WO2022095987A1 - 一种通过灌流培养工艺制备腺病毒载体疫苗的方法 - Google Patents

一种通过灌流培养工艺制备腺病毒载体疫苗的方法 Download PDF

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WO2022095987A1
WO2022095987A1 PCT/CN2021/129182 CN2021129182W WO2022095987A1 WO 2022095987 A1 WO2022095987 A1 WO 2022095987A1 CN 2021129182 W CN2021129182 W CN 2021129182W WO 2022095987 A1 WO2022095987 A1 WO 2022095987A1
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perfusion
cells
adenovirus
cell
culture
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French (fr)
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肖猛
刘云杰
朱涛
许允立
徐灿
李军强
巢守柏
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康希诺生物股份公司
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Priority to EP21888679.4A priority Critical patent/EP4151725A4/en
Priority to US18/011,742 priority patent/US20230313225A1/en
Priority to KR1020227043727A priority patent/KR20230011352A/ko
Priority to CA3182438A priority patent/CA3182438A1/en
Publication of WO2022095987A1 publication Critical patent/WO2022095987A1/zh

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Definitions

  • the invention relates to the technical field of biological products, in particular to a method for culturing adenovirus host cells, a method for producing adenovirus and a method for preparing an adenovirus vector vaccine, in particular a method for preparing an adenovirus vector vaccine by a perfusion culture process.
  • Adenovirus is a non-enveloped DNA virus with the characteristics of susceptibility to infection, wide host range, low toxicity, safe use, large capacity, non-integration, low pathogenicity to humans, and no carcinogenesis. Become one of the most promising gene carriers in gene transfer.
  • the modified adenovirus removes the E1/E3 gene expression cassette, preventing the transcription of functional proteins dependent on the E1 and E3 regions and the subsequent replication of viral DNA and the production of viral coat proteins.
  • E1/E3-deficient adenoviruses can provide E1 /E3 region gene protein was proliferated in cells.
  • Adenovirus has the characteristics of wide host range and high transgene expression ability. At present, up to 300 kinds of human therapeutic or preventive recombinant adenovirus drugs are being used in clinical trials.
  • the widely used adenovirus vectors are human serotype adenovirus Ad5 and Ad26. type 3 and chimpanzee type 3 and 68 adenovirus; a number of vaccines using adenovirus as a vector are also under clinical research.
  • the recombinant adenovirus vector vaccine uses adenovirus as a carrier to recombine the protective antigen gene into the adenovirus genome. After the adenovirus infects the cells, the adenovirus genome together with the protective antigen gene can be injected into the host cell for intracellular expression. Protective antigens that generate humoral and cellular immunity.
  • adenovirus vector vaccines are currently in preclinical or clinical development, such as human immunodeficiency virus (HIV), rabies virus (RV), hepatitis B virus (HBV), hepatitis virus (HCV), dengue virus (DEN), herpes Virus (EB) Ebola and Novel Coronavirus.
  • 293 cells are the most commonly used host cells for adenoviral vector packaging and production.
  • 293 cells are human kidney epithelial cell lines, and there are many derivative strains, such as HEK293, 293T/17, etc.; 293 cells are generally adherent growth, but there are also cell lines adapted to suspension culture.
  • HEK293 cells contain the E1/E3 regions of adenovirus and, therefore, can be used for the production of replication-defective adenovirus vectors.
  • the present invention provides a method for culturing adenovirus host cells, which includes the step of perfusion culture, especially the step of adjusting the perfusion rate according to the cell density (eg, adjusting the perfusion rate through at least two stages).
  • the above method includes the following steps:
  • Perfusion rate of 1-3 VVD vessel volume/day (eg 1, 1.5, 2, 2.5, 3 VVD);
  • the cell density was grown to 5-10 ⁇ 10 6 cells/mL (specifically, 5 ⁇ 10 6 , 6 ⁇ 10 6 , 7 ⁇ 10 6 , 8 ⁇ 10 6 , 9 ⁇ 10 6 , 10 ⁇ 10 6 cells/mL) mL), adjust the perfusion rate to 2-4 VVD (eg, 2, 2.5, 3, 3.5, 4 VVD).
  • VVD eg, 2, 2.5, 3, 3.5, 4 VVD
  • the above-mentioned cell culture is carried out in a bioreactor (eg, a disposable stirred reactor or a torrent reactor).
  • a bioreactor eg, a disposable stirred reactor or a torrent reactor.
  • the above-mentioned perfusion is performed by using a continuous perfusion device; more specifically, the continuous perfusion device adopts an alternate tangential flow cell retention system, wherein the retention pore size of the hollow fiber column is 0.1-0.8 ⁇ m (specifically, 0.1, 0.2, 0.4, 0.6, 0.8 ⁇ m).
  • the liquid exchange rate of the perfusion can be 1-12 L/h (specifically, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 L/h).
  • the temperature of the above cell culture can be 32-38°C (specifically, 32, 33, 34, 35, 36, 37, 38°C).
  • the pH of the above cell culture can be 6.5-7.5 (specifically, 6.5, 6.6, 6.8, 7.0, 7.2, 7.4, 7.5).
  • the dissolved oxygen concentration of the above cell culture can be 30-80% (specifically, such as 30%, 40%, 50%, 60%, 70%, 80%).
  • the CO 2 concentration of the above cell culture can be 4-8% (specifically, 4%, 5%, 6%, 7%, 8%).
  • the stirring speed of the above cell culture can be 30-200 rpm (specifically, 30, 40, 50, 60, 80, 100, 120, 140, 160, 180, 200 rpm).
  • the medium used for the above-mentioned cell culture can be any known medium suitable for the growth of host cells.
  • the above method also includes the step of continuously tracking the consumption of glutamine during the cell culture process, and performing supplementation.
  • the concentration of glutamine is maintained above 2 mM (specifically, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30 mM ).
  • the above method may further include the step of amplifying and culturing the host cells before step (1).
