WO2014015816A1 - 一种去除疫苗中宿主dna的方法 - Google Patents

一种去除疫苗中宿主dna的方法 Download PDF

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WO2014015816A1
WO2014015816A1 PCT/CN2013/080119 CN2013080119W WO2014015816A1 WO 2014015816 A1 WO2014015816 A1 WO 2014015816A1 CN 2013080119 W CN2013080119 W CN 2013080119W WO 2014015816 A1 WO2014015816 A1 WO 2014015816A1
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virus
vaccine
dna
host
rabies
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French (fr)
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杜笑寒
周童
贾芳苗
戚凤春
何荣
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江苏先声药物研究有限公司
江苏先声卫科生物制药有限公司
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Publication of WO2014015816A1 publication Critical patent/WO2014015816A1/zh

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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N2720/12011—Reoviridae
    • C12N2720/12311—Rotavirus, e.g. rotavirus A
    • C12N2720/12334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2760/16011—Orthomyxoviridae
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20111—Lyssavirus, e.g. rabies virus
    • C12N2760/20134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32411—Hepatovirus, i.e. hepatitis A virus
    • C12N2770/32434—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32611—Poliovirus
    • C12N2770/32634—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the field of biomedicine, and in particular to a method for removing host DNA from a vaccine.
  • Rabies is a zoonotic disease caused by rabies virus. It is widely distributed around the world. More than 55,000 people die from rabies every year, and about 95% of them occur in Asia and Africa. Most of the deaths were caused by bites from dogs infected with rabies virus, and 30% to 60% of the victims were children under the age of 15. The prevention and treatment of rabies usually uses a rabies vaccine.
  • virus-infected cell culture supernatants contain the target rabies virus, as well as host cell debris and proteins as well as host DNA. In addition, there is a cell culture medium component. These impurities are all removed. Among them, the key to the safety of rabies vaccine preparation by passage cells is the content of residual DNA.
  • the Chinese Pharmacopoeia stipulates that the rabies vaccine (Vero cells) must have a host DNA residue of no more than 100 pg/dose.
  • the traditional rabies virus purification route is mainly used for ultrafiltration concentration and gel filtration chromatography.
  • gel filtration chromatography is currently the most effective purification method, but the purification effect of this method can not meet the requirements of the new national standard lOOpg/agent of rabies vaccine products.
  • the main reason is that gel filtration chromatography is based on the molecular weight of the organism. The separation is carried out, and the virus is comparable to the molecular weight of the host DNA and cannot be effectively separated in the gel medium.
  • ⁇ is used to remove DNA by adding protamine sulfate to the virus harvesting solution.
  • the principle is that protamine sulfate can bind to DNA, and the conjugate is easy to precipitate. It can be effectively used by centrifugation. In addition to DNA. However, at the same time, protamine is also combined with the virus, and the virus is seriously lost. Usually, the recovery rate is only 25-30%.
  • the nuclease treatment step is added, and the DNA after the digestion is turned into a small fragment, and then subjected to a gel filtration chromatography step to separate most of the DNA and the virus.
  • the action of nuclease is limited by environmental conditions, it is not fully effective. There is still a small part of DNA that cannot be separated from the virus, and the final DNA residue remains at the nanogram (ng) level. The result is only close to national standards, and it is still difficult to reach the level of qualification.
  • an ion exchange chromatography step was introduced during the purification of rabies virus.
  • the first is anion exchange chromatography, which allows DNA and virus to be adsorbed on the medium together, and then the control conditions only elute the virus, enabling DNA in the virus solution.
  • the residual amount was reduced to 500 pg/ml, but the recovery of the virus amount was only about 20%.
  • CN102282253A discloses a method for purifying rabies virus, which can achieve a DNA removal rate of over 95% by using one-step cation exchange chromatography, and a virus recovery rate of more than 70%.
  • the cation exchange medium is a product of MERCK company Fractogel EMD SO 3 " , combined with a subsequent nuclease treatment step and an ultracentrifugation purification step, and the resulting virus solution is formulated into a rabies vaccine, and the DNA residue is contained in an effective dose of 2.5 IU.
  • the amount of virus recovery is less than 20 pg.
  • the virus recovery rate of the whole purification process is about 50%.
  • the purification process of rabies virus disclosed in CN102282253A is too complicated and the production cost is high.
  • CN102282253A discloses a process for purifying rabies virus by ion exchange chromatography, in which a supernatant of a culture of a virus-infected cell is first contacted with a cation exchange color carrier to bind the rabies virus to the support, and then washed from the support. Take off the virus.
