CN1073604A - The method for preparing higher immunogenic hepatitis B vaccine - Google Patents
The method for preparing higher immunogenic hepatitis B vaccine Download PDFInfo
- Publication number
- CN1073604A CN1073604A CN92113651.XA CN92113651A CN1073604A CN 1073604 A CN1073604 A CN 1073604A CN 92113651 A CN92113651 A CN 92113651A CN 1073604 A CN1073604 A CN 1073604A
- Authority
- CN
- China
- Prior art keywords
- vaccine
- high immunogenicity
- preparing
- agarose gel
- sheep
- 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.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 19
- SPSXSWRZQFPVTJ-ZQQKUFEYSA-N hepatitis b vaccine Chemical compound C([C@H](NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CCSC)C(=O)N[C@@H](CC1N=CN=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)OC(=O)CNC(=O)CNC(=O)[C@H](C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@@H](N)CCCNC(N)=N)C1=CC=CC=C1 SPSXSWRZQFPVTJ-ZQQKUFEYSA-N 0.000 title abstract description 5
- 229940124736 hepatitis-B vaccine Drugs 0.000 title abstract description 5
- 230000002163 immunogen Effects 0.000 title description 3
- 230000005847 immunogenicity Effects 0.000 claims abstract description 20
- 229960005486 vaccine Drugs 0.000 claims abstract description 20
- 241001494479 Pecora Species 0.000 claims abstract description 10
- 239000000427 antigen Substances 0.000 claims abstract description 9
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 9
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 9
- 239000011543 agarose gel Substances 0.000 claims abstract description 7
- 210000002966 serum Anatomy 0.000 claims abstract description 7
- 238000001042 affinity chromatography Methods 0.000 claims abstract description 6
- 210000004369 blood Anatomy 0.000 claims abstract description 6
- 239000008280 blood Substances 0.000 claims abstract description 6
- 230000009849 deactivation Effects 0.000 claims abstract description 6
- 238000000502 dialysis Methods 0.000 claims abstract description 6
- 238000002360 preparation method Methods 0.000 claims abstract description 6
- 230000003169 placental effect Effects 0.000 claims abstract description 5
- 230000000694 effects Effects 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 4
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 4
- 238000010521 absorption reaction Methods 0.000 claims description 4
- 102000036639 antigens Human genes 0.000 claims description 4
- 108091007433 antigens Proteins 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 239000012153 distilled water Substances 0.000 claims description 2
- -1 polyoxy ethylene Polymers 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 229920003171 Poly (ethylene oxide) Polymers 0.000 claims 1
- 235000010356 sorbitol Nutrition 0.000 claims 1
- 238000011081 inoculation Methods 0.000 abstract description 9
- 238000000746 purification Methods 0.000 abstract description 8
- 239000000499 gel Substances 0.000 abstract description 6
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 abstract description 5
- 230000000890 antigenic effect Effects 0.000 description 5
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 4
- 230000002779 inactivation Effects 0.000 description 3
- 230000008521 reorganization Effects 0.000 description 3
- 229920002684 Sepharose Polymers 0.000 description 2
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 2
- 238000005374 membrane filtration Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 241000238876 Acari Species 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 230000000951 immunodiffusion Effects 0.000 description 1
- 238000000760 immunoelectrophoresis Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2730/00—Reverse transcribing DNA viruses
- C12N2730/00011—Details
- C12N2730/10011—Hepadnaviridae
- C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
- C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Abstract
Preparation can produce 100% anti-HB by twice inoculation
sThe method of high immunogenicity hepatitis B vaccine, comprise the immune anti-HB of the deutero-height of highly purified sheep
s(ID tires above 1: 4096) combines with agarose gel then to make affinity column, with affinity chromatography purification HB
sAntigen positive human blood or branch puerperium placental blood are clear, through continuous dialysis treatment, from serum, remove trace foreign protein with which, under 98 ℃-100 ℃, said sample is heated 30 minutes with complete deactivation HBV, thereby obtain HB more high immunogenicity, special purification
sAntigen protein, the latter is adsorbed to and can obtains on the gel aluminum hydroxide only can producing 100% anti-HB by twice inoculation
sThe HB vaccine of more high immunogenicity.
