecules partition preferentially into one phase or the other, depending on their characteristics (Hayenga et al., U.S. patent application Ser. No. 09/307,549; Diamond et al., Advances in Biochem. Eng. Biotechn. 1992, 47:89-135).
- However, aqueous two-phase extraction is time consuming, expensive and requires large amounts of chemicals, which must be properly disposed in compliance with environmental regulations. Further, the chemicals used in extraction must be removed from the protein of interest and the two-phase distribution of protein may limit product yield. Finally, two-phase extraction lacks generality since only a limited number of proteins can be purified by this method.
- Microfiltration is another popular method for removing suspended particles from soluble protein solutions. Microfiltration uses membranes that either entrap particles on the membrane surface or within a bed of fibers found within the membrane. However, microfiltration on a process scale is a complicated operation that requires precise optimization of a number of variables such as transmembrane pressure, shear force, flow rate, concentration, pH, ionic strength, etc. Thus, process scale microfiltration frequently requires considerable development time.
- Accordingly, what is needed is a rapid and inexpensive process that removes suspended particles from soluble protein solutions in high yield, particularly on a process scale. Further, such a process should not require the use of expensive capital equipment or large amounts of chemicals that require costly disposal.
- The present invention addresses this need by providing rapid, efficient and inexpensive methods for removing suspended particles from soluble protein solutions. The present invention provides soluble protein solutions, free of suspended particles in high yield, while avoiding the use of expensive capital equipment or chemicals that require expensive disposal.
- The current invention provides a method for removing suspended particles from soluble protein solutions by filtering the soluble protein solution through highly purified diatomaceous earth. Preferably, the highly purified diatomaceous earth is Celpure™ P-1000.
- In one embodiment, the soluble protein solution is a secreted protein solution. In another embodiment, the soluble protein solution is a lysate. In a preferred embodiment, the lysate is a bacterial lysate.
- Preferably, the amount of DNA and endotoxins in a bacterial lysate is reduced. Then, the lysate is filtered through highly purified diatomaceous earth to remove suspended particles, which dramatically reduces lysate turbidity. In one embodiment, the highly purified diatomaceous earth is packed in a filter press.
- In a preferred embodiment, flocculation with polyethyleneimine at between about pH 7.3 and about pH 7.7 reduces the amount of DNA and endotoxins in the lysate. Preferably, the amount of DNA in the lysate is reduced by between about 100-fold and about 150-fold. In one embodiment, the amount of endotoxins in the lysate is reduced by between about 1,000-fold and about 10,000-fold. In another embodiment, the turbidity of the lysate is reduced by between about 200-fold and about 300-fold.
- In another preferred embodiment, the lysate is filtered through highly purified diatomaceous earth with a filter press. In a more specific embodiment, the lysate is stirred with highly purified diatomaceous earth before filtering through the filter press. Preferably, the yield of the soluble protein solution is between about 95% and about 100% after filtration through highly purified diatomaceous earth.
- In yet another preferred embodiment, the lysate is a bacterial lysate containing a heterologous protein that was obtained by expression in bacteria Preferably, the heterologous protein is SY161, which has the amino acid sequence shown in SEQ. ID. NO. 1. In a more specific embodiment, refractile bodies in the lysate are resolubilized. Preferably, the bacteria is E. coli.
- In one embodiment, the cysteine residues of the heterologous protein are blocked. Preferably, the cysteine residues are blocked with an oxidizing agent. More preferably, the oxidizing agent is a mixture of sodium sulfite and sodium tetrathionate. Even more preferably, about a 2:1 ratio of sodium sulfite and sodium tetrathionate are added to the heterologous protein at a pH of between about 7.8 and about 8.2.
- In another embodiment, the blocked cysteine residues of the heterologous protein are deblocked. Preferably, a reducing agent is used to deblock the heterologous protein. More preferably, the reducing agent is dithiothreitol.
-
FIG. 1 provides the amino acid sequence (SEQ ID NO 1) of SY161. - The present invention relates to methods for removing suspended particles from soluble protein solutions. The details for practicing the invention are described in the subsections below.
- 5.1 Sources of Soluble Protein Solutions
- Soluble protein solutions may be prepared by any art-known technique. Thus, for example, soluble protein solutions may be obtained by culturing procaryotes that secrete either wild-type or heterologous proteins, lysis of procaryotes, lysis of procaryotes that express heterologous proteins, lysis of eucaryotes, lysis of eucaryotes expressing heterologous proteins, growing eucaryotes that secrete a soluble protein, dissolving commercially available proteins in solution, etc.
- Procaryotes can provide soluble protein solutions after cell lysis. Alternatively, microorganisms that secrete either wild type or heterologous proteins may be cultured to provide soluble protein solutions. Wild-type prokaryotic cells or those expressing heterologous proteins, can be grown under a variety of conditions known to the skilled artisan. Methods of growing inocula and inoculating culturing medium are known to the skilled artisan and exemplary methods have been described in the art. Preferred media, times, temperatures and pH for culturing microorganisms are also known in the art. Thus, for example, the cells are grown in a medium suitable for growth of such cells, for example, minimal media or complete (i.e., rich) media.
- Soluble protein solutions containing a heterologous protein may be advantageously produced by recombinant DNA technology using techniques well known in the art for expressing genes. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al., “Molecular Cloning,” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., Vols. 1-3: (1989), and periodic updates thereof, and Ausubel et al., eds., 1989, “Current Protocols in Molecular Biology,” Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York. DNA and RNA encoding any heterologous protein may be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in “Oligonucleotide Synthesis”, 1984, Gait, M. J. ed., GIRL Press, Oxford.
