为了应对新型冠状病毒(SARS-CoV-2)引发的世界性大规模病毒感染(COVID-19)(大规模流行病),各个国家/地区都在加速新冠疫苗的研发。虽然现在已经有21种新冠疫苗获得批准,但供应量仍然不够。因此,很多国家/地区选择委托合同生产机构(CMO)进行疫苗生产。新冠疫苗的纯化会使用到各种层析法。下表是已获批上市的新冠疫苗,以及可在其纯化工艺中使用的TOYOPEARL制备层析填料的相关内容。
● 已获批上市的新冠疫苗
企业及研究机构 | 研发产品 / 产品名称 | 种类 | 审批情况* | 国家及地区 |
AstraZeneca、University of Oxford | AZD1222 (ChAdOx1 nCoV-19) | 腺病毒 | 批准 | 英国、欧洲等119个国家 |
CanSino Biologics、Beijing Institute of Biotechnology | Ad5-nCoV | 腺病毒5 | 批准 | 中国等8个国家 |
Gamaleya Research Institute | Sputnik Light/Sputnik V | 腺病毒5, 26 | 批准 | 俄罗斯等70个国家 |
Johnson & Johnson/Janssen Pharmaceuticals | Ad26 CoV2-S | 腺病毒26 | 批准 | 美国等55个国家 |
Serum Institute of India, SpyBiotech | RBD-HBsAg VLP/Covishield | VLP | 批准 | 印度等44个国家 |
Sinovac Biotech | PiCoVacc | 灭活病毒 | 批准 | 中国等37个国家 |
武汉生物制品研究所、Sinopharm | - | 灭活病毒 | 批准 | 中国 |
北京生物制品研究所、Sinopharm | BBIBP-CorV | 灭活病毒 | 批准 | 中国等56个国家 |
Bharat Biotech | BBV152/Covaxin | 灭活病毒 | 批准 | 印度等9个国家 |
Research Institute for Biologial Safety Problem | QazCOVID-In® /QazVac | 灭活病毒 | 批准 | 哈萨克斯坦 |
Chumakov Center | KoviVac | 灭活病毒 | 批准 | 俄罗斯 |
北京民海生物科技 | SARS-CoV-2 Vaccine | 灭活病毒 | 批准 | 中国 |
Shifa Pharmed Industrial Co. | - | 灭活病毒 | 批准 | 伊朗 |
BioNtech、Fosun Pharma、Pfizer | BNT162 | mRNA | 批准 | 美国、欧洲、日本等93个国家 |
Moderna, National Institute of Allergy and Infectious Diseases (NIAID), Takeda | mRNA-1273/TAK-919/Spikevac | mRNA | 批准 | 美国、欧洲、日本等63个国家 |
Center for Genetic Engineering and Biotechnology | CIGB-66 | 蛋白质 | 批准 | 古巴 |
Anfui Zhifei Longcom Biopharm, Inst. Microbiol. Chinese Academy of Sciences | RBD-Dimer/ZIFIVAX | 蛋白质 | 批准 | 中国、乌兹别克斯坦 |
Vektor State Research Center of Virology and Biotechnology | EpiVacCorona | 蛋白质/肽 | 批准 | 俄罗斯、土库曼斯坦 |
Medigen | MVC-COV1901 | 蛋白质 | 批准 | 中国台湾 |
* 以上是截至2021年7月26日的数据,并进行了部分修改。包括“特例批准”和“紧急批准”。
Ref.: WHO, Draft landscape of COVID-19 candidate vaccines, https://www.who.int/publications/m/item/draft-landscape-of-covid-19-candidate-vaccines
Covid-19 Vaccine Tracker; https://covid19.trackvaccines.org/vaccines/
● 可用于纯化新冠疫苗的TOYOPEARL填料示例
疫苗 | 分离模式 | 可使用的TOYOPEARL产品* | 文献编号 |
Spike蛋白疫苗 | SEC | HW-55S | 1-5 |
IEC | GigaCap Q-650M, GigaCap DEAE-650M, SuperQ-650M, NH2-750F | ||
HIC | Butyl-650S, Hexyl-650C | ||
AFC | AF-Chelate 650M (Ni2+ or Co2+),(活化型亲和填料,用于凝集素-AFC) | ||
病毒载体疫苗 | SEC | HW-65F | 6-10 |
IEC | GigaCap Q-650M, GigaCap DEAE-650M, SuperQ-650M | ||
灭活病毒疫苗 | SEC | HW-65F | 11-5 |
IEC | GigaCap Q-650M, GigaCap DEAE-650M, SuperQ-650M, NH2-750F | ||
mRNA/DNA疫苗 | IEC | GigaCap Q-650M, GigaCap DEAE-650M, SuperQ-650M, NH2-750F | 16-20 |