Description
The present invention relates to use affinity chromatography, highly purifiedly contain the tool human albumin in conjunction with active (HSA-BA) proteic HBS (HB of S2 before
s) antigen, can produce 100% anti-HB with preparation
sThe method of hepatitis B vaccine.
In recent years, developed the reorganization HB vaccine of S2 before the coding in that the U.S., France, Japan and other are national, even but behind 3 repeated inoculations, still can not produce 100% anti-HB
s
The present invention illustrates for the first time, if contain the tool human serum albumin in conjunction with active (HSA-BA) the proteic HB of S2 before with the affinity chromatography purification
sAntigen, then generation can produce 100% anti-HB by twice inoculation in a large number
sHigher immunogenic hepatitis B vaccine.
Therefore also just might get rid of fully at exploitation HB
sWhat run in the vaccine exists this greatest problem of non-responder, and the defective of the reorganization HB vaccine that makes with high-tech.
Can carry out highly purified by immunodiffusion method (ID) then by producing the anti-HB of hyperimmunity that tires and be higher than 1: 4096 in the sheep body
sThen it is adsorbed onto with on the highly active agarose gel of CNBr to be formed for the carrier of affinity chromatograph, wherein the concentration range of said CNBr is 50-600mg/ml, be preferably 50-300mg/ml, anti-HBs is every 100ml agarose 8000-10000 unit (ID), and adsorption time is 2-5 hour.
Use 5MMgCl
2To not wash off with said carrier-bound protein material such as anti-HBs fully.From human serum or divide the puerperium placental blood to collect clear to contain the tool human serum albumin in conjunction with activity (PHA tires greater than 1: 64) before the anti-gen(RPHA of S2 tire greater than 1: 512) HBs antigen positive serum (RPHA tires greater than 1: 1024), use then the affinity chromatography purification it.
The sample of said purification was dialysed 2 days continuously under 4 ℃ with saline and distilled water then, so that wherein no longer contain high immune anti-HBs of trace and the deutero-IgG of sheep etc.
In order to make the HBV complete deactivation that is included in the said sample, adding 0.01-0.02%(volume in sample) chemical substance such as polyoxy-ethylene anhydro sorbitol, and in 98-100 ℃ of following heat treatment 30 minutes, and then add 1: 2000 formalin and 1: 10000 merciolate, make said sample 37 ℃ of following inactivations 96 hours afterwards.
By the 0.45 said sample of μ m membrane filtration and make it to be adsorbed onto fully in the gel aluminum hydroxide, to be formed with the more HB vaccine of high immunogenicity.
Embodiment 1
Tire greater than 1 with libanol-ammonium sulfate purification according to known method: 4096(ID tires) the immune anti-HBs of the deutero-height of sheep.Use Sepharose 6B(Phar-macia then, Sweden) carry out gel filtration.300mg/mlCNBr is joined 100mlSepharose 4B(Pharmacia, activates in Sweden), add to tire again be 8000(ID) high immune anti-HBs, under stirring at room, adsorbed 2-5 hour, use 5MMgCl then
2Wash 2 times, 1500ml being contained tire is 1: HSA-BA 512(PHA), 1: 512(RPHA) S2 before, 1: the antigenic human serum of HBs 2048(RPHA) is adsorbed onto (absorption is 5 hours under the room temperature) on the gel column that has connected anti-HBs, uses 5M MgCl
2Eluting contains to obtain 120ml that to tire be 1: 16384(RPHA) the proteic HBs antigenic solution of S2 before.
The gained sample was used saline and distill water dialysis respectively 2 days, and (RPHA) becomes 1: 2048 so that it is tired with the saline dilution, added Tween 80 then and made its final concentration reach 0.02%, again in 98 ℃ of following heat treatments 30 minutes.
For making the HBV complete deactivation, in 98 ℃ with sample heat treatment 30 minutes, add the merciolate of formalin (1: 2000) and 0.01% then, and again in 37 ℃ of hot deactivations 96 hours down.
By the said sample of 0.45 μ m membrane filtration, and make it to be adsorbed by gel aluminum hydroxide fully, the HB vaccine of high immunogenicity is arranged to obtain 450ml.