- A variety of host-expression vector systems may be utilized to express proteins. The expression systems that may be used for purposes of the invention are microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, phasmid DNA or cosmic DNA expression vectors containing a nucleotide sequence encoding the desired protein; yeast (e.g., Saccharomyces, Pichia) transfected with recombinant yeast expression vectors containing a nucleotide sequence encoding the protein of interest; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing a nucleotide sequence encoding the protein of interest; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transfected with recombinant plasmid expression vectors (e.g., Ti plasmid) containing a nucleotide sequence encoding the protein of interest; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3, U937) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
- In eukaryotic systems, a number of selection systems may be used, such as for example, the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell, 11, 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al., 1962, Proc. Natl. Acad. Sci., USA 48, 2026), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell, 22, 817) genes can be employed in tk−, hprt− or aprt− cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci., USA 77, 3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci., USA 78, 1527); gpt, which confers resistance to mycophenolic acid (Mulligan et al., 1981, Proc. Natl. Acad. Sci. USA 78, 2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol. 150, 1); and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene, 30, 147).
- In bacterial systems, as previously mentioned, a number of expression vectors may be selected. Bacteria suitable for the practice of the invention are gram positive and gram negative bacteria In a preferred embodiment, soluble protein solutions are obtained by expression of heterologous proteins in Eschericia coli (“E. coli”) and subsequent cell lysis. The protein can be expressed in a procaryotic cell using expression systems known to those of skill in the art of biotechnology. Expression systems useful for the practice of the current invention are described in U.S. Pat. Nos. 5,795,745; 5,714,346; 5,637,495; 5,496,713; 5,334,531; 4,634,677; 4,604,359; 4,601,980, all of which are incorporated herein by reference in their entirety.
- Procaryotic cells can be grown under a variety of conditions known to the skilled artisan. In one aspect of the current invention, the cells are grown in a medium suitable for growth of such cells, for example, minimal media or complete (i.e., rich) media.
- Staphylokinase is a potent, uniquely fibrin-selective thrombolytic agent that has substantial therapeutic value in the treatment of acute myocardial infarction and ischemic stroke (Collen, Nature Medicine, 1998, 279). The staphylokinase gene has been cloned from a variety of sources including a Staphylococcus aureus strain and has been expressed in E. coli (Sako et al., Mol. Gen. Genet., 1983, 271; Behnke et al., Mol. Gen. Genet., 1987, 528; Colleen et al., Fibrinolysis 6, 203). A number of natural variants of the staphylokinase gene from Staphylococcus aureus have been isolated (Kim et al., Thrombosis Research, 1997, 387).
- SY161 is a staphylokinase analogue that differs at thirteen amino acids from the amino acid sequence of the native protein, while retaining significant in vivo thrombolytic activity (Collen et al., Circulation 102, 1766, 2000). A modification of SY161 (i.e., attachment of polyethylene glycol to the lone cysteine in the protein) that increases half-life and decreases immunogenicity is currently in clinical trials for treatment of myocardial infarction and peripheral arterial occlusion. A preferred soluble protein solution that may be used in practicing the current invention may be obtained by lysis of E. coli strains that express SY161.
- 5.2. Removing Suspended Particles from Soluble Protein Solutions
- The current invention removes suspended particles from soluble protein solutions. Soluble protein solutions may be obtained from cells, cell homogenates, disrupted cells, etc. and can be prepared in a variety of ways. For example, a paste of frozen dead cells may be prepared, living cells may be frozen or living cells may be used directly in the method of the current invention.
- The method of the current invention relates to filtering suspended particles from soluble protein solutions. Suspended particles are frequently formed when cells are lysed and may include insoluble precipitates along with cell debris. Further, suspended particles are often found in solutions of soluble proteins secreted either by microorganisms or eucaryotes. Suspended particles are often difficult to remove from soluble protein solutions because of their small size.
- In one embodiment, suspended particles are removed from a soluble protein solution obtained by secretion. In another embodiment, suspended particles are removed from a soluble protein solution obtained by cell lysis.
- In a preferred embodiment, bacterial cells are lysed to form soluble protein solutions. In a specific embodiment, E. coli cells that express a heterologous protein, such as SY161, are lysed. In another embodiment, E. coli cells that express wild type protein are lysed.
- A number of methods well-known in the art may be used to lyse bacterial cells such as bead mills, osmotic shock, freeze fracture and enzymatic treatment. Preferably, a high pressure homogenizer, such as a microfluidizer, is used to lyse bacterial cells.
- Lysis of bacterial cells releases substantial amounts of DNA and endotoxins into the lysate. Preferably, the amount of DNA and endotoxins in the bacterial lysate are reduced prior to removing suspended particles. Many methods for removing DNA and endotoxins from bacterial lysates are known to those of skill in the art. These methods, include but are not limited to, ammonium sulfate precipitation, anion exchange chromatography or filtration.
- In a preferred embodiment, DNA and endotoxins in a bacterial lysate are reduced by flocculation. Preferably, flocculation is performed with polyethyleneimine at between about pH 7.3 and about pH 7.7. Other flocculation methods are known to those of skill in the art. Typically, flocculation reduces the amount of DNA in the lysate by between about 100-fold to about 150-fold and the amount of endotoxins by between about 1,000-fold and about 10,000-fold, as measured by conventional DNA threshold methods and Limulus Amoebocyte Lysate (LAL) methods, respectively.
- Flocculant and suspended particles are then removed from lysate by filtration through highly purified diatomaceous earth. Diatomaceous earth (i.e., kieselguhr), is a light colored, porous sedimentary rock composed of the silaceous shells of diatoms, which are unicellar aquatic plants of microscopic size. When well hardened, diatomaceous earth is commonly called diatomite.