  • This process is mainly to remove host DNA residues in the virus solution, but the amount of virus is lost due to the virus's first adsorption and re-elution.
  • both the virus and the host DNA are adsorbed on the support, and the subsequent elution of the virus and DNA also leads to incomplete DNA removal.
  • the object of the present invention is to provide a method for removing host DNA in a vaccine, comprising the following steps: Step 1) adjusting the pH value and salt concentration of the supernatant of the virus-infected host cell supernatant to make the pH between 6 and 8, salt concentration Between 0.2 ⁇ 0.5mol/L;
  • Step 2) The adjusted host cell supernatant concentrate is sufficiently contacted with the anion exchange medium.
  • the cell culture supernatant concentrate is clarified and concentrated before contact with the medium, and the adjusted supernatant concentration is between 6 and 8 and the salt concentration is between 0.2 and 0.5 mol/L.
  • the pH of the supernatant concentrate is preferably between 7 and 8, more preferably between 7 and 7.8; and the salt concentration is preferably from 0.3 to 0.5 mol/L.
  • the pH range is the active tolerance interval of rabies virus, and the biological activity of the virus is greatly affected by this interval.
  • the salt concentration interval is the preferred interval for virus recovery and DNA removal. When the salt concentration is lower than 0.2M/L, the virus recovery rate is only 8%. When the salt concentration is higher than 0.5M/L, the DNA removal rate decreases. To 70%.
  • the anion exchange color medium is selected from agarose microspheres coupled with diethylaminoethyl (DEAE), quaternary ammonium salt (Q), or other anionic effect media.
  • DEAE diethylaminoethyl
  • Q quaternary ammonium salt
  • the anion exchange chromatography used in the present invention does not need to rely on a specific filler, and any medium having an anionic effect can be applied to the process of the present invention.
  • Q Sepharose FF and DEAE Sepharose FF are the most common ion exchange chromatography media.
  • Q Sepharose FF is a strong anion exchange medium
  • DEAE Sepharose FF is a weak anion medium, both of which can be used in the process of the present invention and the host DNA removal effect is good.
  • the host cell is a Vero cell and the vaccine selected is a rabies vaccine.
  • Vero cells are an ideal vaccine production substrate: clear genetic background, stable karyotype, no exogenous factor contamination, no tumorigenicity within 160 generations, suitable for large-scale culture, can be produced by bioreactor, ensuring large-scale vaccine cells Homogenization and safety.
  • Vaccines currently developed, studied and produced using Vero cells as host cells include, but are not limited to, rabies vaccines, influenza vaccines, hemorrhagic fever vaccines, sputum liver vaccines, Japanese encephalitis vaccines, rotavirus vaccines, SARS vaccines, and the like.
  • CHO cells and Vero cells are recognized by the Chinese Medicine Administration as a passage cell for the production of biological products.
  • the vaccine includes, but is not limited to, a rabies vaccine, a polio vaccine, a Japanese encephalitis vaccine, a hail vaccination vaccine, a hemorrhagic fever vaccine, an influenza vaccine, a SARS vaccine or a rotavirus vaccine
  • the host cells include but Not limited to CHO cells and Vero cells
  • the method of the present invention is used to adjust the pH and salt concentration of the virus-infected host cell supernatant concentrate to a pH between 6 and 8, and the salt concentration is 0.2 to 0.5 mol. Between /L; the adjusted host cell supernatant concentrate can be fully contacted with the anion exchange medium to achieve the purpose of removing the host DNA in the vaccine.
  • the amount of virus detected in the cell culture supernatant concentrate is at least 60% before the treatment, preferably at least 80% before the treatment, and the virus amount is detected by the ELISA method for the virus. Detection of G protein component # ⁇ .
  • the DNA removal rate in the supernatant concentrate after contact with the medium is 99% or more, preferably 99.5% or more, more preferably 99.9% or more, and DNA detection is carried out by dot blot hybridization.
  • the method of removing the rabies vaccine host DNA preferably further comprises an endonuclease treatment step after the anion exchange chromatography step.
  • the endonuclease may be selected from endonucleases such as Benzonase.
  • the digested virus solution was purified by gel filtration chromatography to collect a purified absorption peak containing a viral component, and the gel filtration medium was a 4-6% cross-linked agarose microsphere.
  • the amount of virus detected in the gel filtration chromatography purification solution was 78% or more before chromatography, and some were as high as 95% or more.
  • the residual amount of host DNA in the gel filtration chromatography purification solution was less than 40 pg/ml.
