CN102633898A - Optimization method of Hib polysaccharide and protein binding process - Google Patents
Optimization method of Hib polysaccharide and protein binding process Download PDFInfo
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- CN102633898A CN102633898A CN2012101213345A CN201210121334A CN102633898A CN 102633898 A CN102633898 A CN 102633898A CN 2012101213345 A CN2012101213345 A CN 2012101213345A CN 201210121334 A CN201210121334 A CN 201210121334A CN 102633898 A CN102633898 A CN 102633898A
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- 150000004676 glycans Chemical class 0.000 title claims abstract description 57
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 57
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 57
- 238000009739 binding Methods 0.000 title claims abstract description 40
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 30
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 28
- 238000005457 optimization Methods 0.000 title claims abstract description 11
- 230000008569 process Effects 0.000 title abstract description 4
- 239000000126 substance Substances 0.000 claims abstract description 26
- 238000006243 chemical reaction Methods 0.000 claims description 29
- 239000000243 solution Substances 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 238000002360 preparation method Methods 0.000 claims description 14
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 12
- 230000004913 activation Effects 0.000 claims description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 238000009795 derivation Methods 0.000 claims description 10
- 238000005516 engineering process Methods 0.000 claims description 10
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 6
- XFXPMWWXUTWYJX-UHFFFAOYSA-N Cyanide Chemical compound N#[C-] XFXPMWWXUTWYJX-UHFFFAOYSA-N 0.000 claims description 5
- 229920002684 Sepharose Polymers 0.000 claims description 5
- 238000000502 dialysis Methods 0.000 claims description 5
- 238000004321 preservation Methods 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 5
- 239000000376 reactant Substances 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 238000011146 sterile filtration Methods 0.000 claims description 5
- 238000000108 ultra-filtration Methods 0.000 claims description 5
- 239000008215 water for injection Substances 0.000 claims description 5
- 229960000814 tetanus toxoid Drugs 0.000 abstract description 15
- 230000035484 reaction time Effects 0.000 abstract description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 238000007796 conventional method Methods 0.000 abstract 1
- 238000011031 large-scale manufacturing process Methods 0.000 abstract 1
- ZWLIYXJBOIDXLL-UHFFFAOYSA-N decanedihydrazide Chemical compound NNC(=O)CCCCCCCCC(=O)NN ZWLIYXJBOIDXLL-UHFFFAOYSA-N 0.000 description 23
- 229960005486 vaccine Drugs 0.000 description 16
- 229960004443 hemophilus influenzae b vaccines Drugs 0.000 description 8
- 102000014914 Carrier Proteins Human genes 0.000 description 7
- 108010078791 Carrier Proteins Proteins 0.000 description 7
- 150000001718 carbodiimides Chemical class 0.000 description 5
- 230000003053 immunization Effects 0.000 description 5
- 238000002649 immunization Methods 0.000 description 5
- 241000606768 Haemophilus influenzae Species 0.000 description 4
- 229920000642 polymer Polymers 0.000 description 4
- 238000001514 detection method Methods 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- 241000194017 Streptococcus Species 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 206010025482 malaise Diseases 0.000 description 2
- 230000008520 organization Effects 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 206010027202 Meningitis bacterial Diseases 0.000 description 1
- 206010027241 Meningitis haemophilus Diseases 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 201000009904 bacterial meningitis Diseases 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 229940047650 haemophilus influenzae Drugs 0.000 description 1
- 229940045808 haemophilus influenzae type b Drugs 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 229940031937 polysaccharide vaccine Drugs 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000003335 steric effect Effects 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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Abstract
The invention discloses an optimization method of Hib polysaccharide and protein binding process, and the method comprises the following steps of: derivating polysaccharide, and preparing polysaccharide-TT (tetanus toxoid) conjugate. The invention provides a novel bridging substance combined by polysaccharide and vector protein, and the novel bridging substance can be used for more effectively performing binding reaction by changing a bridge molecule based on a conventional method, so that the binding rate of polysaccharide and vector protein is improved, the binding reaction time is shortened, the yield of qualified conjugate of the binding reaction is improved to a certain extent, the binding reaction has high repeatability, large-scale production is facilitated, and the production cost is reduced.
Description
Technical field
The present invention relates to the optimization method of a kind of Hib polysaccharide and protein binding technology.