- Diatomaceous earth has been used in a number of different situations, including but not limited to, separation, adsorption, support and functional filler applications (Breese, (1994) “Industrial Minerals and Rocks,” 6th ed., Littleton, Colo.: Society for Mining, Metallurgy and Exploration, pp. 397-412). Diatomaceous earth is known in the art as a filtration aid and has been used, for example, in the processing of oils, beverages, solvents and chemicals on an industrial scale.
- Diatomaceous earth may be obtained in a variety of different grades and purity. A significant problem with the use of diatomaceous earth typically available from commercial suppliers, in biological applications, is leaching of significant amounts of impurities from the diatomaceous earth into biological solutions.
- Preferably, highly purified diatomaceous earth is used to practice the current invention. Methods for preparing highly purified diatomaceous earth have been described in the art (Shiuh et al, U.S. Pat. No. 5,656,568, which is herein incorporated by reference). Preferably, Celpure™ (most preferably, Celpure™ P-1000) a grade of highly purified diatomaceous earth is used to practice the current invention (Advanced Minerals, Inc., 130 Castilian Drive, Santa Barbara, Calif., 93117).
- Highly purified diatomaceous earth such as Celpure™ has the following general characteristics: extremely high purity, low density, low soluble impurity content, low total impurity content, and high brightness (Shiuh et al., U.S. Pat. No. 5,656,568). The highly purified diatomaceous earth (i.e., Celpure™) used to practice the invention should have been leached in appropriate media (e.g., by acid treatment) to remove soluble impurities, have a total SiO2 content of at least about 95% and a silica specific volume of greater than about 3.4 (Shiuh et al., U.S. Pat. No. 5,656,568).
- The bead size of the highly purified diatomaceous earth used to practice the current invention is typically determined by the volume of the soluble protein solution. While small bead sizes may provide reasonable protein recovery, the amount of back pressure generated is unacceptable when large volumes of soluble protein are filtered. Generally, larger beads provide a better filter cake and lower back pressure and are preferred for at least these reasons.
- Preferably, the lysate is stirred with highly purified diatomaceous earth before filtration through a filter press. Typically, the yield of soluble protein solution after filtration through highly purified diatomaceous earth is between about 95% and about 100%, as measured by quantitative reverse phase HPLC.
- In some situations, protection of cysteine residues in the soluble protein solution (preferably, a bacterial lysate containing a heterologous protein expressed in bacteria) may be desirable. Cysteine protection may prevent protein intermolecular or intramolecular disulfide bond formation and/or undesirable sulfhydryl oxidation. Preferably, the soluble protein solution is treated with a sulfhydryl protecting group, which may be selected from the many reagents that have been described in the art (see e.g., Greene et al, “Protective Groups in Organic Synthesis”, Chapter 6, John Wiley & Sons). Appropriate sulfhydryl protecting groups include, but are not limited to, disulfides, sulfenyl compounds, thiocarbamates, thiocarbonates, thioesters, thioethers, etc.
- In an exemplary embodiment, the sulfhydryl groups of cysteine residues of the soluble protein solution (preferably, a bacterial lysate containing a heterologous protein) are blocked by oxidation to a disulfide or sulfenyl group. Preferably, sulfonation with sodium sulfate and sodium tetrathionate is used to block the sulfhydryl groups. Other methods for forming sulfonates are known to the skilled artisan. Ideally, about a 2:1 ratio of sodium sulfite and sodium tetrathionate are added to the soluble protein solution, which is adjusted to a pH of between about 7.8 and about 8.2. Preferably, when the soluble protein solution is a bacterial lysate containing a heterologous protein, the sulfhydryl groups of cysteine-residues are protected after cell lysis and before flocculation.
- The cysteine protecting group should also be readily removable. Many methods for converting disulfides, sulfenyl compounds, thiocarbamates, thiocarbonates, thioesters, thioethers, etc. to the free thiol have been described in the art (see e.g., Greene et al., “Protective Groups in Organic Synthesis”, Chapter 6, John Wiley & Sons). In an exemplary embodiment, when the cysteine residues in the soluble protein solution have been protected by sulfonation, they are deblocked with a reducing agent. Many reducing agents are known in the art and include, but are not limited to, sodium borohydride, mercaptans (e.g., 2-mercaptoethanol, methythioglycoloate, 3-mercapto-1,2-propanediol, 3-mercaptoproprionic acid, dithioerythritol and dithiothreitol), tri-n-butyl phosphine, hydrogen in the presence of noble metal catalysts and alkali in liquid ammonia. Preferably, dithiothreitol is used to deprotect the cysteine residues of the soluble protein solution when a sulfonate has been used as the protecting group. The cysteine protecting group may be removed after lysate flocculation (e.g., when the soluble protein solution is a bacterial lysate containing a heterologous protein) or any other subsequent purification step.
- In some cases, substantial amounts of heterologous protein may be precipitated within the bacterial cell as refractile bodies. In this situation, cell lysis will provide a lysate that contains substantial amounts of refractile bodies. Preferably, these refractile bodies are resolubilized and the resulting heterologous protein restored to active form, prior to removing suspended particles from the lysate. Otherwise, large quantities of the heterologous protein will be removed during filtration, which greatly reduces the overall yield of the process. Methods for resolubilizing refractile bodies and restoring the resulting heterologous protein to active form are known to the skilled artisan (see, e.g., Jones et al., U.S. Pat. No. 4,512,922). Preferably, refractile bodies are resolubilized and restored to active form prior to lysate flocculation.
- 5.3 Processing of the Soluble Protein Solution Following Removal of Suspended Particles
- Soluble protein solutions may be further processed, for example, in order to provide a soluble protein solution of a higher level of purity. The level of purity required will depend on the potential use of the protein. For example, therapeutic uses will typically require extensive further purification following application of the method of the current invention.