Embodiment 2
With 150mg/mlCNBr activation 200ml Sepharose 4B, (England) highly purified anti-HBs is to make affinity column for DE52, Whatman with the DEAE cellulose to add 1200 units (ID) then.
2000ml contains 1 with the affinity chromatography purification: HSA-BA 1024(PHA), 1: 2048(RPHA) S2 before, 1: antigenic minute puerperium placental blood of HBs 32768(RPHA) is clear, to obtain the HBs antigenic solution (1: 65536RPHA) that 230ml contains pre-s2 protein.Continuously, add polysorbas20 then and make its final concentration reach 0.015% with saline and distill water dialysis sample, and in 98 ℃ of following heat inactivations 40 minutes.
Then, inactivation, aseptic filtration and with gel aluminum hydroxide absorption have the HB vaccine of hyperimmunization originality to obtain 1800ml once more.
To the analytical data of the said vaccine composition that makes according to the inventive method and produce anti-HBs and render a service as follows.
1. component analysis data (sample of direct purification after gel aluminum hydroxide absorption):
1) .HBs is antigenic tires: greater than 1: 2048(RPHA);
2) pre-s2 protein is tired: greater than 1: 512(RPHA);
3) HSA-BA tires: greater than 1: 128(PHA);
4) total protein concentration: be less than 0.006%(60 μ g/ml);
5) the deutero-anti-HBs of sheep: its available RIA or PHA method detect; Even exist with trace, also be only limited to few to 5mIU/ml can the ignorance amount;
6) content of the normal IgG composition that produces of sheep: even exist with trace, also be only limited to few to below 0.00000034% can the ignorance amount;
7) normal human serum albumen: immunoelectrophoresis is determined as feminine gender.
2. produce the effect of anti-HBs
1) effect of the anti-HBs of generation in the inoculator
In 30 high immunogenicity HB vaccination persons, (1.0ml is respectively inoculated in growing up more than 15 years old at every turn in inoculation for the second time, the child respectively inoculates 0.25-0.5ml at every turn, two minor ticks one month, being seeded in the 90th day for the third time carries out) after, viewing duration does not show any pair of effect, anti-HBs do not occur, enzyme value (GPT, GOT) fluctuation do not appear yet.
On the other hand, as shown in table 1, producing 100% anti-HBs after the inoculation for the second time.
2) by the reorganization HB vaccine of S2 and the situation that high immunogenicity HB vaccine produces anti-HBs before the coding
Find, though vaccine (Takeda, TGP-943T that the yeast that produces with recombinant DNA technology is derived, Japan, 1989) contain pre-s2 protein, but only producing 47% anti-HBs after the inoculation for the second time, even also only producing 92% anti-HBs(table 2 after the inoculation for the third time).
Table 1
The effect that high immunogenicity HBs vaccine produces anti-HBs
Yet, proved that only doing twice inoculation according to the high immunogenicity HB vaccine of the inventive method generation promptly can solve the problem that has non-responder fully.
Though described the present invention with reference to particular, its detailed content is not construed as limiting the invention; Obviously, can do various changes that are equal to or improvement within the scope of the present invention to those skilled in the art.
Claims (7)
1, the method for preparing high immunogenicity HB vaccine with affinity chromatography is characterized in that said method comprises the deutero-anti-HB of high immunogenicity sheep
sCombine with the preparation affiliation carrier with agarose gel, absorption, eluting contain the tool human serum albumin in conjunction with active (HBA-BA) before the proteic human serum formula of S2 divide the puerperium placental blood clear, remove with trace with continuous dialysis process and to be contained in wherein protein, and under 98-100 ℃ high-temperature the step of complete deactivation HBV.
2, according to the preparation high immunogenicity HB of claim 1
sThe method of vaccine is characterised in that the anti-HB that derives the sheep that makes high immunogenicity
sCombine with in the step for preparing affiliation carrier said anti-HB with agarose gel
sTire and remain on 1: 4096(ID).
3,, be characterised in that the anti-HB that derives the sheep that makes high immunogenicity according to the method for preparing high immunogenicity HB vaccine of claim 1 or 2
sCombine in the step with the preparation affiliation carrier the anti-HB that is adsorbed by the 100ml agarose gel with agarose gel
sGross activity in 8000-10000 unit (ID) scope.
4, according to claim 1,2 or 3 the method for preparing high immunogenicity HB vaccine, be characterised in that the anti-HB that derives the sheep that makes high immunogenicity
sCombine with in the step for preparing affiliation carrier with agarose gel, human serum or the clear HSA-BA of branch puerperium placental blood tire 1: 64(PHA), preS 2 antigen is 1: 512(RPHA), and HB
sAntigen valence is 1: 1024(RPHA).
5, according to claim 1,2,3 and 4 preparation high immunogenicity HB
sThe method of vaccine is characterised in that in remove the proteinic step that wherein comprises with trace from sample, and dialysis process is to dialyse 2-4 days in 0.85-1% saline continuously, and dialysis is 1-2 days in distilled water.
6, according to claim 1,2,3,4 or 5 the method for preparing high immunogenicity HB vaccine, the step that is characterised in that complete deactivation HBV is to carry out more than 30 minutes under 98-100 ℃.
7,, be characterised in that when heat treatment to stablizing HB according to claim 1,2,3,4,5 or 6 the method for preparing high immunogenicity HB vaccine
sAntigen protein to wherein adding as chemical substances such as polyoxy ethylene anhydro sorbitols, so that its final concentration reaches 0.01-0.002%, and then is heat-treated.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KP297991 | 1991-12-01 | ||
| KP91-2979 | 1991-12-01 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1073604A true CN1073604A (en) | 1993-06-30 |
Family
ID=19198156
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN92113651.XA Pending CN1073604A (en) | 1991-12-01 | 1992-11-30 | The method for preparing higher immunogenic hepatitis B vaccine |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN1073604A (en) |
| DE (1) | DE4240386C2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113631192A (en) * | 2019-03-26 | 2021-11-09 | 富士胶片株式会社 | Pharmaceutical composition for inhibiting production of hepatitis B virus protein, pharmaceutical composition for treating hepatitis B, and screening method |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2781160B1 (en) * | 1998-07-03 | 2000-08-18 | Pasteur Merieux Serums Vacc | USE OF AN AMPHIPATHIC COMPOUND TO ADJUST A SUBUNIT VACCINE |
| CN103111270B (en) * | 2013-02-26 | 2015-06-10 | 王业富 | Adsorbing material of hepatitis B antigen protein and preparation method of material |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4816564A (en) * | 1986-01-31 | 1989-03-28 | Merck & Co., Inc. | Method for producing hepatitis B virus proteins in yeast |
| FR2608052B1 (en) * | 1986-12-10 | 1990-04-13 | Pasteur Vaccins | PROCESS FOR THE PREPARATION OF A HEPATITIS B VACCINE AND VACCINE OBTAINED |
| EP0310178A1 (en) * | 1987-09-30 | 1989-04-05 | Merck & Co. Inc. | Recovery of pres2+S antigen |
| US4963483A (en) * | 1987-10-13 | 1990-10-16 | Merck & Co., Inc. | Method for producing hepatitis B virus proteins in yeast |
| US4855055A (en) * | 1988-09-28 | 1989-08-08 | National Science Council | Isolation and purification pre-S2 containing hepatitis B virus surface antigen by chemical affinity chromatography |
-
1992
- 1992-11-30 CN CN92113651.XA patent/CN1073604A/en active Pending
- 1992-12-01 DE DE4240386A patent/DE4240386C2/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113631192A (en) * | 2019-03-26 | 2021-11-09 | 富士胶片株式会社 | Pharmaceutical composition for inhibiting production of hepatitis B virus protein, pharmaceutical composition for treating hepatitis B, and screening method |
Also Published As
| Publication number | Publication date |
|---|---|
| DE4240386C2 (en) | 1994-07-07 |
| DE4240386A1 (en) | 1993-06-17 |
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| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C01 | Deemed withdrawal of patent application (patent law 1993) | ||
| WD01 | Invention patent application deemed withdrawn after publication |