  • the purified liquid obtained by the above method is incubated with a single dose and lyophilized, and the lyophilized vaccine host is detected.
  • the final purified virus solution was seeded in a single dose and then lyophilized.
  • the effective dose of the lyophilized vaccine is 8 IU (tested by NIH method)
  • the host DNA residue detected by dot blot hybridization is not higher than 10 pg.
  • the method for removing the more advantageous rabies vaccine host DNA provided by the present invention has been carefully explored by anion exchange chromatography, and it has been unexpectedly found that the rabies virus is not suitable for being adsorbed and then eluted. Therefore, the inventor uses the method. Strictly control the idea of anion exchange conditions, by adjusting the pH and salt concentration of the culture supernatant of the virus-infected host cells, only the host DNA is adsorbed, and the virus is almost free from loss. The effect of DNA,
  • the present invention has the following advantages:
  • the host DNA removal rate is good, can reach 99.95%, and better can reach 99.99%; 2) the virus loss is low, and the total virus recovery rate is above 50%;
  • Figure 2 shows the results of Western blot analysis of various components of the virus.
  • FIG. 3 is a photograph of a rabies virus electronic display after treatment by the method of the present invention.
  • DETAILED DESCRIPTION OF THE INVENTION The present invention discloses a method for removing host DNA in a vaccine, which can be borrowed by those skilled in the art. In view of the content of this paper, the process parameters are appropriately improved. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the method of the present invention has been described by the preferred embodiments, and it is obvious that the method and application described herein can be modified or appropriately modified and combined without departing from the scope of the present invention. Invention technology.
  • FIG. 1 the specific process of the method of the present invention is shown in FIG. 1.
  • the method for producing the rabies vaccine is taken as an example, and the method of the present invention is further described in detail:
  • ⁇ Use a 0.65um pore size filter to filter and clarify the virus-infected Vero cell culture harvest solution, remove a large number of cell debris and large particulate matter caused by virus lysis, and the virus particle size is about 200nm, which can be easily worn.
  • the sieve is permeable, and cell debris and large particulate matter are isolated to clarify the viral fluid.
  • the clarified virus solution is bulky, which is not conducive to the next purification operation. Therefore, the clarified virus solution is concentrated by ultrafiltration method, and the concentration ratio is generally 20-30 times.
  • the molecular weight of the ultrafiltration membrane used is 300-1000 KD. between.
  • the anion exchange medium is an agarose microsphere coupled to a DEAE or Q ligand, or other medium having an anion effect.
  • the virus solution is subjected to secondary ultrafiltration and concentration, and the ultrafiltration membrane package used has a molecular weight cutoff of 300-1000 KD.
  • virus inactivation The virus solution was inactivated with ⁇ -propiolactone 1:4000 for 24 hours, and hydrolyzed at 37 °C for 2 hours to degrade ⁇ -propiolactone.
  • the virus solution was treated with benzonase and allowed to stand at 30-37 ° C for 20 hours.
  • the virus solution is purified by gel filtration column chromatography to remove the heteroprotein, small fragment DNA and residual nuclease.
  • the gel filtration medium is a 4-6% cross-linked agarose microsphere.
  • the invention adopts an electron microscope to observe the morphology of the virus particles, and shows that the virus particles are bullet-shaped, the particle size is about 200 nm, which is consistent with the characteristics of rabies virus; and the Western blot detection is also performed on each link of the whole process (Fig. 2
  • Fig. 3 The results showed that the virus components did not change from the virus harvested liquid to the purified liquid, as shown in Fig. 3, which shows that the method for removing DNA of the present invention and the subsequent purification method preferably maintain the integrity of the virus particles, and the virus is not Cause damage.
  • the method for removing rabies vaccine host DNA provided by the present invention was carried out in a scale test of 1200 ml, 12 L, 30 L and a test of 300 L scale, respectively.
  • the host DNA was efficiently removed during the purification of the virus, and the DNA removal rate reached 99.95% or more; and the virus was efficiently recovered, and the total virus recovery rate was over 50%; and the removal of the foreign protein reached the current detection limit. the following.
  • the present invention will be further described in detail below with reference to specific embodiments.
  • the sample was loaded onto a 1 L Sepharose 4FF gel column, and the column buffer was used.
  • the liquid was PBS, and the linear elution peak was collected at a linear flow rate of 90 cm/h.
  • the residual DNA and virus amount were detected.
  • the DNA residue was 80 pg/ml, the removal rate was 99.95%, and the virus recovery rate was 55%.
  • the sample was applied to a 1 L Sepharose 4FF gel column.