- Any protein purification methods known to the skilled artisan may be used for further purification. Such techniques have been extensively described in “New Protein Techniques: Methods in Molecular Biology,” Walker, J. M., ed., Humana Press, Clifton, N.J., 1988; and Protein Purification: Principles and Practice, 3rd. Ed., Scopes, R. K., Springer-Verlag, New York, N.Y., 1987. In general, techniques including, but not limited to, ammonium sulfate precipitation, centrifugation, ion exchange chromatography, affinity chromatography, gel filtration, reverse-phase chromatography (and the HPLC or FPLC forms thereof), and adsorption chromatography may be used to further purify a soluble protein solution.
- The following examples are provided to further illustrate the current invention but are not intended to limit the scope of the current invention in any way.
- 6.1. Lysis of E. coli Cells Expressing SY161
- SY161 may be produced in E. coli strain TG1 transformed with plasmid pMc5-SY161-S3C. This clone represents 13 mutations from the original Staphylokinase gene subcloned from Staphylococcus aureus.
- The E. coli cells expressing SY161 were harvested by centrifugation and stored at −70° C. prior to use. The frozen cell paste was broken into pieces and suspended in about 7.0 volumes (weight/volume) of lysate buffer (50 mM sodium phosphate, pH 9.5 containing 5 mM EDTA) using an overhead mixer set at between about 500 RPM to about 1000 RPM. Mixing was continued until the cell paste was completely suspended in the lysate buffer. A microfluidizer unit was assembled by connecting the required air pressure lines, coolant lines and hoses. The microfluidizer was then purged with lysate buffer and the pressure was adjusted to between about 13,000 psi to about 14,000 psi. The suspended cell paste was transferred to a pressure vessel, which was then sealed and adjusted to a pressure of about 30 psi. A stainless steel in-line filter was then connected to the bottom of the pressure vessel to prevent large cell clumps from clogging the microfluidizer. A feed line was attached to the pressure vessel containing the suspended cell paste. The homogenizer was turned on, the feed valve was opened and the pressure of the system was allowed to equilibrate until it was between about 13,000 psi to about 14,000 psi. The once-lysed cell suspension was collected in a clean tank and the system was rinsed with appropriate quantities of lysate buffer. The above procedure was then repeated to provide a twice-lysed cell suspension containing SY161.
- 6.2. Lysate Sulfonation
- The lysate prepared in Section 5.1 was stirred until well suspended. If necessary, the pH of the lysate was adjusted to about 8.0±0.2 with either dilute acid or base. The target lysate volume may be calculated by multiplying the weight of the cell paste used in the procedure above by 10. Lysate buffer was added with stirring until the desired volume was reached. The amount of sulfitolysis stock solution that was added to the lysate may be readily approximated by multiplying the lysate volume by 0.05 (the stock solution was a mixture of 200 mg/ml sodium sulfite and 100 mg/ml sodium tetrathionate). The appropriate amount of sulfitolysis stock solution was added to the lysate, which was mixed for about 4.0 hours at room temperature until sulfonation of SY161 was complete.
- 6.3. Lysate Flocculation
- A 10% phosphoric acid was slowly added with mixing to the sulfonated lysate prepared in Section 5.2 until the lysate reached a pH of about 7.5±0.2. A 5% (w/w) polyethyleneimine (“PEI”) stock solution was prepared by diluting 50% PEI to 5% and adjusting the pH to about 7.5±0.2 with hydrochloric acid. The volume of PEI stock solution used for flocculation may be estimated by dividing the volume of sulfonated lysate by 25. The appropriate amount of PEI stock solution was added to the lysate to provide a final PEI concentration of about 0.2%. The flow rate of PEI addition was a critical parameter and may be calculated by multiplying the volume of pH adjusted sulfonated lysate by 0.8, which provided an appropriate flow rate in milliliter per minute. If PEI was added at too rapid of a rate the product protein was co-flocculated, which significantly reduced the yield of the process. The calculated volume of 5% PEI was added to the sulfonated lysate at the flow rate calculated from the formula provided above. The sulfonated lysate was gently stirred during PEI addition, although vortexing or foaming was avoided.
- 6.4. Lysate Filtration
- The amount of highly purified diatomaceous earth (e.g., Celpure™ P-1000) added to the lysate prepared in Section 5.3 may be estimated by multiplying the volume of the lysate in liters by 0.06, which provided a Celpure™ P-1000 concentration of about 6%. Table 1 provides the relationship among the bead size, percentage of activity and product recovery. Small beads such as Celpure™ P-65 provided reasonable recovery but generated higher back-pressure, which is unacceptable in large scale. Generally, larger beads provided a better filter cake and preferred for this reason. The calculated amount of Celpure™ P-1000 was added to the lysate with mixing. Importantly, mixing should be done at the lowest rate necessary to provide a suspension of Celpure™ P-1000.
TABLE 1 Test Activity (HU/Assay) Recovery (%) Total Lysate (%) 43.46 100 4% Celpure ™ P-65 37.51 86.31 6% Celpure ™ P-65 27.85 64.09 2% Celpure ™ P-300 36.74 84.54 4% Celpure ™ P-300 33.11 76.18 6% Celpure ™ P-300 40.62 93.47 4% Celpure ™ P-1000 35.48 81.63 6% Celpure ™ P-1000 48.52 111.65 - The filter press was prepared as follows. Fresh filter pads (preferably, filter cloth septums from Nylon filter cloth S/46412-4-CHS made by Komline-Sanderson) were installed and a filter press (preferably, a Begerow BECO-ASF 4000 filter press) was rinsed and equilibrated. It should be noted that Nylon filter cloth was critical to filtration of the lysate through highly purified diatomaceous earth. The hold-up volume of the filter press may be estimated at this time. The amount of Celpure™ P-1000 pre-coat used in the filter press may be calculated by multiplying the filtration surface area, in square meters, by 1000 (each filter sheet is 0.14 m2). The required amount of Celpure™ P-1000 was suspended in approximately 50 liters of filtration buffer (i.e., 50 mM sodium phosphate, pH 7.5) and the filter press was pre-coated by circulating the Celpure™ P-1000 suspension through the filter press until the suspension became clear. Lysate filtration commenced immediately at a flow rate of between about 5 and about 10 liters per minute. The filtrate and filter cake wash were collected and any liquid remaining in the filter press was removed by flushing with compressed air. The filter cake retained cell debris such as suspended particles and flocculated material. The turbidity of the lysate was reduced from about 1800 Nephelometric Turbidity Units (“NTU”) to less than about 10 NTU. The yield of SY161 was between about 95% and about 100%. The clear solution was directly used in further applications.