Background technology
(Haemophilus Influenzae Hi) is still the main pathogenic bacterium that cause human affecting conditions to hemophilus influenzae up to now, and wherein (Haemophilus Influenzae type b Hib) causes the overwhelming majority by b type hemophilus influenzae.The Hib meningitis occupies the first place of bacterial meningitis, has characteristics such as sickness rate height, case fatality rate height and disability rate height, and its age of onset is little, and the infant infection rate is high, and age of onset mainly concentrated on below 5 years old, especially below 2 years old.According to World Health Organization's report in 2006, the whole world caused 3,000,000 routine serious diseases every year at least, and wherein about 38.6 ten thousand people are dead, become a global big bus hygienic issues.In developing country, the Hib pneumonia is occupied critical role in Children's Infectious Diseases, accounts for 20% of all children Streptococcus, is the important death cause of developing country's children Streptococcus.Show that in 48 researchs of 22 countries in Asia Hib is the non-tuberculous meningitic primary pathogenesis of 2/3 children.
Vaccination is unique public health measure of the serious case of the most Hib of prevention.Hib natural infection antibody horizontal is low among 3～5 years old children of China, is that Hib infects the high risk population.Therefore, there is necessity of using the Hib vaccine in China.
First kind of Hib polysaccharide vaccine went on the market in the U.S. in April, 1985, and this vaccine is applicable to 2～5 years old children.For the children less than 18 monthly ages, the efficient actual of the vaccine of this employing PRP is zero, becomes the biggest obstacle that this vaccine uses.Show that according to epidemiology survey children just need protection before 6 monthly ages.
The succeeding in developing of combined vaccine solved vaccine invalid problem in the infant.Combined vaccine has stronger immunogenicity and better immune effect than vaccine in the past.Combined vaccine was put on market in 1987, was primarily aimed at above children of 18 monthly ages.Between coming years, three kinds of new vaccines appear on the market very soon, have adopted different albumen as conjugated protein.These novel vaccines are very effective in the infant.Even in the baby at little age, use the Hib vaccine, also be safely and effectively.
At the beginning of the nineties in last century, countries such as West Europe, America include the Hib combined vaccine in conventional immunization programs for children planning, and the sickness rate of Hib affecting conditions reduces rapidly or disappears.At present, The World Health Organization (WHO) just is being devoted to promote it is included in other national immunization procedures.Global having comprised that 94 countries in America, European most of areas and Australia include the Hib vaccine in the immunization programs for children plan to 2004; Other has 15 countries under the subsidy of global vaccine and immune alliance (GAVI), to use this vaccine, accounts for the half the of global All countries and regions usage quantity.To 2007, there have been 112 countries to include the planned immunization of Hib vaccine in, 3 country's parts are included the Hib vaccine in.But account for Asia in the world 1/2 at population, have only few countries, the Hib vaccine is brought sth. into the plan in the immunity like Malaysia and Mongolian (GAVI subsidy) etc.China is owing to lack the epidemiologic data of the Hib disease of system, and the Hib vaccine is not included the scope of Chinese planned immunization as yet in.Only there is the part city to begin to promote the use of the Hib vaccine and obtained effect preferably.Because vaccine price is higher, does not have ability to buy in rural area and city middle and low income family at all, thereby limited the generally use of vaccine.
In the process of Hib combined vaccine preparation, Hib capsular polysaccharide and protein carrier coupling are critical step.At present the combining method of widespread is: with connecting AH after the CNBr activation Hib capsular polysaccharide, the bridging effect that the Hib capsular polysaccharide passes through AH under the katalysis of carbodiimide then combines with carrier proteins.But the qualified binding substances yield of this combining method is very low, and at present therefore the Hib capsular polysaccharide yield of this combining method only 4～6% in the industry, for improving qualified binding substances yield, improves being of practical significance to combined process.
Summary of the invention
The object of the invention promptly is to overcome the deficiency of prior art, and the optimization method of a kind of Hib polysaccharide and protein binding technology is provided, through using sebacic dihydrazide (SDH) as polysaccharide and the new bridging thing of carrier proteins bonded; On original method,, association reaction is more effectively carried out through changing bridging molecule; Then improve the combination rate of polysaccharide and carrier proteins; Shorten the association reaction time, and the qualified binding substances yield of association reaction is to a certain degree improved, association reaction repeatability is very high; Help scale operation, reduced production cost.
The objective of the invention is to realize through following technical scheme: the optimization method of a kind of Hib polysaccharide and protein binding technology, it comprises following polysaccharide derivation, polysaccharide-two steps of TT binding substances preparation, wherein, described polysaccharide derivation comprises following substep:
S11: in refining Hib capsular polysaccharide, add water for injection, be diluted to the polysaccharide soln that final concentration is 1～2mg/ml;
S12: add 0.1～0.4 times to the CNBr of polysaccharide quality, carry out activation treatment;
S13: activation adds 3～5 times to the SDH of polysaccharide quality after accomplishing, and carries out linked reaction;
S14: the reactant of linked reaction gained is sub-packed in the dialysis tubing; And the NaCl solution that places 0.02～0.08mol/L dialyses 2～3 times, dialyses 8～12 hours at every turn, removes remaining prussiate; Ultrafiltration and concentration obtains polysaccharide-SDH verivate to original volume then;
Described polysaccharide-TT binding substances preparation comprises following substep:
S21: in polysaccharide-SDH derivative solution, adding isopyknic concentration is the TT solution of 1～2mg/ml, and mixing evenly was placed in the ice bath 25～35 minutes, and bath temperature is controlled to be 2～8 ℃;
S22: after ice bath was accomplished, adding EDAC powder to final concentration was 15～25mmol/L, and keeping pH is 4.5～4.9, reacted 1.5～2.5h down in 2～8 ℃ temperature condition, obtained the intermediate reaction thing;
S23: the intermediate reaction thing through the separation and purification of Sepharose 4FF gel column, is collected KD value less than 0.3 elutriant, and the elutriant of collecting is carried out Sterile Filtration, obtain polysaccharide-TT binding substances, and place 2～8 ℃ of preservations down.
Wherein, SDH is the abbreviation of sebacic dihydrazide, and TT is the abbreviation of tetanus toxoid protein, and EDAC is the abbreviation of carbodiimide, and CNBr is a cyanogen bromide.
The invention has the beneficial effects as follows: the present invention provides the optimization method of a kind of Hib polysaccharide and protein binding technology, through using sebacic dihydrazide (SDH) as polysaccharide and the new bridging thing of carrier proteins bonded, on original method; Through changing bridging molecule; Association reaction is more effectively carried out, improve the combination rate of polysaccharide and carrier proteins then, shorten the association reaction time; And the qualified binding substances yield of association reaction is to a certain degree improved; Association reaction repeatability is very high, helps scale operation, has reduced production cost.
Embodiment
Below in conjunction with embodiment the present invention is done further description, but protection scope of the present invention is not limited to the following stated.
Embodiment 1:
The optimization method of a kind of Hib polysaccharide and protein binding technology, it comprises following polysaccharide derivation, two steps of polysaccharide-TT (tetanus toxoid protein) binding substances preparation, wherein, described polysaccharide derivation comprises following substep:
S11: in the Hib capsular polysaccharide, add water for injection, be diluted to the solution that final concentration is 1mg/ml;
S12: add 0.1 times to the CNBr of polysaccharide quality, carry out activation treatment;
S13: activation adds 5 times of SDH to the polysaccharide quality (sebacic dihydrazide) after accomplishing, and carries out linked reaction;
S14: the reactant of linked reaction gained is sub-packed in the dialysis tubing; And place the NaCl solution of 0.02mol/L to dialyse 3 times, and dialysed 8 hours at every turn, remove remaining prussiate; Ultrafiltration and concentration obtains polysaccharide-SDH (sebacic dihydrazide) verivate to original volume then;
The preparation of described polysaccharide-TT (tetanus toxoid protein) binding substances comprises following substep:
S21: in polysaccharide-SDH (sebacic dihydrazide) derivative solution, adding isopyknic concentration is TT (tetanus toxoid protein) solution of 1mg/ml, mixes to be placed in the ice bath 25 minutes, and bath temperature is controlled to be 2 ℃;
S22: after ice bath was accomplished, adding EDAC (carbodiimide) powder to final concentration was 15mmol/L, and keeping pH is 4.9, reacted 2.5h down in 8 ℃ temperature condition, obtained the intermediate reaction thing;
S23: the intermediate reaction thing through the separation and purification of Sepharose 4FF gel column, is collected KD value less than 0.3 elutriant, and the elutriant of collecting is carried out Sterile Filtration, obtain polysaccharide-TT (tetanus toxoid protein) binding substances, and place 2 ℃ of preservations down.
Detection of the present invention is following:
Sampling is also pressed official method and is detected polysaccharide content, protein content, polymer conjugate content, relative molecular mass, and preparation 3 crowdes of polysaccharide-TT (tetanus toxoid protein) binding substances detects among the present invention, and its detected result is as shown in table 1 below:
Polysaccharide content, protein content, polymer conjugate content, relative molecular mass detected result in table 1 polysaccharide-TT binding substances
After each item calibrating; Three batches of binding substancess that obtain all meet the pharmacopeia requirement, calculate the polysaccharide yield and find, the polysaccharide yield of the qualified binding substances that obtains through present method is between 9～10%; With the yield compared of traditional combining method 4～6%, it combines yield to improve a lot.
Sebacic dihydrazide is called for short SDH, and structural formula is H
2NNH-CO-(CH
2)
8-CO-HNNH
2, molecular weight is 230.20.White crystalline powder, 186～190 ℃ of fusing points, hydrazide group content 51.4%.Have similar physics and chemical property with AH; But in its molecular structure, carbochain more helps overcoming polysaccharide molecule and carrier proteins molecule space steric effect than the carbon chain length of AH; Association reaction is carried out more easily, improve the combination rate of polysaccharide and carrier proteins then.
Prove through test of many times,, shortened the association reaction time through using sebacic dihydrazide (SDH) as polysaccharide and protein bound bridging thing; Traditional method generally needs the reaction that ability is accomplished in 3～4 hours; The present invention will shorten to 1.5～2.5 hours the time, and many batches of binding substances each item verification results that obtain are stable, and be in the intermediate value of pharmacopeia regulation; Association reaction repeatability is very high, helps scale operation.
Embodiment 2:
The optimization method of a kind of Hib polysaccharide and protein binding technology, it comprises following polysaccharide derivation, two steps of polysaccharide-TT (tetanus toxoid protein) binding substances preparation, wherein, described polysaccharide derivation comprises following substep:
S11: in the Hib capsular polysaccharide, add water for injection, be diluted to the solution that final concentration is 1.5mg/ml;
S12: add 0.4 times to the CNBr of polysaccharide quality, carry out activation treatment;
S13: activation adds 4 times of SDH to the polysaccharide quality (sebacic dihydrazide) after accomplishing, and carries out linked reaction;
S14: the reactant of linked reaction gained is sub-packed in the dialysis tubing; And place the NaCl solution of 0.08mol/L to dialyse 2 times, and dialysed 12 hours at every turn, remove remaining prussiate; Ultrafiltration and concentration obtains polysaccharide-SDH (sebacic dihydrazide) verivate to original volume then;
The preparation of described polysaccharide-TT (tetanus toxoid protein) binding substances comprises following substep:
S21: in polysaccharide-SDH (sebacic dihydrazide) derivative solution, adding isopyknic concentration is TT (tetanus toxoid protein) solution of 1.5mg/ml, mixes to be placed in the ice bath 35 minutes, and bath temperature is controlled to be 8 ℃;
S22: after ice bath was accomplished, adding EDAC (carbodiimide) powder to final concentration was 20mmol/L, and keeping pH is 4.7, reacted 2.0h down in 5 ℃ temperature condition, obtained the intermediate reaction thing;
S23: the intermediate reaction thing through the separation and purification of Sepharose 4FF gel column, is collected KD value less than 0.3 elutriant, and the elutriant of collecting is carried out Sterile Filtration, obtain polysaccharide-TT (tetanus toxoid protein) binding substances, and place 5 ℃ of preservations down.
Detect: sampling is also pressed official method and is detected polysaccharide content, protein content, polymer conjugate content, relative molecular mass, and preparation is 3 batches among the present invention, and its detection is of table 1 among the embodiment 1.
Embodiment 3:
The optimization method of a kind of Hib polysaccharide and protein binding technology, it comprises following polysaccharide derivation, two steps of polysaccharide-TT (tetanus toxoid protein) binding substances preparation, wherein, described polysaccharide derivation comprises following substep:
S11: in the Hib capsular polysaccharide, add water for injection, be diluted to the solution that final concentration is 2mg/ml;
S12: add 0.25 times to the CNBr of polysaccharide quality, carry out activation treatment;
S13: activation adds 3 times of SDH to the polysaccharide quality (sebacic dihydrazide) after accomplishing, and carries out linked reaction;
S14: the reactant of linked reaction gained is sub-packed in the dialysis tubing; And place the NaCl solution of 0.05mol/L to dialyse 2.5 times, and dialysed 10 hours at every turn, remove remaining prussiate; Ultrafiltration and concentration obtains polysaccharide-SDH (sebacic dihydrazide) verivate to original volume then;
The preparation of described polysaccharide-TT (tetanus toxoid protein) binding substances comprises following substep:
S21: in polysaccharide-SDH (sebacic dihydrazide) derivative solution, adding isopyknic concentration is TT (tetanus toxoid protein) solution of 2mg/ml, mixes to be placed in the ice bath 30 minutes, and bath temperature is controlled to be 5 ℃;
S22: after ice bath was accomplished, adding EDAC (carbodiimide) powder to final concentration was 25mmol/L, and keeping pH is 4.5, reacted 1.5h down in 2 ℃ temperature condition, obtained the intermediate reaction thing;
S23: the intermediate reaction thing through the separation and purification of Sepharose 4FF gel column, is collected KD value less than 0.3 elutriant, and the elutriant of collecting is carried out Sterile Filtration, obtain polysaccharide-TT (tetanus toxoid protein) binding substances, and place 8 ℃ of preservations down.
Detect: sampling is also pressed official method and is detected polysaccharide content, protein content, polymer conjugate content, relative molecular mass, and preparation is 3 batches among the present invention, and its detection is of table 1 among the embodiment 1.
Claims (1)
1. the optimization method of Hib polysaccharide and protein binding technology, it is characterized in that: it comprises following polysaccharide derivation, polysaccharide-two steps of TT binding substances preparation, and wherein, described polysaccharide derivation comprises following substep:
S11: in the Hib capsular polysaccharide, add water for injection, be diluted to the solution that final concentration is 1～2 mg/ml;
S12: add 0.1～0.4 times to the CNBr of polysaccharide quality, carry out activation treatment;
S13: activation adds 3～5 times to the SDH of polysaccharide quality after accomplishing, and carries out linked reaction;
S14: the reactant of linked reaction gained is sub-packed in the dialysis tubing; And the NaCl solution that places 0.02～0.08mol/L dialyses 2～3 times, dialyses 8～12 hours at every turn, removes remaining prussiate; Ultrafiltration and concentration obtains polysaccharide-SDH verivate to original volume then;
Described polysaccharide-TT binding substances preparation comprises following substep:
S21: in polysaccharide-SDH derivative solution, adding isopyknic concentration is the TT solution of 1～2mg/ml, even being placed in the ice bath 25～35 minutes that be mixed, and bath temperature is controlled to be 2～8 ℃;
S22: after ice bath was accomplished, adding EDAC powder to final concentration was 15～25mmol/L, and keeping pH is 4.5～4.9, reacted 1.5～2.5h down in 2～8 ℃ temperature condition, obtained the intermediate reaction thing;
S23: the intermediate reaction thing through the separation and purification of Sepharose 4FF gel column, is collected KD value less than 0.3 elutriant, and the elutriant of collecting is carried out Sterile Filtration, obtain polysaccharide-TT binding substances, and place 2～8 ℃ of preservations down.
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| CN105031643A (en) * | 2015-08-31 | 2015-11-11 | 成都欧林生物科技股份有限公司 | Preparation method and application of meningococcal A capsular polysaccharide conjugate vaccine |
| CN105031642A (en) * | 2015-08-31 | 2015-11-11 | 成都欧林生物科技股份有限公司 | Meningococcal A capsular polysaccharide conjugate vaccine and preparation method thereof |
| CN105056228A (en) * | 2015-08-31 | 2015-11-18 | 成都欧林生物科技股份有限公司 | Method for preparing group A meningococcal capsular polysaccharide conjugate vaccine |
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| WO2017036141A1 (en) * | 2015-08-31 | 2017-03-09 | 成都欧林生物科技股份有限公司 | Preparation method and use of group a meningococcal capsular polysaccharide conjugate vaccine |
| CN106344915A (en) * | 2016-08-29 | 2017-01-25 | 成都欧林生物科技股份有限公司 | Preparation method of group A meningococcal capsular polysaccharide conjugate vaccine |
| CN111855827A (en) * | 2019-04-24 | 2020-10-30 | 岛津企业管理（中国）有限公司 | Method for determining the binding rate of polysaccharide protein binding sites in polysaccharide protein conjugate vaccines |
| CN111855827B (en) * | 2019-04-24 | 2022-09-16 | 岛津企业管理（中国）有限公司 | Method for determining polysaccharide protein binding site binding rate in polysaccharide protein binding vaccine |
| CN111871396A (en) * | 2020-08-03 | 2020-11-03 | 中国科学院过程工程研究所 | A kind of albumin electrostatic affinity dual mode chromatography medium and its preparation method and application |
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| CN102633898B (en) | 2013-11-27 |
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