  • the column buffer was PBS, and the linear flow rate was 90 cm. /h, The virus elution peak was collected, and the residual DNA and virus amount were detected.
  • the DNA residue was 60 pg/ml, the removal rate was 99.98%, and the virus recovery rate was 48%.
  • the Mg 2+ concentration was 1-2 mmol/L, and the final concentration of 90 IU/ml Benzonase nuclease was added for 20 hours; the sample was applied to a 5 L Sepharose 4FF gel column, and the column buffer was PBS, linear flow rate. At 90 cm/h, the virus elution peak was collected, and the residual DNA and virus amount were detected. The DNA residue was 50 pg/ml, the removal rate was 99.99%, and the virus recovery rate was 45%.
  • Enzyme treatment for 20 hours; loading onto 80 L Sepharose 4FF gel column, column buffer was PBS, linear flow rate was 90 cm/h, virus elution peak was collected, residual DNA and virus amount were detected, and DNA residue was 80pg/ml, the removal rate was 99.95%, and the virus recovery rate was 51%.

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Abstract

本发明公开了一种去除疫苗中宿主DNA的方法，包括调整病毒感染的宿主细胞上清浓缩液的pH值和盐浓度，使pH值在pH 6〜8之间，盐浓度在0.2〜0.5mol/L之间；使调整后的宿主细胞上清浓缩液与阴离子交换介质充分接触。采用本发明所述方法处理后，病毒量检测结果显示细胞培养上清浓缩液中检测的病毒量至少是处理前的60%，上清浓缩液中DNA的去除率在99%以上，最终的纯化病毒液制备冻干疫苗检测到宿主DNA残留量不高于10pg，完全符合疫苗中宿主DNA残留量标准，具有良好的工业应用前景。

Description

一种去除疫苗中宿主 DNA的方法 本申请要求于 2012 年 7 月 27 日提交中国专利局、 申请号为 201210263908.2、 发明名称为"一种去除疫苗中宿主 DNA的方法 "的中国专利 申请的优先权， 其全部内容通过引用结合在本申请中。 技术领域
本发明涉及生物医药领域， 特别涉及一种去除疫苗中宿主 DNA的方法。
背景技术
狂犬病是一种由狂犬病病毒引起的人畜共患疾病，在全球分布广泛，每年 死于狂犬病的人数超过 5.5万人， 其中约 95%发生在亚洲和非洲。 大多数死亡 事件均是被狂犬病毒感染的狗咬伤所致，受害者中 30%〜60%为 15岁以下儿童。 狂犬病的预防和治疗通常釆用狂犬病疫苗。
在狂犬疫苗生产中， 经病毒感染的细胞培养上清液中含有目标物狂犬病 毒， 而且还有宿主细胞碎片和蛋白以及宿主 DNA。 另外， 还有细胞培养基组 份。 这些杂质都是需要去除的。 其中， 以传代细胞制备狂犬疫苗安全性的关键 是细胞残余 DNA的含量。 中国药典规定狂犬疫苗 （Vero细胞） 的宿主 DNA 残留量不得高于 lOOpg/剂。