- 7.1 Lysis of E. coli cells that do not Express a Heterologous Protein
- E. coli null cells (E. coli TGI, pMc5-8 (Δ clone)) for expression of SY161 were harvested by centrifugation and stored at −70° C. prior to use. The frozen cell paste was suspended in about 7.0 volumes (weight/volume) of lysate buffer (50 mM sodium phosphate buffer, pH 9.5, containing 5 mM EDTA). The frozen cell paste was stirred for about 0.5 hour with a Silverson Lab Mixer Emulsifier (Model L4R) at about 3,000 rpm to resuspend the cells. A microfluidizer (Model 110Y) was connected to compressed air and the cooling chamber was filled with ice. The homogenizer was purged with lysate buffer and the pressure was adjusted to between about 13,000 psi to about 14,000 psi. The suspended cells were fed into a homogenizer and lysed under the operational pressure of between about 13,000 psi to about 14,000 psi. The once-lysed cell suspension was collected in a clean container and the system was rinsed with appropriate quantities of lysate buffer. The above procedure was then repeated to provide a twice-lysed cell suspension containing E. coli host cell proteins.
- 7.2 Lysate Flocculation
- 10% phosphoric acid was slowly added to the lysate prepared in Section 7.1 with mixing, until the lysate pH reached a pH of about 7.5±0.2. A 5% (w/w) PEI stock solution was prepared by diluting 50% PEI to 5% and adjusting the pH to 7.5±0.2 with hydrochloric 35 acid. The appropriate amount of PEI stock solution was then added to provide a final PEI concentration of about 0.2%. The flow rate of PEI addition was calculated by multiplying the volume of the lysate by 0.8, which provided an appropriate flow rate in milliliter per minute. If PEI was added too rapidly, the E. coli proteins can be co-flocculated which would significantly reduce the yield of the process. The lysate was gently stirred during the PEI addition, although vortexing or foaming was avoided.
- 7.3 Lysate Filtration
- The amount of highly purified diatomaceous earth (e.g., Celpure™ P-1000) added to the flocculated lysate was estimated by multiplying the volume of the flocculated lysate in liters by 0.09, which provides a Celpure™ P-1000 concentration of 9%. The calculated amount of Celpure™ P-1000 (about 450 g of Celpure™ P-1000 was added to about 5L of lysate) was added to the lysate with mixing.
- A Komline-Sanderson Avery Filter Press, Model 177 Laboratory Filter Press and Nylon filter cloth were used in the filtration process. The system hold-up volume was about 1.8 L. The lysate-DE mixture was recycled by pumping the mixture through the filter press with a Sandpiper pump, (model PB ½-A) and maintaining the air pressure between about 25 psi and 40 psi until the filtrate was cleared. The filtrate was then directed with an outlet tube to a clean container. The hold-up liquid was removed by connecting the filter press to compressed air. An SDS-PAGE gel indicated that no protein species were lost during flocculation and filtration. However, most DNA and endotoxin were removed from the lysate.
- The addition of PEI was critical for removing suspended particles from the soluble protein solution as shown in Table 2. Table 2 illustrates endotoxin removal and removal of suspended particles from lysate with and without PEI addition.
- Removal of suspended particles in the lysate was conveniently monitored by turbidity measurements of the lysate using an HACH 2100 AN turbidimeter. Endotoxin removal was measured by LAL.
TABLE 2 Comparison of an E. coli lysate after PEI addition and filtration with an E. coli lysate after filtration without PEI addition. Endotoxin Turbidity (NTU) Endotoxin Unit Dilu- Total per ml Line Exp Sample tion Reading NTU (EU/ml) 1 1 Lysate 15 125 1875 >3,000,000 2 1 Lysate after 1 7 7 30 PEI addition and filtration 3 2 Lysate 15 175 2625 >3,000,000 4 2 Lysate after 1 130 130 3,000,000 filtration. - The turbidity reading of the lysate was 1875 NTU, while the amount of endotoxins was greater than about 3,000,000 EU/ml (see
line 1, Table 2) prior to addition of PEI and filtration inExperiment 1. After filtration, the amount of endotoxins (i.e., 30 EU) and suspended particles (i.e., 7 NTU) were greatly reduced (compareline 1 to line 2, Table 2) inExperiment 1. - The turbidity reading of the lysate was 2625 NTU, while the amount of endotoxins were greater than about 3,000,000 EU/ml (see line 3, Table 3) prior to filtration in Experiment 2 (no PEI added). After filtration, the amount of endotoxins (i.e., 3,000,000 EU) and suspended particles (i.e., 130 NTU) were reduced (compare line 3 to line 4, Table 2) but were significantly greater than when PEI was added to the lysate in Experiment 1 (compare line 2 and line 4, Table 2). Thus, PEI addition may increase the removal of suspended particles by about 10-20-fold and reduce the amount of endotoxin in the filtrate by about 10,000-fold.