HIC | PPG-600M, Phenyl-600M/650M, Phenyl FT-750F, Butyl-600M/650M, Hexyl-650C | ||
MXC | Ca++Pure-HA® (羟基磷灰石) | ||
AFC | (Oligo-dT-AFC等无相关产品) |
* 根据具体情况,也可使用不同粒径等级的产品(S, M, C, F等级等)。还可使用高速制备TSKgel® PW系列填料。
在疫苗纯化中,一般会使用尺寸排阻层析(SEC)、离子交换层析(IEC;主要是阴离子交换层析)和疏水层析(HIC),而在Spike蛋白疫苗的纯化中,在纯化末端添加了His-Tag标记的重组蛋白质时,可使用金属螯合亲和层析(IMAC),在纯化mRNA/DNA疫苗时,还可使用羟基磷灰石等多(混合)模式层析(MMC, MXC)填料。
● 用于工艺开发和筛选的预装柱SkillPakTM
在工艺开发的早期阶段需要进行制备填料的筛选和分离条件探讨/优化,使用容量1 mL或5 mL的预装柱SkillPak,可更加迅速、准确地进行评估。套装产品包含不同模式的各种层析柱,非常有利于层析工艺开发。
(产品网页:https://www.separations.asia.tosohbioscience.com/productjp/process/prosdev)
● 参考文献 (注)参考文献中并未记述疫苗的层析纯化、生产的详细内容。
1. J.-H. Tian et al., SARS-CoV-2 spike glycoprotein vaccine candidate NVX-CoV2373 immunogenicity in baboons and protection in mice, Nature Communications (2021)12:372, https://doi.org/10.1038/s41467-020-20653-8
2. Q.-D. Su et al., Recombinant SARS-CoV-2 RBD with a built in T helper epitope induces strong neutralization antibody response, Vaccine, 39, (2021) 1241-1247, https://doi.org/10.1016/j.vaccine.2021.01.044
3. L. Dai et al., A Universal Design of Betacoronavirus Vaccines against COVID-19, MERS, and SARS, Cell. 2020 Aug 6; 182(3): 722–733.e11., doi: 10.1016/j.cell.2020.06.035, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7321023/
4. W.-H. Chen et al, Optimization of the production process and the characterization of yeast-expressed SARS-CoV recombinant receptor-binding domain (RBD219-N1), a SARS vaccine candidate, J. Pharmaceutical Sciences, 106 (2017) 1961-1970, https://www.sciencedirect.com/science/article/abs/pii/S0022354917302733?via%3Dihub
5. W.-H. Chen et al., Yeast-expressed recombinant protein of the receptor-binding domain in SARS-CoV spike protein with deglycosylated forms as a SARS vaccine candidate, Human Vaccine & Immunotherapeutics10:3 648-658 (2014), https://dx.doi.org/10.4161/hv.27464
6. European Medicines Agency Assessment report, Covid-19 vaccine, AstraZeneca, EMA/94907/2021, https://www.ema.europa.eu/en/documents/assessment-report/vaxzevria-previously-covid-19-vaccine-astrazeneca-epar-public-assessment-report_en.pdf
7. European Medicines Agency Assessment report, Covid-19 vaccine, Covid-19 Vaccine Janssen, EMA/158424/2021, https://www.ema.europa.eu/en/documents/assessment-report/covid-19-vaccine-janssen-epar-public-assessment-report_en.pdf
8. China Patent CN111218459B (2020); Recombinant novel coronavirus vaccine taking human replication defective adenovirus as carrier, Claim 14. The method of claim 9, wherein the purification method of step (6) is Source 30Q chromatography.