传统的狂犬病毒纯化路线主要釆用超滤浓缩和凝胶过滤色谱法。其中， 凝 胶过滤色谱法是目前最有效的纯化方法，但是该方法的纯化效果并不能满足狂 犬疫苗制品新的国家标准 lOOpg/剂的要求，主要原因是凝胶过滤色谱法是根据 生物分子量大小进行分离的， 而病毒与宿主 DNA的分子量不相上下， 不能在 凝胶介质中被有效分离。
此外， 釆用在病毒收获液中添加硫酸鱼精蛋白的方法去除 DNA, 原理是 硫酸鱼精蛋白可以与 DNA结合， 结合物容易沉淀， 使用离心的方法能有效去 除 DNA。 但是同时鱼精蛋白也与病毒结合， 病毒损失严重， 通常回收率只有 25-30%。
此外， 在超滤浓缩后加入核酸酶处理步骤， 经过酶切以后的 DNA变成了 小片段， 然后再经过凝胶过滤色谱步骤， 使大部分 DNA和病毒分离开。 但是 由于核酸酶的作用受环境条件限制，不能完全发挥效果，仍有极小一部分 DNA 不能和病毒分开， 最终 DNA残留量还停留在纳克（ng )级水平。 结果只是接 近国家标准， 仍然 ^艮难达到合格水平。
近几年，在狂犬病毒的纯化过程中引入了离子交换色谱步骤， 首先是阴离 子交换色谱， 让 DNA和病毒一起被吸附在介质上， 然后控制条件仅对病毒洗 脱， 能够使病毒液中 DNA的残留量降低至 500pg/ml, 但是病毒量的回收率只 有大约 20%。
CN102282253A公开了一种狂犬病病毒纯化方法 ,使用一步阳离子交换色 谱法能够使 DNA的去除率达到 95%以上， 而病毒回收率在 70%以上。 所述的 阳离子交换介质是 MERCK公司的产品 Fractogel EMD SO3" , 结合后续的核酸 酶处理步骤和超速离心纯化步骤， 最终得到的病毒液配制成的狂犬疫苗，在含 有 2.5IU有效剂量里 DNA残留量小于 20pg。整个纯化过程病毒回收率在 50% 左右。 然而， CN102282253A公开的狂犬病病毒纯化工艺过于复杂， 生产成本 高。
CN102282253A公开了釆用离子交换色谱纯化狂犬病病毒的工艺，先将被 病毒感染的细胞的培养物的上清液与阳离子交换色语担体接触，以便让狂犬病 毒结合在担体上， 随后从担体上洗脱该病毒。这样的工艺流程主要是为了去除 病毒液中的宿主 DNA残留， 但是由于病毒先吸附再洗脱的做法使得病毒量损 失较大。 而且病毒和宿主 DNA都吸附在担体上， 先后洗脱病毒和 DNA的做 法也导致了 DNA去除不彻底的后果。
因此开发一种既能有效去除 DNA残留从而达到国家药典标准又能提高病 毒回收率的纯化工艺非常重要。 发明内容
本发明的目的在于提供一种去除疫苗中宿主 DNA的方法， 包括： 步骤 1 )调整病毒感染的宿主细胞上清浓缩液的 pH值和盐浓度， 使 pH 值在 6〜8之间 , 盐浓度在 0.2〜0.5mol/L之间；
步骤 2 )使调整后的宿主细胞上清浓缩液与阴离子交换介质充分接触。 细胞培养上清浓缩液在与介质接触之前要进行澄清和浓缩 ,调整后的上清 浓缩液 pH值在 6〜8之间， 盐浓度在 0.2〜0.5mol/L之间。 上清浓缩液 pH值优 选在 7〜8之间， 进一步优选在 7〜7.8之间； 盐浓度优选 0.3〜0.5mol/L。
pH值的区间是狂犬病毒的活性耐受区间， 越过这个区间病毒生物活性会 受到很大影响。 盐浓度区间则是病毒回收和 DNA去除的优选区间， 当盐浓度 低于 0.2M/L时， 病毒的回收率只有 8%; 而当盐浓度高于 0.5M/L时， DNA的 去除率下降到 70%。
阴离子交换色语介质选自偶联二乙胺基乙基 ( DEAE )、 季铵盐（ Q )为配 基的琼脂糖微球或者是其他具有阴离子效果的介质。本发明釆用的阴离子交换 层析并不需要依赖特定的填料，任何具有阴离子效果的介质都可以运用到本发 明的工艺中。
在本发明的实施例中釆用了 Q Sepharose FF和 DEAE Sepharose FF这两种 为最普遍的因离子交换色谱介质。 Q Sepharose FF为强阴离子交换介质， DEAE Sepharose FF是弱阴离子介质， 两者均可用于本发明的工艺中而且去除宿主 DNA效果良好。
在本发明的具体实施方式中， 所述宿主细胞为 Vero细胞， 所选用疫苗为 狂犬疫苗。 Vero细胞是一种理想的疫苗生产基质： 遗传背景清楚， 核型稳定， 无外 源因子污染， 160代以内没有致瘤性，适合大规模培养，可用生物反应器生产， 保证了疫苗大批量细胞的均质性和安全性。 目前用 Vero细胞作为宿主细胞开 发、 研究和生产的疫苗包括但不限于狂犬疫苗、 流感疫苗、 出血热疫苗、 曱肝 疫苗、 乙脑疫苗、 轮状病毒疫苗、 SARS疫苗等等。