- The present invention is not to be limited in scope by the exemplified embodiments which are intended as illustrations of single aspects of the invention and any sequences which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.
- All publications cited herein are incorporated by reference in their entirety.
Claims (7)
1-23. (canceled)
24. A method for removing suspended particles from a soluble protein solution comprising the step of:
filtering the soluble protein solution through highly purified diatomaceous earth, thereby providing a clarified soluble protein solution;
reducing the amount of DNA and endotoxins, wherein the protein solution is a lysate; and,
purifying the soluble protein solution.
25. A method for removing suspended particles from a soluble protein solution comprising the step of:
filtering the soluble protein solution through highly purified diatomaceous earth, thereby providing a clarified soluble protein solution;
reducing the amount of DNA and endotoxins, wherein the protein solution is a lysate, wherein the soluble protein solution is stirred with a highly purified diatomaceous earth before filtering through a filter press.
30. The method of one of claims 24 or 25 wherein the protein comprises cysteine residues and those residues are protected by treating the residues with a protecting group selected from the group consisting of disulfides, sulfenyl compounds, thiocarbamates, thioesters, and thioethers.
31. The method of one of claims 24 or 25 wherein the protein comprises a cysteine residue and the residue is blocked by oxidation.
32. The method of claim 24 further comprising the step of removing the protecting group.
33. The method of claim 25 further comprising the step of deblocking the residue with a reducing agent.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/873,801 US20060020120A1 (en) | 2000-10-19 | 2004-06-21 | Methods for removing suspended particles from soluble protein solutions |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US24196700P | 2000-10-19 | 2000-10-19 | |
US09/792,789 US6995246B1 (en) | 2000-10-19 | 2001-02-22 | Methods for removing suspended particles from soluble protein solutions |
US10/873,801 US20060020120A1 (en) | 2000-10-19 | 2004-06-21 | Methods for removing suspended particles from soluble protein solutions |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/792,789 Division US6995246B1 (en) | 2000-10-19 | 2001-02-22 | Methods for removing suspended particles from soluble protein solutions |
Publications (1)
Publication Number | Publication Date |
---|---|
US20060020120A1 true US20060020120A1 (en) | 2006-01-26 |
Family
ID=35730950
Family Applications (4)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/792,789 Expired - Lifetime US6995246B1 (en) | 2000-10-19 | 2001-02-22 | Methods for removing suspended particles from soluble protein solutions |
US10/698,230 Abandoned US20060021937A1 (en) | 2000-10-19 | 2003-10-31 | Methods for removing suspended particles from soluble protein solutions |
US10/698,238 Abandoned US20060142551A1 (en) | 2000-10-19 | 2003-10-31 | Methods for removing suspended particles from soluble protein solutions |
US10/873,801 Abandoned US20060020120A1 (en) | 2000-10-19 | 2004-06-21 | Methods for removing suspended particles from soluble protein solutions |
Family Applications Before (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/792,789 Expired - Lifetime US6995246B1 (en) | 2000-10-19 | 2001-02-22 | Methods for removing suspended particles from soluble protein solutions |
US10/698,230 Abandoned US20060021937A1 (en) | 2000-10-19 | 2003-10-31 | Methods for removing suspended particles from soluble protein solutions |
US10/698,238 Abandoned US20060142551A1 (en) | 2000-10-19 | 2003-10-31 | Methods for removing suspended particles from soluble protein solutions |
Country Status (1)
Country | Link |
---|---|
US (4) | US6995246B1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111246938A (en) * | 2017-10-17 | 2020-06-05 | 赛多利斯史泰迪生物技术有限责任公司 | Diatomaceous earth compositions having low endotoxin content |
CN114225542A (en) * | 2021-12-17 | 2022-03-25 | 呼和浩特市草原绿野生物工程材料有限公司 | Method for efficiently removing toxin in newborn bovine serum |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1379673A4 (en) * | 2001-04-20 | 2006-02-15 | Du Pont | A product removal process for use in a biofermentation system |
CN101522086A (en) * | 2006-10-03 | 2009-09-02 | 佐治亚-太平洋消费产品有限合伙公司 | Easy load sheet product dispenser |
AU2007351548B2 (en) * | 2006-11-01 | 2012-08-16 | Biogen Ma Inc. | Method of isolating biomacromolecules using low pH and divalent cations |
Citations (25)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3852496A (en) * | 1973-09-04 | 1974-12-03 | Corning Glass Works | Treatment of whey with immobilized lactase and glucose isomerase |
US3984539A (en) * | 1973-12-13 | 1976-10-05 | Boen Tie Khouw | Bovine immunoglobulin isolation process |
US4000121A (en) * | 1973-01-30 | 1976-12-28 | Baxter Travenol Laboratories, Inc. | Production of antisera comprising fractionating plasma or serum with an ethylene oxide-polyoxypropylene block copolymer |
US4460689A (en) * | 1982-04-15 | 1984-07-17 | Merck & Co., Inc. | DNA Cloning vector TG1, derivatives, and processes of making |
US4512922A (en) * | 1982-12-22 | 1985-04-23 | Genentech, Inc. | Purification and activity assurance of precipitated heterologous proteins |