  • the passage density of the above-mentioned scaled-up culture is 1-4 ⁇ 10 6 cells/mL (specifically, such as 1 ⁇ 10 6 , 2 ⁇ 10 6 , 3 ⁇ 10 6 , and 4 ⁇ 10 6 cells/mL).
  • the passage time of the above-mentioned scale-up culture is 48-90 hours (specifically, 48, 60, 72, 84, and 90 hours).
  • the temperature of the above-mentioned scale-up culture can be 32-38°C (specifically, 32, 33, 34, 35, 36, 37, 38°C).
  • the CO 2 concentration of the above cell culture can be 4-8% (specifically, 4%, 5%, 6%, 7%, 8%).
  • the stirring speed of the above cell culture can be 100-170 rpm (specifically, 100, 110, 120, 130, 140, 150, 160, 170 rpm).
  • the above-mentioned host cell is a cell capable of packaging adenovirus.
  • the above-mentioned host cells can be grown in adherent culture or in suspension culture, especially those that have been adapted to suspension culture.
  • the above-mentioned host cells are 293 cells, such as, but not limited to, HEK293 cells, HEK293.CS cells, and the like.
  • the above-mentioned adenoviruses may be replication-type or replication-deficient, especially replication-deficient adenoviruses.
  • the above-mentioned adenovirus can be a human adenovirus (eg, human adenovirus type 5 (AdHu5 type), AdHu4, AdHu7, AdHu11, AdHu26, AdHu55 type, etc.), animal adenovirus vectors such as chimpanzee adenovirus vectors (eg, chimpanzee adenovirus 68 type (AdC68), AdC3, etc.); in one embodiment of the present invention, the above-mentioned adenovirus is AdHu5.
  • human adenovirus eg, human adenovirus type 5 (AdHu5 type), AdHu4, AdHu7, AdHu11, AdHu26, AdHu55 type, etc.
  • animal adenovirus vectors such as chimpanzee adenovirus vectors (eg, chimpanzee adenovirus 68 type (AdC68), AdC3, etc.); in one embodiment of the present invention, the above-menti
  • the above-mentioned adenovirus is a recombinant adenovirus comprising an encoding foreign gene, for example, for gene therapy, for vaccine and the like.
  • the above-mentioned adenovirus comprises the structural protein (such as one or more of S protein, M protein, E protein, and N protein) gene (full-length or partial sequence) of SARS-CoV-2, especially the S protein gene .
  • structural protein such as one or more of S protein, M protein, E protein, and N protein
  • SARS-CoV-2 full-length or partial sequence
  • the above-mentioned adenovirus is an AdHu5 type comprising the S protein gene of SARS-CoV-2.
  • the present invention also provides a method for producing adenovirus, comprising the steps of:
  • the above-mentioned step (a) can adopt the steps of the above-mentioned adenovirus host cell culture method of the present invention.
  • the production method of the above-mentioned adenovirus comprises the following steps:
  • the perfusion rate is 1-3VVD (eg 1, 1.5, 2, 2.5, 3VVD);
  • the cell density was grown to 5-10 ⁇ 10 6 cells/mL (specifically, 5 ⁇ 10 6 , 6 ⁇ 10 6 , 7 ⁇ 10 6 , 8 ⁇ 10 6 , 9 ⁇ 10 6 , 10 ⁇ 10 6 cells/mL) mL), adjust the perfusion rate to 2-4VVD (eg 2, 2.5, 3, 3.5, 4VVD);
  • the host cell density of the virus inoculated in step (3) reaches 10-50 ⁇ 10 6 cells/mL (specifically, such as 10 ⁇ 10 6 , 20 ⁇ 10 6 , 30 ⁇ 10 6 , 40 ⁇ 10 6 , 50 ⁇ 106 /mL).
  • the culture methods include: perfusion culture, batch culture, fed batch culture, etc., especially perfusion culture; more specifically, the perfusion rate can be 1-3VVD (specifically, 1, 1.5 , 2, 2.5, 3VVD).
  • the medium used for the culture can be any known medium suitable for adenovirus amplification and production, such as CD293 medium, SFM4HEK293 medium, Ex-Cell293 medium, etc.
  • the medium used in the above-mentioned medium is suitable for all.
  • the above-mentioned production method of adenovirus may further comprise step (5): harvesting the virus.
  • the above-mentioned adenoviruses may be replication-type or replication-deficient, especially replication-deficient adenoviruses.
  • the above-mentioned adenovirus can be a human adenovirus (eg, human adenovirus type 5 (AdHu5 type), AdHu4, AdHu7, AdHu11, AdHu26, AdHu55 type, etc.), animal adenovirus vectors such as chimpanzee adenovirus vectors (eg, chimpanzee adenovirus 68 type (AdC68), AdC3, etc.); in one embodiment of the present invention, the above-mentioned adenovirus is AdHu5 type.
  • human adenovirus eg, human adenovirus type 5 (AdHu5 type), AdHu4, AdHu7, AdHu11, AdHu26, AdHu55 type, etc.
  • animal adenovirus vectors such as chimpanzee adenovirus vectors (eg, chimpanzee adenovirus 68 type (AdC68), AdC3, etc.); in one embodiment of the present invention, the above-
  • the above-mentioned adenovirus is a recombinant adenovirus comprising an encoding foreign gene, for example, for gene therapy, for vaccine and the like.
  • the above-mentioned adenovirus comprises the structural protein (such as one or more of S protein, M protein, E protein, and N protein) gene (full-length or partial sequence) of SARS-CoV-2, especially the S protein gene .
  • structural protein such as one or more of S protein, M protein, E protein, and N protein
  • SARS-CoV-2 full-length or partial sequence
  • the above-mentioned adenovirus is an AdHu5 type comprising the S protein gene of SARS-CoV-2.
  • the present invention also provides a method for preparing an adenovirus vector vaccine, which comprises the steps of the above-mentioned adenovirus host cell culture method or the above-mentioned adenovirus production method of the present invention.
  • the above-mentioned adenovirus vector contains the structural protein (such as one or more of S protein, M protein, E protein, and N protein) gene (full-length or partial sequence) of SARS-CoV-2, especially the S protein Gene.
  • structural protein such as one or more of S protein, M protein, E protein, and N protein
  • SARS-CoV-2 full-length or partial sequence of SARS-CoV-2, especially the S protein Gene.
  • the above-mentioned adenovirus vector is an AdHu5 type adenovirus vector comprising the S protein gene of SARS-CoV-2.
  • the above-mentioned preparation method of the adenovirus vector vaccine may further comprise the step of preparing the adenovirus into a suitable preparation.
  • the above formulation is an injection.
  • the above formulation is a formulation for mucosal administration, for example, nasal drops, aerosols, sprays, powder aerosols, powders, gels, microspheres, liposomes, membranes doses, suspensions, etc.
  • the technical solution provided by the present invention is a technical method for culturing adenovirus host cells (eg, 293 cells) at high density through a perfusion culture process.
  • the selected culture conditions and perfusion conditions are suitable for the growth of adenovirus host cells (such as 293 cells), which can achieve higher cell densities and virus infection than batch and conventional perfusion processes.
  • the technical solution provided by the present invention can cultivate cells to a high density and obtain a high titer virus harvest liquid, and purify the upstream perfusion culture liquid by a combination of lysis, clarification and column chromatography, effectively removing various impurities and obtaining high titer. pure stock solution;
  • the technical solution provided by the present invention achieves high-density growth of adenovirus host cells (eg, 293 cells), and at the same time increases the single-cell yield of the infected virus and the specific activity of the virus harvest solution.
  • adenovirus host cells eg, 293 cells
  • Figure 1 shows the comparison of the highest density of 293 cells in perfusion culture and batch culture.
  • Figure 2 shows the comparison of the specific activity of adenovirus stock solution cultured by perfusion process and batch process.
  • the SARS-CoV-2 gene and its structural proteins can be retrieved by techniques known in the art.
  • the SARS-CoV-2 gene can be shown in GenBank: MT419849.1
  • its structural proteins: S protein, E protein , M protein gene can be shown in GenBank: 21387-25208, 26069-26296, 26347-27015 of MT419849.1.
  • Perfusion process 1 After the 293 cells were recovered, they were expanded and inoculated into the fermenter. When the cell density reached 1 ⁇ 10 6 cells/mL, the perfusion was started, and the perfusion rate was 2VVD. Continue to cultivate the cell density to 5 ⁇ 10 6 cells/mL, and adjust the perfusion rate to 3VVD.
  • Perfusion process 2 After the 293 cells were recovered, they were expanded and inoculated into the fermenter. When the cell density reached 1 ⁇ 10 6 cells/mL or 5 ⁇ 10 6 cells/mL, the perfusion was started. The perfusion rate was maintained constant at 1VVD or 3VVD throughout the culture.
  • Perfusion process 3 After the 293 cells were recovered, they were expanded and inoculated into the fermenter. When the cell density reached 1 ⁇ 10 6 cells/mL or 5 ⁇ 10 6 cells/mL, the perfusion was started. The perfusion rate was maintained constant at 2VVD or 4VVD throughout the culture.
  • the reactor parameters are as follows: the culture temperature is 37°C, the pH is adjustable at 6.5-7.5, the dissolved oxygen concentration is 30-80%, and the stirring speed is adjustable at 30-40rpm.
  • the method of perfusion process 1 can obtain higher cell density, and the cell viability is also higher than that of process 2 and process 3.
  • Example 2 Comparison of cell density and viability between perfusion culture process and batch process culture
  • Experiment 2 The perfusion process 1 of Example 1 was used, that is, after the 293 cells were recovered, they were expanded and inoculated into the fermentor. When the cell density reached 1 ⁇ 10 6 cells/mL, the perfusion was started, and the perfusion rate was 2VVD. Continue to cultivate the cell density to 5 ⁇ 10 6 cells/mL, and adjust the perfusion rate to 3VVD.
  • Experiment 1 After the 293 cells were recovered, they were expanded and inoculated into the fermenter. When the cell density reached 1 ⁇ 10 6 cells/mL, the perfusion was started, and the perfusion rate was 2VVD. Continue to cultivate the cell density to 5 ⁇ 10 6 cells/mL, and adjust the perfusion rate to 3VVD. The concentration of glutamine was not added in the perfusion process.
  • Experiment 2 After the 293 cells were recovered, they were expanded and inoculated into the fermenter. When the cell density reached 1 ⁇ 10 6 cells/mL, the perfusion was started, and the perfusion rate was 2VVD. Continue to cultivate the cell density to 5 ⁇ 10 6 cells/mL, and adjust the perfusion rate to 3VVD. The glutamine concentration in the perfusion process was monitored, and glutamine was supplemented to maintain the glutamine concentration to 2 mM.
  • Experiment 3 After the 293 cells were recovered, they were expanded and inoculated into the fermenter. When the cell density reached 1 ⁇ 10 6 cells/mL, the perfusion was started, and the perfusion rate was 2VVD. Continue to cultivate the cell density to 5 ⁇ 10 6 cells/mL, and adjust the perfusion rate to 3VVD. The glutamine concentration in the perfusion process was monitored, and glutamine was supplemented to maintain the glutamine concentration to 10 mM.
  • Experiment 4 After the 293 cells were recovered, they were expanded and inoculated into the fermenter. When the cell density reached 1 ⁇ 10 6 cells/mL, the perfusion was started, and the perfusion rate was 2VVD. Continue to cultivate the cell density to 5 ⁇ 10 6 cells/mL, and adjust the perfusion rate to 3VVD. The glutamine concentration in the perfusion process was monitored, and glutamine was supplemented to maintain the glutamine concentration to 20 mM.
  • the reactor parameters in the above experiments 1-4 were as follows: the culture temperature was 37°C, the pH was adjustable at 6.5-7.5, the dissolved oxygen concentration was adjustable at 30-80%, and the stirring speed was adjustable at 30-40rpm.
  • Test 1 Test 2 Trial 3 Test 4 Glutamine concentration 0 2mM 10mM 20mM
  • Example 4 The highest density comparison between perfusion culture and batch culture of 293 cells
  • Test 2 The perfusion culture process of the perfusion process of Example 3 was adopted, that is, after the 293 cells were recovered, they were expanded and inoculated into the fermenter. When the cell density reached 1 ⁇ 10 6 cells/mL, the perfusion was started, and the perfusion flow rate was 2VVD. . Continue to cultivate the cell density to 5 ⁇ 10 6 cells/mL, and adjust the perfusion rate to 3VVD. The glutamine concentration in the perfusion process was monitored, and glutamine was supplemented to maintain the glutamine concentration to 2 mM.
  • Fig. 1 show that the maximum culture density of batch culture is 3.8 ⁇ 10 6 cells/mL.
  • the continuous perfusion device can continuously supplement fresh medium to the reactor to replace the cell metabolic waste in the reactor and maintain sufficient nutrients for the cells. 100 times the culture, has obvious advantages.
  • Example 5 Virus yield of perfusion culture process
  • Test 1 perfusion process: perfusion culture to a cell density of 10 ⁇ 10 6 cells/mL, inoculate a recombinant novel coronavirus (adenovirus vector) seed (prepared according to the known prior art), monitor the glutamine concentration in the medium, and replenish The concentration of glutamine was added to more than 3mM, and the perfusion rate was selected as 1VVD or 3VVD. After the incubation, the virus harvest solution was taken to measure the titer by enzyme-linked immunosorbent assay.
  • adenovirus vector recombinant novel coronavirus
  • Experiment 2 Batch process: batch culture inoculation, no glutamine supplementation, and no perfusion after inoculation.
  • the single cell yield can be increased by about 10 times by using the perfusion culture method, and the production capacity of a single tank can be increased by nearly 100 times.
  • the adenovirus harvest solution of perfusion culture and batch culture was purified, the titer and the number of virus particles were determined, and the specific activity was calculated. The results are shown in Figure 2.

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Abstract

提供了一种通过灌流培养工艺制备腺病毒载体疫苗的方法。该方法包括腺病毒宿主细胞的培养步骤，特别是根据细胞密度至少通过两个阶段调节灌流速率的步骤。该方法在实现腺病毒宿主细胞高密度生长的同时，提高了感染后的病毒的单细胞产量，以及病毒收获液的比活。

Description

一种通过灌流培养工艺制备腺病毒载体疫苗的方法 技术领域
本发明涉及生物制品技术领域，具体涉及一种腺病毒宿主细胞的培养方法、腺病毒的生产方法以及腺病毒载体疫苗的制备方法，特别是通过灌流培养工艺制备腺病毒载体疫苗的方法。
背景技术
腺病毒是一种无包膜DNA病毒，具有易感染、宿主范围广、毒性低、使用安全、容纳量大、非整合性、对人致病性小且不诱导癌变等特点，使得腺病毒载体成为基因转移中最有前途的基因载体之一。
改造后的腺病毒去除了E1/E3基因表达盒，阻止了依赖E1区和E3区功能蛋白的转录与随后病毒DNA的复制及病毒外壳蛋白的产生，E1/E3缺失的腺病毒在能够提供E1/E3区基因蛋白的细胞中得到增值。
腺病毒具有宿主范围广、高转基因表达能力的特点，目前有高达300种人用治疗或者预防性重组腺病毒药物正用于临床试验，广泛应用的腺病毒载体是人血清型腺病毒Ad5、Ad26型和黑猩猩3、68型腺病毒；以腺病毒作为载体的疫苗也有多个处于临床研究中。
重组腺病毒载体疫苗是以腺病毒作为载体，将保护性抗原基因重组到腺病毒基因组中，腺病毒感染细胞后，可以将腺病毒基因组连同保护性抗原基因一起注入宿主细胞，从而在细胞内表达保护性抗原，产生体液和细胞免疫。目前有多个腺病毒载体疫苗处于临床前或者临床中，如人体免疫缺陷病毒(HIV)、狂犬病毒(RV)、乙肝病毒(HBV)、病肝病毒(HCV)、登革热病毒(DEN)、疱疹病毒(EB)埃博拉病以及新型冠状病毒。
293细胞是最常用的腺病毒载体包装和生产用宿主细胞。293细胞是人 肾上皮细胞系，有多种衍生株，比如HEK293，293T/17等；293细胞一般为贴壁生长，但是也有适应悬浮培养的细胞系。HEK293细胞含有腺病毒的E1/E3区，因此，可以用于复制缺陷型腺病毒载体的生产。
现有技术中，293细胞生产腺病毒受限于细胞密度低和细胞产毒少导致产量少，细胞密度低的主要原因是细胞培养受营养供给、氧气、代谢影响很难对293细胞进行高密度培养。受感染细胞与正常细胞生长、营养物质的竞争，以及受染细胞的状态等导致产毒不高。虽然定期补加新鲜培养基，用于维持营养环境，可以提高细胞的生长，但是这种生长有限。
发明内容
为克服现有技术的不足，本发明提供一种腺病毒宿主细胞的培养方法，其包括灌流培养的步骤，特别是根据细胞密度调节灌流速率(例如至少通过两个阶段调节灌流速率)的步骤。
具体地，上述方法包括如下步骤：
(1)接种宿主细胞，进行细胞培养；
(2)细胞密度达到1～5×10 6个/mL(具体如1×10 6、2×10 6、3×10 6、4×10 6、5×10 6个/mL)后开启灌流，灌流速率为1-3VVD(容器体积/天)(例如1、1.5、2、2.5、3VVD)；
(3)细胞密度生长至5～10×10 6个/mL(具体如5×10 6、6×10 6、7×10 6、8×10 6、9×10 6、10×10 6个/mL)后，调节灌流速率至2-4VVD(例如2、2.5、3、3.5、4VVD)。
具体地，上述细胞培养在生物反应器(例如一次性搅拌式反应器或激流式反应器)中进行。
具体地，上述灌流通过利用连续灌流装置进行；更具体地，该连续灌流装置采用交替切向流细胞截留系统，其中，中空纤维柱截留孔径为0.1～0.8μm(具体如0.1、0.2、0.4、0.6、0.8μm)。
具体地，上述灌流的换液速率可以为1～12L/h(具体如1、2、3、4、5、6、7、8、9、10、11、12L/h)。
具体地，上述细胞培养的温度可以为32-38℃(具体如32、33、34、35、36、37、38℃)。
具体地，上述细胞培养的pH可以为6.5-7.5(具体如6.5、6.6、6.8、7.0、7.2、7.4、7.5)。
具体地，上述细胞培养的溶氧浓度可以为30-80％(具体如30％、40％、50％、60％、70％、80％)。
具体地，上述细胞培养的CO 2浓度可以为4-8％(具体如4％、5％、6％、7％、8％)。
具体地，上述细胞培养的搅拌速度可以为30-200rpm(具体如30、40、50、60、80、100、120、140、160、180、200rpm)。
具体地，上述细胞培养所用培养基可以为任何已知适宜宿主细胞生长的培养基。
具体地，上述方法还包括在细胞培养过程中持续追踪谷氨酰胺的消耗情况，并进行补充的步骤。
具体地，在上述细胞培养中，谷氨酰胺的浓度维持在2mM以上(具体如2、3、4、5、6、7、8、9、10、12、14、16、18、20、30mM)。
具体地，上述方法还可以包括在步骤(1)之前对宿主细胞进行放大培养的步骤。
具体地，上述放大培养的传代密度为1～4×10 6个/mL(具体地如1×10 6、2×10 6、3×10 6、4×10 6个/mL)。
具体地，上述放大培养的传代时间为48-90小时(具体如48、60、72、84、90小时)。
具体地，上述放大培养的温度可以为32-38℃(具体如32、33、34、35、36、37、38℃)。
具体地，上述细胞培养的CO 2浓度可以为4-8％(具体如4％、5％、6％、7％、8％)。
具体地，上述细胞培养的搅拌速度可以为100-170rpm(具体如100、110、120、130、140、150、160、170rpm)。
具体地，上述宿主细胞为能够包装腺病毒的细胞。
具体地，上述宿主细胞可以贴壁培养生长或悬浮培养生长，特别是已适应悬浮培养。
在本发明的一个实施方式中，上述宿主细胞为293细胞，例如，但不限于，HEK293细胞、HEK293.CS细胞等。
具体地，上述腺病毒可以为复制型或复制缺陷型，特别是复制缺陷型腺病毒。
具体地，上述腺病毒可以为人腺病毒(例如人腺病毒5型(AdHu5型)、AdHu4、AdHu7、AdHu11、AdHu26、AdHu55型等)、动物腺病毒载体如黑猩猩腺病毒载体(例如黑猩猩腺病毒68型(AdC68)、AdC3等)；在本发明的一个实施例中，上述腺病毒为AdHu5。
在本发明的一个实施方式中，上述腺病毒为包含编码外源基因的重组腺病毒，例如用于基因治疗、用于疫苗等。
具体地，上述腺病毒包含SARS-CoV-2的结构蛋白(如S蛋白、M蛋白、E蛋白、N蛋白中的一种或多种)基因(全长或部分序列)，特别是S蛋白基因。
在本发明的一个实施例中，上述腺病毒为包含SARS-CoV-2的S蛋白基因的AdHu5型。
本发明还提供一种腺病毒的生产方法，其包括如下步骤：
(a)培养腺病毒宿主细胞；
(b)接种病毒，培养。
具体地，上述步骤(a)可采用本发明上述腺病毒宿主细胞的培养方法 的步骤。
在本发明的一个实施方式中，上述腺病毒的生产方法包括如下步骤：
(1)接种宿主细胞，进行细胞培养；
(2)细胞密度达到1～5×10 6个/mL(具体如1×10 6、2×10 6、3×10 6、4×10 6、5×10 6个/mL)后开启灌流，灌流速率为1-3VVD(例如1、1.5、2、2.5、3VVD)；
(3)细胞密度生长至5～10×10 6个/mL(具体如5×10 6、6×10 6、7×10 6、8×10 6、9×10 6、10×10 6个/mL)后，调节灌流速率至2-4VVD(例如2、2.5、3、3.5、4VVD)；
(4)接种病毒，培养。
具体地，上述病毒接种在步骤(3)的宿主细胞密度达到10～50×10 6个/mL(具体如10×10 6、20×10 6、30×10 6、40×10 6、50×10 6个/mL)后进行。
具体地，上述步骤(4)中，培养方式包括：灌流培养、批次培养、补料批次培养等，特别是灌流培养；更具体地，灌流速率可以为1-3VVD(具体如1、1.5、2、2.5、3VVD)。
具体地，上述步骤(4)中，培养所用培养基可以为任何已知适宜腺病毒扩增与生产的培养基，例如CD293培养基、SFM4HEK293培养基、Ex-Cell293培养基等，本发明实施例中所采用的培养基均适用上述培养基。
具体地，上述步骤(4)中的其他培养条件具有上述腺病毒宿主细胞的培养方法中相应条件的定义。
具体地，上述腺病毒的生产方法还可以包括步骤(5)：收获病毒。
具体地，上述腺病毒可以为复制型或复制缺陷型，特别是复制缺陷型腺病毒。
具体地，上述腺病毒可以为人腺病毒(例如人腺病毒5型(AdHu5型)、AdHu4、AdHu7、AdHu11、AdHu26、AdHu55型等)、动物腺病毒载体如黑猩猩腺病毒载体(例如黑猩猩腺病毒68型(AdC68)、AdC3等)；在本发明的一个实施例中，上述腺病毒为AdHu5型。
在本发明的一个实施方式中，上述腺病毒为包含编码外源基因的重组腺病毒，例如用于基因治疗、用于疫苗等。
具体地，上述腺病毒包含SARS-CoV-2的结构蛋白(如S蛋白、M蛋白、E蛋白、N蛋白中的一种或多种)基因(全长或部分序列)，特别是S蛋白基因。
在本发明的一个实施例中，上述腺病毒为包含SARS-CoV-2的S蛋白基因的AdHu5型。
本发明还提供一种腺病毒载体疫苗的制备方法，其包括本发明上述腺病毒宿主细胞的培养方法或上述腺病毒的生产方法的步骤。
具体地，上述腺病毒载体包含SARS-CoV-2的结构蛋白(如S蛋白、M蛋白、E蛋白、N蛋白中的一种或多种)基因(全长或部分序列)，特别是S蛋白基因。
在本发明的一个实施例中，上述腺病毒载体为包含SARS-CoV-2的S蛋白基因的AdHu5型腺病毒载体。
具体地，上述腺病毒载体疫苗的制备方法还可以包括将腺病毒制备成合适的制剂步骤。
在本发明的一个实施方式中，上述制剂为注射剂。
在本发明另一个实施方式中，上述制剂为黏膜给药制剂，例如，滴鼻剂、气雾剂、喷雾剂、粉雾剂、粉末剂、凝胶剂、微球剂、脂质体、膜剂、混悬剂等。
本发明的有益效果包括如下：
1.本发明提供的技术方案是一种通过灌流培养工艺高密度培养腺病毒宿主细胞(例如293细胞)的技术方法。所选培养条件和灌流条件适合腺病毒宿主细胞(例如293细胞)生长，经该技术方法可以得到比批次和常规灌流工艺更高的细胞密度和病毒感染，通过灌流培养和截留装置，可以持续补充新鲜培养基至反应器从而实时带走代谢废物，实现了细胞高密度培养，接 种腺病毒后病毒滴度极大提高；
2.本发明提供的技术方案可以培养细胞至高密度并得到高滴度病毒收获液，通过裂解、澄清加柱层析相结合的方法纯化上游灌流培养料液，有效去除了各种杂质，得到高纯度原液；
3.本发明提供的技术方案实现了腺病毒宿主细胞(例如293细胞)高密度生长的同时，提高了感染后的病毒的单细胞产量，以及病毒收获液的比活。
附图说明
图1所示为灌流培养与批次培养293细胞最高密度对比图。
图2所示为灌流工艺培养与批次工艺培养腺病毒原液比活对比图。
具体实施方式
除非另有定义，本发明中所使用的所有科学和技术术语具有与本发明涉及技术领域的技术人员通常理解的相同的含义。
SARS-CoV-2的基因及其各结构蛋白可通过本领域公知技术检索得到，例如，SARS-CoV-2的基因可以如GenBank:MT419849.1所示，其各结构蛋白：S蛋白、E蛋白、M蛋白基因可以为GenBank:MT419849.1的21387-25208、26069-26296、26347-27015所示。
本文所引用的各种出版物、专利和公开的专利说明书，其公开内容通过引用整体并入本文。
下面将结合本发明实施例，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1灌流法培养293细胞工艺参数确认
灌流工艺1：293细胞复苏后，扩增并接种到发酵罐，当细胞密度达到1×10 6个/mL后开启灌流，灌流速率为2VVD。继续培养细胞密度生长至5×10 6个/mL，灌流速率调整为3VVD。
灌流工艺2：293细胞复苏后，扩增并接种到发酵罐，当细胞密度达到1×10 6个/mL或5×10 6个/mL后开启灌流。整个培养过程中，灌流速率维持恒定，在1VVD或3VVD。
灌流工艺3：293细胞复苏后，扩增并接种到发酵罐，当细胞密度达到1×10 6个/mL或5×10 6个/mL后开启灌流。整个培养过程中，灌流速率维持恒定，在2VVD或4VVD。
以上3种工艺中，反应器参数为：培养温度37℃，pH为6.5-7.5可调，溶氧浓度30-80％，搅拌速度30-40rpm可调。
检测不同灌流工艺培养293细胞的密度和活率，结果见表1-3。
表1 灌流工艺培养293细胞的密度和活率(灌流工艺1)
接种密度 1×10 6个/mL 备注
第一阶段灌流速度 2VVD  
第二阶段灌流速度 3VVD 细胞生长到5×10 6个/mL开启
密度 11×10 7个/mL  
活率 98％  
结果表明，当细胞密度达到1×10 6个/mL后开启灌流，灌流速率为2VVD。继续培养细胞密度生长至5×10 6个/mL，灌流速率调整3VVD，细胞的最高生长密度可达到11×10 7个/mL。
表2 灌流工艺培养293细胞的密度和活率(灌流工艺2)
Figure PCTCN2021129182-appb-000001
Figure PCTCN2021129182-appb-000002
结果表明，当细胞密度达到1×10 6个/mL或者5×10 6个/mL后开启灌流，灌流速率全程为1VVD或者3VVD，细胞的最高生长密度可达到7×10 7个/mL。
表3 灌流工艺培养293细胞的密度和活率(灌流工艺3)
Figure PCTCN2021129182-appb-000003
结果表明：当细胞密度达到1×10 6个/mL或者5×10 6个/mL后开启灌流，灌流速率全程为2VVD或者4VVD，细胞的最高生长密度可达到70×10 6个/mL。
综合上述结果，采用灌流工艺1的方式可以培养得到更高的细胞密度，细胞的活率也高于工艺2和工艺3。
实施例2：灌流培养工艺和批次工艺培养的细胞密度和活率对比
试验1：采用本领域常规的批次培养工艺进行培养。
试验2：采用实施例1的灌流工艺1，即293细胞复苏后，扩增并接种到发酵罐，当细胞密度达到1×10 6个/mL后开启灌流，灌流速率为2VVD。继续培养细胞密度生长至5×10 6个/mL，灌流速率调整为3VVD。
试验1和2在培养过程中其他参数(如培养温度、pH溶氧浓度、搅拌速度等)基本相同，检测培养结束后的细胞密度和活率，结果见表4。
表4 批次工艺和灌流工艺培养293细胞的活率和密度
  批次工艺-试验1 灌流工艺-试验2
密度 3.8×10 6个/mL 13×10 7个/mL
活率 96％ 98％
结果表明，采用灌流工艺可以培养得到更高的细胞密度，细胞密度、活率高于批次工艺。
实施例3：谷氨酰胺对灌流工艺中细胞生产的影响
试验1：293细胞复苏后，扩增并接种到发酵罐，当细胞密度达到1×10 6个/mL后开启灌流，灌流速率为2VVD。继续培养细胞密度生长至5×10 6个/mL，灌流速率调整为3VVD。灌流工艺中不补加谷氨酰胺的浓度。
试验2：293细胞复苏后，扩增并接种到发酵罐，当细胞密度达到1×10 6个/mL后开启灌流，灌流速率为2VVD。继续培养细胞密度生长至5×10 6个/mL，灌流速率调整为3VVD。监控灌流工艺中谷氨酰胺浓度，补加谷氨酰胺，维持谷氨酰胺的浓度到2mM。
试验3：293细胞复苏后，扩增并接种到发酵罐，当细胞密度达到1×10 6个/mL后开启灌流，灌流速率为2VVD。继续培养细胞密度生长至5×10 6个/mL，灌流速率调整为3VVD。监控灌流工艺中谷氨酰胺浓度，补加谷氨酰胺，维持谷氨酰胺的浓度到10mM。
试验4：293细胞复苏后，扩增并接种到发酵罐，当细胞密度达到1×10 6个/mL后开启灌流，灌流速率为2VVD。继续培养细胞密度生长至5×10 6个/mL，灌流速率调整为3VVD。监控灌流工艺中谷氨酰胺浓度，补加谷氨酰胺，维持谷氨酰胺的浓度到20mM。
以上试验1-4中反应器参数为：培养温度37℃，pH为6.5-7.5可调，溶氧浓度为30-80％可调，搅拌速度30-40rpm可调。
检测培养结束后的最高细胞密度和活率，结果见表5。
表5 不同灌流工艺培养293细胞的密度和活率
  试验1 试验2 试验3 试验4
谷氨酰胺浓度 0 2mM 10mM 20mM
密度 11×10 7个/mL 29×10 7个/mL 30×10 7个/mL 30×10 7个/mL
活率 98％ 99％ 99％ 99％
结果表明，采用灌流工艺时补加谷氨酰胺的浓度可以培养得到更高的细胞密度，细胞密度、活率高于不补加谷氨酰胺工艺。
实施例4：灌流培养与批次培养293细胞最高密度对比
试验1：采用本领域常规的批次培养工艺进行培养。
试验2：采用实施例3的灌流工艺试验2的灌流培养工艺，即293细胞复苏后，扩增并接种到发酵罐，当细胞密度达到1×10 6个/mL后开启灌流，灌流速率为2VVD。继续培养细胞密度生长至5×10 6个/mL，灌流速率调整为3VVD。监控灌流工艺中谷氨酰胺浓度，补加谷氨酰胺，维持谷氨酰胺的浓度到2mM。
检测培养结束后的最高细胞密度和活率，结果见图1。
图1结果表明：批次培养最高培养密度至3.8×10 6个/mL。而灌流培养可通过连续灌流装置，持续补充新鲜培养基至反应器置换反应器内细胞代谢废物，维持细胞充足营养，灌流工艺培养的细胞密度最高达33×10 7个/mL，约是批次培养的100倍，具有明显优势。
实施例5：灌流培养工艺病毒产量
试验1：灌流工艺：灌流培养至细胞密度达10×10 6个/mL接种重组新型冠状病毒(腺病毒载体)毒种(根据已知现有技术制备)，监控培养基中谷氨酰胺浓度，补加谷氨酰胺的浓度到3mM以上，灌流速率选择1VVD或3VVD两种。培养结束后，取病毒收获液采用酶联免疫法测定滴度。
试验2：批次工艺：批次培养接毒，不补加谷氨酰胺，接毒后不进行灌流。
试验1-2在培养过程中其他参数基本相同。结果见表4。
表6 灌流与批次培养293细胞产毒量比较
Figure PCTCN2021129182-appb-000004
Figure PCTCN2021129182-appb-000005
对比灌流培养与批次培养单细胞产毒量，采用灌流培养方式可以得到更高单细胞产量增加约10倍，单罐产能扩大近100倍。
实施例6：灌流培养工艺病毒比活更高的研究
分别对灌流培养及批次培养腺病毒收获液进行纯化，测定滴度和病毒颗粒数，计算比活。结果如图2所示。
结果显示：批次培养得到腺病毒原液比活为3.8％，灌流培养得到腺病毒原液比活为7.1％，灌流培养腺病毒可以得到更多活病毒颗粒，优于批次培养。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换等，均应包含在本发明的保护范围之内。
本发明中描述的前述实施例和方法可以基于本领域技术人员的能力、经验和偏好而有所不同。
本发明中仅按一定顺序列出方法的步骤并不构成对方法步骤顺序的任何限制。

Claims (16)

  1. 一种腺病毒宿主细胞的培养方法，其包括如下步骤：
    (1)接种宿主细胞，进行细胞培养；
    (2)细胞密度达到1×10 6～5×10 6个/mL后开启灌流，灌流速率为1-3VVD；
    (3)细胞密度生长至5×10 6～10×10 6个/mL后，调节灌流速率至2-4VVD。
  2. 如权利要求1所述的方法，其特征在于，所述灌流通过利用连续灌流装置进行；所述连续灌流装置采用交替切向流细胞截留系统，其中，中空纤维柱截留孔径为0.1～0.8μm。
  3. 如权利要求1所述的方法，其特征在于，所述细胞培养中，谷氨酰胺的浓度维持在2mM以上。
  4. 如权利要求1-3任一项所述的方法，其特征在于，所述宿主细胞为293细胞。
  5. 如权利要求4所述的方法，其特征在于，所述宿主细胞为HEK293细胞或HEK293.CS细胞。
  6. 一种腺病毒的生产方法，其包括如下步骤：
    (1)接种宿主细胞，进行细胞培养；
    (2)细胞密度达到1×10 6～5×10 6个/mL后开启灌流，灌流速率为1-3VVD；
    (3)细胞密度生长至5×10 6～10×10 6个/mL后，调节灌流速率至2-4VVD；
    (4)接种病毒，培养。
  7. 如权利要求6所述的方法，其特征在于，步骤(4)中，所述培养为灌流培养，灌流速率为1-3VVD。
  8. 如权利要求6或7所述的方法，其特征在于，所述灌流通过利用连续灌流装置进行；所述连续灌流装置采用交替切向流细胞截留系统，其中，中空纤维柱截留孔径为0.1～0.8μm。
  9. 如权利要求6所述的方法，其特征在于，所述培养中，谷氨酰胺的浓度维持在2mM以上。
  10. 如权利要求6所述的方法，其特征在于，所述宿主细胞为293细胞。
  11. 如权利要求10所述的方法，其特征在于，所述宿主细胞为HEK293细胞、HEK293.CS细胞。
  12. 如权利要求6所述的方法，其特征在于，所述腺病毒为人腺病毒或黑猩猩腺病毒。
  13. 如权利要求6所述的方法，其特征在于，所述腺病毒选自：AdHu5、AdHu4、AdHu7、AdHu11、AdHu26、AdHu55、AdC68、AdC3。
  14. 如权利要求6所述的方法，其特征在于，所述腺病毒为包含编码外源基因的重组腺病毒。
  15. 如权利要求14所述的方法，其特征在于，所述腺病毒包含SARS-CoV-2的结构蛋白基因，所述结构蛋白选自：S蛋白、M蛋白、E蛋白、N蛋白中的一种或多种。
  16. 一种腺病毒载体疫苗的制备方法，其包括权利要求1-15任一项所述的方法的步骤。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102391995A (zh) * 2011-11-24 2012-03-28 陈志南 一种杂交瘤细胞无血清高密度悬浮灌流培养工艺
WO2013012770A2 (en) * 2011-07-19 2013-01-24 Wenlin Huang Process for producing recombinant human endostatin adenovirus
WO2019055796A1 (en) * 2017-09-15 2019-03-21 Bristol-Myers Squibb Company ONLINE BIOMASS CAPACITY MONITORING DURING LARGE SCALE PRODUCTION OF POLYPEPTIDES OF INTEREST
CN110964688A (zh) * 2019-12-18 2020-04-07 杭州奕安济世生物药业有限公司 动物细胞灌流培养的方法
CN112094814A (zh) * 2020-11-09 2020-12-18 康希诺生物股份公司 一种通过灌流培养工艺制备腺病毒载体疫苗的方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101028514A (zh) * 2006-06-30 2007-09-05 辽宁成大生物股份有限公司 一种应用生物反应器生产人用疫苗的方法
CN101423820B (zh) * 2008-11-28 2012-02-01 浙江大学 基于骨髓间充质干细胞的骨组织分阶段灌流培养方法
BR102016004653B1 (pt) * 2016-03-02 2018-09-25 Inst De Tecnologia Do Parana processo compacto de produção de vacina antirrábica veterinária utilizando células bhk-21, vírus pv e método de perfusão
CN110894494B (zh) * 2019-11-22 2022-09-27 广西梧州制药（集团）股份有限公司 一种大规模高密度悬浮培养293细胞高产腺病毒的方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013012770A2 (en) * 2011-07-19 2013-01-24 Wenlin Huang Process for producing recombinant human endostatin adenovirus
CN102391995A (zh) * 2011-11-24 2012-03-28 陈志南 一种杂交瘤细胞无血清高密度悬浮灌流培养工艺
WO2019055796A1 (en) * 2017-09-15 2019-03-21 Bristol-Myers Squibb Company ONLINE BIOMASS CAPACITY MONITORING DURING LARGE SCALE PRODUCTION OF POLYPEPTIDES OF INTEREST
CN110964688A (zh) * 2019-12-18 2020-04-07 杭州奕安济世生物药业有限公司 动物细胞灌流培养的方法
CN112094814A (zh) * 2020-11-09 2020-12-18 康希诺生物股份公司 一种通过灌流培养工艺制备腺病毒载体疫苗的方法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. MT419849.1
LI YOU, ZHOU HANG, LI JINCAI, ZHANG YUBIN: "Development and Applications of Mammalian Cell Perfusion Culture Technologies", CHINESE MEDICINAL BIOTECHNOLOGY, ZHONGGUO YIYAO SHENGWU JISHU XIEHUI, CN, vol. 10, no. 3, 10 June 2015 (2015-06-10), CN , pages 267 - 270, XP055928860, ISSN: 1673-713X, DOI: 10.3969/cmba.j.issn.1673-713X.2015.03.014 *
See also references of EP4151725A4
VALÉRIE CORTIN ET AL.: "High-titer adenovirus vector production in 293S cell perfusion culture", BIOTECHNOL PROG, vol. 20, no. 3, 2 October 2004 (2004-10-02), XP002317549, ISSN: 8756-7938, DOI: 10.1021/bp034237l *
ZHANG QIONGQIONG, FANG MINGYUE, LI JUNJIE, CAO RONGYUE: "Development and optimization of perfusion process for mammalian cell culture", CHINESE JOURNAL OF BIOTECHNOLOGY, vol. 36, no. 6, 25 June 2020 (2020-06-25), pages 1041 - 1050, XP055928854, DOI: 10.13345/j.cjb.190431 *

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US20230313225A1 (en) 2023-10-05
EP4151725A4 (en) 2024-03-13
CA3182438A1 (en) 2022-05-12
CN112094814B (zh) 2021-08-24
KR20230011352A (ko) 2023-01-20
CN112094814A (zh) 2020-12-18

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