https://www.incopat.com/detail/init2?formerQuery=3eQEo0gaDTgd8BXMtGFAiWr4kAd0KKkg&local=en
9. Sputnik V Press release, April 28, 2021, It uses a 4-stage purification technology that includes two stages of chromatography and two stages of tangential flow filtration.
https://sputnikvaccine.com/newsroom/pressreleases/sputnik-v-statement-on-brazilian-health-regulator-anvisa-s-decision-to-postpone-authorization/
10. Pharmar's Almanac, April 23, 2020 PAO-M04-20-CL-002, Preliminary scalable downstream processes have been designed, including
concentration and buffer exchange of clarified extract capture and polishing chromatography.
https://www.pharmasalmanac.com/articles/applying-rapid-countermeasure-preparedness-to-development-of-a-novel-covid-19-vaccine
11. Q. Gao et al., Rapid development of an inactivated vaccine for SARS-CoV-2, Science, 03 Jul. 2020: Vol. 369, Issue 6499, pp. 77-81,
DOI: 10.1126/science.abc1932 "
12. Wang et al., Cell 182 (2020) 713-721, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275151/pdf/main.pdf
13. Y.-F. Yao et al., Protective Efficacy of Inactivated Vaccine against SARS-CoV-2 Infectionin Mice and Non-Human Primates, Virologica Sinica, (2021), https://doi.org/10.1007/s12250-021-00376-w
14. B. Ganneru et al., E valuation of S afety and I mmunogenic ity of an Adjuvanted, TH-1 Ske wed, Whole Virion 1 Inac tiv atedSARS-CoV -2
Vaccine, BBV152, https://www.biorxiv.org/content/10.1101/2020.09.09.285445v2.full
15. E. Qin et al., Vaccine 24 (2006) 1028-1034, Immunogenicity and protective efficacy in monkeys of purified inactivated Vero-cell SARS vaccine, https://doi.org/10.1016/j.vaccine.2005.06.038
16. Z. Kis et al., HOW TO MAKE ENOUGH VACCINE FOR THE WORLD IN ONE YEAR, May 26 (2021),
https://mkus3lurbh3lbztg254fzode-wpengine.netdna-ssl.com/wp-content/uploads/mRNA-vaccine-roadmap-May-26-final.pdf
17. A. Schmidt et al., Digital twin of mRNA-based SARS-CoV-19 vaccine manufacturing towards autonomous operation for improvements in speed, scale, robustness, flexibility and real-time release testing, Processes 2021, 9, 748. https://doi.org/10.3390/pr9050748
18. A. B. Vogel et al., BNT162b vaccines protect rhesus macaques from SARS-CoV-2, Nature 592 (2021) 283,
https://doi.org/10.1038/s41586-021-03275-y
19. K. S. Corbett et al., SARS-Cov-2 mRNA vaccine design enabled by prototype pathogen preparedness, Nature 586 (2020) 567,
https://www.nature.com/articles/s41586-020-2622-0
20. E. Prompetchara et al., DNA vaccine candidate encording SARS-CoV-2 spike proteins elicited potent humoral and Th1 cell-mediated immune response in mice, PLOS ONE, March 22, 2021, https://doi.org/10.1371/journal.pone.0248007
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超高纯度纯化单克隆抗体——使用疏水层析填料纯化
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