US4601980A (en) * | 1979-07-05 | 1986-07-22 | Genentech Inc. | Microbial expression of a gene for human growth hormone |
US5334531A (en) * | 1986-05-07 | 1994-08-02 | Eniricerche S.P.A | Plasmid vector for expression in bacillus and used for cloning the structural gene which codes for the human growth hormone and a method of producing the hormone |
US5496713A (en) * | 1992-09-09 | 1996-03-05 | Mitsui Toatsu Chemicals, Inc. | Process for producing 20 kD human growth hormone |
US5518917A (en) * | 1992-05-18 | 1996-05-21 | Solvay Enzymes, Inc. | Bacillus proteolyticus species which produce an alkaline protease |
US5561064A (en) * | 1994-02-01 | 1996-10-01 | Vical Incorporated | Production of pharmaceutical-grade plasmid DNA |
US5637495A (en) * | 1983-07-15 | 1997-06-10 | Bio-Technology General Corp. | Plasmids for production of human growth hormone or polypeptide analog thereof, hosts containing the plasmids, products manufactured thereby, and related methods |
US5656568A (en) * | 1995-08-11 | 1997-08-12 | Advanced Minerals Corporation | Highly purified biogenic silica product |
US5714346A (en) * | 1993-02-22 | 1998-02-03 | Sumitomo Pharmaceuticals Company, Limited | Process for production of human growth hormone using Bacillus Brevis |
US5747633A (en) * | 1993-11-18 | 1998-05-05 | Toyo Seikan Kaisha, Ltd. | Resin composition having improved mechanical properties and bio-disintegrating property and containers comprising thereof |
US5917022A (en) * | 1994-02-16 | 1999-06-29 | Csl Limited | Process for removing endotoxins |
US6008328A (en) * | 1994-10-13 | 1999-12-28 | Amgen Inc. | Method for purifying keratinocyte growth factors |
US6162904A (en) * | 1997-12-24 | 2000-12-19 | Alpha Therapeutic Corporation | Manufacturing method for intraveneously administrable immune globulin and resultant product |
US6197571B1 (en) * | 1998-01-16 | 2001-03-06 | Agaricus Laboratories Co., Ltd. | Protein polysaccharide 0041 |
US6274371B1 (en) * | 1994-09-14 | 2001-08-14 | Qiagen Gmbh | Process and device for the isolation of cell components, such as nucleic acids, from natural sources |
US6313285B1 (en) * | 1999-07-23 | 2001-11-06 | Genentech, Inc. | Purification of plasmid DNA |
US20010044136A1 (en) * | 1999-12-22 | 2001-11-22 | Lander Russel Jackson | Process for the scaleable purification of plasmid DNA |
US20010053366A1 (en) * | 1997-07-31 | 2001-12-20 | Mapleson Bridget Kathleen | Method of removing endotoxin from vaccines |
US6365147B1 (en) * | 1999-10-13 | 2002-04-02 | New Jersey Institute Of Technology | Methods for removing endotoxins from biological solutions using immobilized metal affinity chromatography |
US6468534B1 (en) * | 2000-09-21 | 2002-10-22 | 4Life Research, Lc | Methods for obtaining transfer factor from avian sources, compositions including avian-generated transfer factor, and methods of use |
US6504012B2 (en) * | 1999-05-20 | 2003-01-07 | Alpha Therapeutic Corporation | Method for preparing dual virally inactivated immune globulin for intravenous administration |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4923967A (en) * | 1988-09-26 | 1990-05-08 | Eli Lilly And Company | Purification and refolding of recombinant proteins |
JP4113580B2 (en) * | 1994-02-07 | 2008-07-09 | キアゲン ゲゼルシャフト ミット ベシュレンクテル ハフツング | Method for reducing or removing endotoxin |
-
2001
- 2001-02-22 US US09/792,789 patent/US6995246B1/en not_active Expired - Lifetime
-
2003
- 2003-10-31 US US10/698,230 patent/US20060021937A1/en not_active Abandoned
- 2003-10-31 US US10/698,238 patent/US20060142551A1/en not_active Abandoned
-
2004
- 2004-06-21 US US10/873,801 patent/US20060020120A1/en not_active Abandoned
Patent Citations (28)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4000121A (en) * | 1973-01-30 | 1976-12-28 | Baxter Travenol Laboratories, Inc. | Production of antisera comprising fractionating plasma or serum with an ethylene oxide-polyoxypropylene block copolymer |
US3852496A (en) * | 1973-09-04 | 1974-12-03 | Corning Glass Works | Treatment of whey with immobilized lactase and glucose isomerase |
US3984539A (en) * | 1973-12-13 | 1976-10-05 | Boen Tie Khouw | Bovine immunoglobulin isolation process |
US4634677A (en) * | 1979-07-05 | 1987-01-06 | Genentech, Inc. | Plasmid capable of expressing human growth hormone |
US5795745A (en) * | 1979-07-05 | 1998-08-18 | Genentech, Inc. | Human growth hormone |
US4601980A (en) * | 1979-07-05 | 1986-07-22 | Genentech Inc. | Microbial expression of a gene for human growth hormone |
US4604359A (en) * | 1979-07-05 | 1986-08-05 | Genentech, Inc. | Microbial expression of a gene for human growth hormone |
US4460689A (en) * | 1982-04-15 | 1984-07-17 | Merck & Co., Inc. | DNA Cloning vector TG1, derivatives, and processes of making |
US4512922A (en) * | 1982-12-22 | 1985-04-23 | Genentech, Inc. | Purification and activity assurance of precipitated heterologous proteins |
US5637495A (en) * | 1983-07-15 | 1997-06-10 | Bio-Technology General Corp. | Plasmids for production of human growth hormone or polypeptide analog thereof, hosts containing the plasmids, products manufactured thereby, and related methods |
US5334531A (en) * | 1986-05-07 | 1994-08-02 | Eniricerche S.P.A | Plasmid vector for expression in bacillus and used for cloning the structural gene which codes for the human growth hormone and a method of producing the hormone |
US5518917A (en) * | 1992-05-18 | 1996-05-21 | Solvay Enzymes, Inc. | Bacillus proteolyticus species which produce an alkaline protease |