CHO细胞与 Vero细胞被 WHO是经中国药品监督管理局认可， 用于生物 制品的生产的传代细胞。本领域技术人员理解， 所述疫苗包括但不限于狂犬疫 苗、 脊髓灰质炎疫苗、 乙脑疫苗、 曱肝疫苗、 出血热疫苗、 流感疫苗、 SARS 疫苗或轮状病毒疫苗， 所述宿主细胞包括但不限于 CHO细胞与 Vero细胞， 利 用本发明所述方法处理， 调整病毒感染的宿主细胞上清浓缩液的 pH值和盐浓 度， 使 pH值在 6〜8之间， 盐浓度在 0.2〜0.5mol/L之间； 使调整后的宿主细胞 上清浓缩液与阴离子交换介质充分接触， 均可达到去除疫苗中宿主 DNA的目 的。
釆用本发明所述方法处理后，细胞培养上清浓缩液中检测的病毒量至少是 处理前的 60%, 优选至少是处理前的 80%以上， 病毒量检测是釆用 ELISA方 法对病毒的 G蛋白组分 #丈的检测。
接触介质后上清浓缩液中 DNA的去除率在 99%以上， 优选 99.5%以上， 更优选 99.9%以上， DNA检测釆用斑点杂交法。
所述狂犬疫苗宿主 DNA的去除方法， 在阴离子交换色谱步骤之后优选还 包含核酸内切酶处理步骤。 核酸内切酶可以选自 Benzonase等品牌的内切酶。
酶切后的病毒液经过凝胶过滤层析纯化， 收集含有病毒成分的纯化吸收 峰， 凝胶过滤介质是 4-6%交联度的琼脂糖微球。 凝胶过滤层析纯化液中检测 的病毒量是层析前的 78%以上， 有的高达 95%以上。 凝胶过滤层析纯化液中 宿主 DNA残留量小于 40pg/ml。 上述方法得到的纯化液以单剂量配苗并进行冻干， 检测到冻干疫苗宿主
DNA残留量 < 10pg/剂。
最终的纯化病毒液以单剂量配苗， 然后进行冻干。 冻干疫苗有效剂量为 8IU ( NIH法检测） 时， 用斑点杂交法检测宿主 DNA残留量不高于 10pg。
本发明提供的更具优势的狂犬疫苗宿主 DNA的去除方法， 通过对阴离子 交换色谱进行了仔细的实验摸索，意外的发现狂犬病毒不适合被吸附后再洗脱 下来， 为此， 发明人釆用严格控制阴离子交换条件的思路， 通过调整病毒感染 的宿主细胞培养上清浓缩液的 pH值和盐浓度， 仅仅使宿主 DNA被吸附， 而 病毒几乎不受损失， 这一思路达到非常好的去除宿主 DNA的效果，
与现有技术相比， 本发明具有如下优点：
1 )宿主 DNA去除率好， 能够达到 99.95%, 更优的能达到 99.99%; 2 ) 病毒损失低， 总的病毒回收率在 50%以上；
3 )避免使用价格昂贵的耗材和设备， 生产成本大大降低；
4 )避免使用操作复杂的技术和方法， 使生产过程大大简化；
5 )全过程各项技术结合紧密， 运行流畅， 易于放大进行工业化大规模生 产。 附图说明 图 1为本发明所述方法流程图。
图 2为对病毒各组分的 Western blot检测结果。
图 3为本发明所述方法处理后的狂犬病毒电子显啟镜照片。 具体实施方式 本发明公开了一种去除疫苗中宿主 DNA的方法， 本领域技术人员可以借 鉴本文内容， 适当改进工艺参数实现。 特别需要指出的是， 所有类似的替换和 改动对本领域技术人员来说是显而易见的， 它们都被视为包括在本发明。本发 明的方法已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内 容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合， 来实 现和应用本发明技术。
在本发明的实施例中， 本发明所述方法的具体流程如图 1 , 下面以 Vero 细胞生产狂犬疫苗为例， 对本发明所述方法作进一步的详细说明：
1. 澄清步骤
釆用 0.65um孔径的套筒式滤器对病毒感染的 Vero细胞培养收获液做过滤 澄清，去除因细胞被病毒裂解产生的大量细胞碎片和大颗粒物质， 病毒颗粒大 小在 200nm左右， 能够很容易穿透滤器的筛孔， 而细胞碎片和大颗粒物质被 隔离， 使病毒液澄清。
2. 浓缩步骤
澄清后的病毒液体积大， 不利于下一步的纯化操作， 所以釆用超滤方法对 澄清病毒液浓缩， 浓缩倍数一般在 20-30倍， 使用的超滤膜包截留分子量在 300-1000KD之间。
3. 阴离子交换介质
与离子交换介质充分接触之前， 调整病毒浓缩液的 pH值在 6-8之间， 盐 浓度在 0.2-0.5mol/之间， 然后进行阴离子交换处理， 收集处理后病毒液。 所述 的阴离子交换介质是耦联 DEAE或 Q配基的琼脂糖微球， 或者是其他具有阴 离子效果的介质。
4. 二次超滤浓缩
经过离子交换色语后， 为了减轻下一步操作负担， 同时也为了降低生产成 本， 对病毒液进行二次超滤浓缩， 所用超滤膜包截留分子量 300-1000KD。
5. 病毒灭活 釆用 β-丙内酯 1 :4000稀释对病毒液灭活 24小时， 37°C水解 2小时降解 β- 丙内酯。
6. 核酸酶处理
釆用 benzonase酶处理病毒液， 30-37°C静置 20小时。
7. 凝胶过滤色谱法纯化
酶处理后病毒液经过凝胶过滤柱层析纯化， 去除杂蛋白、 小片段 DNA和 残留核酸酶。 所述凝胶过滤介质为 4-6%交联度的琼脂糖微球。
本发明对最终纯化液做了电子显微镜观察病毒颗粒形态，显示病毒颗粒呈 子弹状， 颗粒大小在 200nm左右， 符合狂犬病毒特征； 同时也对整个工艺的 每个环节做了 Western blot检测（图 2 ), 结果显示从病毒收获液到纯化液， 病 毒组分没有发生改变， 见图 3 , 说明本发明的去除 DNA的方法以及后续的纯 化方法比较好的保持了病毒颗粒的完整性， 对病毒没有造成损害。
本发明提供的去除狂犬疫苗宿主 DNA的方法分别进行了 1200ml、 12L、 30L规模试验和 300L规模的实验。 在病毒的纯化过程中均高效地去除了宿主 DNA, DNA去除率达到 99.95%以上； 并且有效地回收了病毒， 总的病毒回收 率在 50%以上； 而且外源蛋白的去除到了目前的检测限度以下。 为了使本领域的技术人员更好地理解本发明的技术方案，下面结合具体实 施例对本发明作进一步的详细说明。 实施例 1:
取 1200ml狂犬病毒收获液，用 0.65um孔径的套筒滤器澄清过滤，超滤浓 缩 30倍， 用磷酸盐緩冲液调整 pH为 7.8 , 盐摩尔浓度为 0.4mol/L, 与 50ml Q Sepharose FF介质充分接触 15分钟； 然后对介质处理后的病毒液超滤浓缩 3 倍； 用 β-丙内酯终浓度 1 :4000灭活 24小时； 灭活液 37°C水解 2小时； 用氯 化镁溶液调整 Mg2+浓度为 l-2mmol/L,加入终浓度为 90IU/ml的 Benzonase核 酸酶， 处理 20小时； 上样到 1 L Sepharose 4FF凝胶层析柱中， 层析柱緩冲液 为 PBS, 线性流速 90cm/h收取病毒洗脱峰， 检测残留 DNA和病毒量， DNA 残留量为 80pg/ml, 去除率为 99.95%, 病毒回收率 55%。
Figure imgf000009_0001
实施例 2:
取 1200ml狂犬病毒收获液，用 0.65um孔径的套筒滤器澄清过滤，超滤浓 缩 30倍， 用磷酸盐緩冲液调整 pH为 7.0, 盐摩尔浓度为 0.32mol/L, 与 50ml DEAE Sepharose FF介质充分接触 15分钟； 然后对介质处理后的病毒液超滤 浓缩 3倍； 用 β-丙内酯终浓度 1:4000灭活 24小时； 灭活液 37°C水解 2小时； 用氯化镁溶液调整 Mg2+浓度为 l-2mmol/L,加入终浓度为 90IU/ml的 Benzonase 核酸酶， 处理 20小时； 上样到 1 L Sepharose 4FF凝胶层析柱中， 柱緩冲液为 PBS, 线性流速 90cm/h, 收取病毒洗脱峰， 检测残留 DNA和病毒量， DNA 残留量为 60pg/ml, 去除率为 99.98%, 病毒回收率 48%。 病毒收率 病毒总收率 DNA去除率 DNA总去除率 病毒收获液 100%
浓缩液 90%
离子交换液 65% 99.90%
二次浓缩液 100% 48.40% 99.98%
灭活液 93%
酶处理液 89% 70%
凝胶过滤液 100% 20% 实施例 3:
取 12L狂犬病毒收获液， 用 0.65um孔径的套筒滤器澄清过滤， 超滤浓缩 30倍， 用碑酸盐緩冲液调整 pH为 7.5 , 盐摩尔浓度为 0.375mol/L, 与 500ml Q Sepharose FF介质充分接触 15分钟； 然后对介质处理后的病毒液超滤浓缩 3 倍； 用 β-丙内酯终浓度 1 :4000灭活 24小时； 灭活液 37°C水解 2小时； 用氯 化镁溶液调整 Mg2+浓度为 l-2mmol/L,加入终浓度为 90IU/ml的 Benzonase核 酸酶，处理 20小时；上样到 5 L Sepharose 4FF凝胶层析柱中，柱緩冲液为 PBS , 线性流速 90cm/h, 收取病毒洗脱峰， 检测残留 DNA和病毒量， DNA残留量 为 50pg/ml, 去除率为 99.99%, 病毒回收率 45%。
Figure imgf000010_0001
实施例 4:
取 30L狂犬病毒收获液， 用 0.65um孔径的套筒滤器澄清过滤， 超滤浓缩
30倍， 用磷酸盐緩冲液调整 pH为 7.6, 盐摩尔浓度为 0.35mol/L, 与 1 L Q Sepharose FF介质充分接触 20分钟； 然后对介质处理后的病毒液超滤浓缩 5 倍； 用 β-丙内酯终浓度 1 :4000灭活 24小时； 灭活液 37°C水解 2小时； 用氯 化镁溶液调整 Mg2+浓度为 l-2mmol/L,加入终浓度为 90IU/ml的 Benzonase核 酸酶， 处理 20小时； 上样到 10 L Sepharose 4FF凝胶层析柱中， 柱緩冲液为 PBS , 线性流速 90cm/h, 收取病毒洗脱峰， 检测残留 DNA和病毒量， DNA 残留量为 80pg/ml, 去除率为 99.96%, 病毒回收率 50%t
Figure imgf000011_0001
实施例 5:
300L狂犬病毒收获液， 用 0.65um孔径的套筒滤器澄清过滤， 超滤浓缩
30倍， 用碑酸盐緩冲液调整 pH为 7.0, 盐摩尔浓度为 0.4mol/L, 与 10L DEAE Sepharose FF介质充分接触 20分钟； 然后对介质处理后的病毒液超滤浓缩 5 倍； 用 β-丙内酯终浓度 1 :4000灭活 24小时； 灭活液 37°C水解 2小时； 用氯 化镁溶液调整 Mg2+浓度为 l-2mmol/L,加入终浓度为 90IU/ml的 Benzonase核 酸酶， 处理 20小时； 上样到 80 L Sepharose 4FF凝胶层析柱中， 柱緩冲液为 PBS , 线性流速 90cm/h, 收取病毒洗脱峰， 检测残留 DNA和病毒量， DNA 残留量为 80pg/ml, 去除率为 99.95%, 病毒回收率 51%。
Figure imgf000011_0002
本发明提出的一种去除疫苗中宿主 DNA的方法已通过实施例进行了描 述，相关技术人员明显能在不脱离本发明内容、精神和范围内对本文所述的去 除疫苗中宿主 DNA的方法进行改动或适当变更与组合， 来实现本发明技术。 特别需要指出的是，所有相类似的替换和改动对本领域技术人员来说是显而易 见的， 它们都被视为包括在本发明的精神、 范围和内容中。

Claims

权 利 要 求
1. 一种去除疫苗中宿主 DNA的方法， 包括以下步骤：
步骤 1 )调整病毒感染的宿主细胞上清浓缩液的 pH值和盐浓度， 使 pH 值在 6〜8之间， 盐浓度在 0.2〜0.5mol/L之间；
步骤 2 )使调整后的宿主细胞上清浓缩液与阴离子交换介质充分接触。
2. 根据权利要求 1的方法， 所述盐浓度为在 0.3〜0.5mol/L之间。
3. 根据权利要求 1的方法， 所述上清浓缩液的 pH值选自 7-8或 7-7.8。
4. 根据权利要求 1-3任一项所述的方法，所述阴离子交换色语介质选自偶 联二乙胺基乙基或者季铵盐为配基的琼脂糖微球。
5. 根据权利要求 1的方法， 所述宿主细胞选自 Vero细胞或 CHO细胞。
6. 根据权利要求 1的方法， 所述疫苗选自狂犬疫苗、 脊髓灰质炎疫苗、 乙脑疫苗、 曱肝疫苗、 出血热疫苗、 流感疫苗、 SARS疫苗或轮状病毒疫苗。
7. 根据权利要求 1的方法， 步骤 2 )之后还包含核酸内切酶处理步骤。
8. 根据权利要求 7中所述的方法， 所述核酸内切酶为 Benzonase。
9. 根据权利要求 7或 8所述的方法， 酶切处理后的病毒液经过凝胶过滤 层析纯化。
10. 根据权利要求 9的方法， 所述凝胶过滤介质是 4-6%交联度的琼脂糖 微球。
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