US5496713A (en) * | 1992-09-09 | 1996-03-05 | Mitsui Toatsu Chemicals, Inc. | Process for producing 20 kD human growth hormone |
US5714346A (en) * | 1993-02-22 | 1998-02-03 | Sumitomo Pharmaceuticals Company, Limited | Process for production of human growth hormone using Bacillus Brevis |
US5747633A (en) * | 1993-11-18 | 1998-05-05 | Toyo Seikan Kaisha, Ltd. | Resin composition having improved mechanical properties and bio-disintegrating property and containers comprising thereof |
US5561064A (en) * | 1994-02-01 | 1996-10-01 | Vical Incorporated | Production of pharmaceutical-grade plasmid DNA |
US5917022A (en) * | 1994-02-16 | 1999-06-29 | Csl Limited | Process for removing endotoxins |
US6274371B1 (en) * | 1994-09-14 | 2001-08-14 | Qiagen Gmbh | Process and device for the isolation of cell components, such as nucleic acids, from natural sources |
US6008328A (en) * | 1994-10-13 | 1999-12-28 | Amgen Inc. | Method for purifying keratinocyte growth factors |
US5656568A (en) * | 1995-08-11 | 1997-08-12 | Advanced Minerals Corporation | Highly purified biogenic silica product |
US20010053366A1 (en) * | 1997-07-31 | 2001-12-20 | Mapleson Bridget Kathleen | Method of removing endotoxin from vaccines |
US6162904A (en) * | 1997-12-24 | 2000-12-19 | Alpha Therapeutic Corporation | Manufacturing method for intraveneously administrable immune globulin and resultant product |
US6197571B1 (en) * | 1998-01-16 | 2001-03-06 | Agaricus Laboratories Co., Ltd. | Protein polysaccharide 0041 |
US6504012B2 (en) * | 1999-05-20 | 2003-01-07 | Alpha Therapeutic Corporation | Method for preparing dual virally inactivated immune globulin for intravenous administration |
US6313285B1 (en) * | 1999-07-23 | 2001-11-06 | Genentech, Inc. | Purification of plasmid DNA |
US6365147B1 (en) * | 1999-10-13 | 2002-04-02 | New Jersey Institute Of Technology | Methods for removing endotoxins from biological solutions using immobilized metal affinity chromatography |
US20010044136A1 (en) * | 1999-12-22 | 2001-11-22 | Lander Russel Jackson | Process for the scaleable purification of plasmid DNA |
US6468534B1 (en) * | 2000-09-21 | 2002-10-22 | 4Life Research, Lc | Methods for obtaining transfer factor from avian sources, compositions including avian-generated transfer factor, and methods of use |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111246938A (en) * | 2017-10-17 | 2020-06-05 | 赛多利斯史泰迪生物技术有限责任公司 | Diatomaceous earth compositions having low endotoxin content |
CN114225542A (en) * | 2021-12-17 | 2022-03-25 | 呼和浩特市草原绿野生物工程材料有限公司 | Method for efficiently removing toxin in newborn bovine serum |
Also Published As
Publication number | Publication date |
---|---|
US6995246B1 (en) | 2006-02-07 |
US20060021937A1 (en) | 2006-02-02 |
US20060142551A1 (en) | 2006-06-29 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CA2167910C (en) | Aqueous multiple-phase isolation of polypeptide | |
AU549721B2 (en) | Process for recovering human ifn-beta from a transformed microorganism | |
EP0778893B1 (en) | Bacterial production of Interferon-beta polypeptide | |
WO1999063076A1 (en) | Novel method of large scale plasmid purification | |
EP1194445B1 (en) | Methods for purifying recombinant growth hormone antagonist proteins using aqueous two-phase extraction | |
US6995246B1 (en) | Methods for removing suspended particles from soluble protein solutions | |
CA2067790A1 (en) | Process for enriching or cleaning biomolecules | |
US4705848A (en) | Isolation of bioactive, monomeric growth hormone | |
EP0288280A2 (en) | Production of proteins in active forms | |
EP1403274A1 (en) | Process for the purification of recombinant proteins from complex media and purified proteins obtained thereby | |
JP2001525672A (en) | Methods for increasing the yield of recombinant proteins | |
CN100387614C (en) | Method of inclusion body protein renaturation and purification at the same time | |
EP2502633A1 (en) | Recombinant plasmid DNA pMSIN4, encoding a hybrid polypeptide comprising human insulin precursor, E. coli strain BL21 (DE3) / pMSIN4 - producer of recombinant human insulin, the method for the recombinant human insulin production | |
EP1390522B1 (en) | Acetate-free purification of plasmid dna on hydroxyapatite | |
EP1439191B1 (en) | Process for the purification of bacterially expressed proteins | |
US7157562B1 (en) | Bioprocess for the production of recombinant anti-botulinum toxin antibody | |
AU2002308753A1 (en) | Acetate-free purification of plasmid DNA on hydroxyapatite | |
EP1575967B1 (en) | Process for the extraction and isolation of insulin from recombinant sources | |
US6114510A (en) | Process for the purification of recombinant human interleukin-8 | |
WO1993003134A1 (en) | Process for isolating and purifying recombinant interleukin-7 | |
JPH07184680A (en) | Method for recovering periplasmic protein | |
CN112159466A (en) | Preparation method of recombinant grass carp interleukin-6 active protein | |
JPH06253842A (en) | Preparation of plasmid dna | |
WO1994002625A1 (en) | Process for isolating recombinant polypeptides | |
JPH01233299A (en) | Solubilization of insoluble protein |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: N.V. ORGANON,NETHERLANDS Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:AKZO NOBEL N.V.;REEL/FRAME:018816/0737 Effective date: 20070112 Owner name: N.V. ORGANON, NETHERLANDS Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:AKZO NOBEL N.V.;REEL/FRAME:018816/0737 Effective date: 20070112 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |