WO2023045140A1 - 一种提高宿主细胞中抗体表达量的方法 - Google Patents

一种提高宿主细胞中抗体表达量的方法 Download PDF

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WO2023045140A1
WO2023045140A1 PCT/CN2021/139489 CN2021139489W WO2023045140A1 WO 2023045140 A1 WO2023045140 A1 WO 2023045140A1 CN 2021139489 W CN2021139489 W CN 2021139489W WO 2023045140 A1 WO2023045140 A1 WO 2023045140A1
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antibody
uridine
expression
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钱卫珠
戴建新
寇庚
黄卫红
侯盛
郭怀祖
徐进
陈微
耿网锁
朱亮
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上海迈泰君奥生物技术有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the invention belongs to the field of biotechnology, specifically, the invention provides a method for increasing the expression level of an antibody in a host cell, and belongs to the technical field of antibody expression.
  • Nuclear factor kappa-B ligand receptor activator (Receptor Activator of nuclear factor kappa-B ligand, RANKL) is a member of the tumor necrosis factor superfamily and is a key factor for the activation of osteoclast differentiation. It can induce the activation of osteoclast production and inhibit the apoptosis of osteoclasts. It can be used for Osteoporosis treatment.
  • the RANKL/RANK/OGP system is a typical pathway for RANKL to participate in bone remodeling.
  • RANKL expressed by osteoblasts combines with RANK of osteoclast precursor cells, and RANK binds with downstream tumor necrosis factor-related receptor 6 (TRAF6) to initiate bone destruction.
  • TNF6 tumor necrosis factor-related receptor 6
  • Osteoprotegerin was discovered by Amgen scientists in the 1990s. It is a soluble form of RANK and an inducible receptor for RANKL. It can bind to RANKL on osteoblasts, thereby blocking the interaction of RANKL/RANK. Combining effects that weaken the entire system.
  • RANKL/RANK pathway can promote bone metastasis by activating osteoclasts, and inhibiting RANKL can prevent osteolysis and tumor growth caused by tumor metastasis.
  • RANKL antibodies also have application value in tumor treatment.
  • Denosumab (commercial name Prolia) was approved by the FDA for the treatment of osteoporosis in women after menopause. Loose monoclonal antibody.
  • Denosumab gained another indication for the prevention of bone metastasis of solid tumors, with a trade name of Xgeva.
  • Denosumab received indications such as giant cell tumor of bone, malignant hypercalcemia, and multiple myeloma bone-related events.
  • Prolia and Xgeva contributed $1.968 billion and $1.575 billion to Amgen, respectively.
  • Osteoporosis is a metabolic disease caused by a combination of endocrine, nutritional, immune, and genetic factors, and its incidence is high in the elderly, postmenopausal women, and patients with malignant tumors.
  • National Osteoporosis Foundation approximately 54 million American adults are currently at risk for osteoporosis.
  • my country's large-scale epidemiological survey shows that the total prevalence of osteoporosis in China is 12.4%, and the total number has exceeded 160 million.
  • the global osteoporosis market is about 10 billion. Although it is much lower than the tumor market, the competition in this market is not as fierce as the tumor market.
  • Several blockbuster drugs have been born.
  • the global treatment of osteoporosis mainly includes inhibiting bone resorption (bisphosphonates, selective estrogen receptor modulators, calcitonin, RANKL inhibitor monoclonal antibody) and increasing bone formation (parathyroid hormone), etc. .
  • Denosumab is the first monoclonal antibody drug for osteoporosis, which brings new blood to the osteoporosis market and also challenges traditional small molecule drugs.
  • the other two monoclonal antibodies for osteoporosis Romosozumab and Blosozumab, are in the late stage of clinical trials. Both drugs target sclerostin.
  • the invention provides a method for increasing the expression level of an antibody in a host cell, by adding one or more trace elements to a serum-free medium to significantly increase the expression level of the antibody.
  • the invention provides a method for increasing the expression of the antibody in the host cell, which is characterized in that one or more trace elements are added to the serum-free medium.
  • the host cell is a CHO cell.
  • the trace elements include metal elements.
  • the concentration of the manganese ions is 2.5-100 ⁇ M
  • the concentration of the copper ions is 20-320 ⁇ M
  • the concentration of the ferric ions is 20-160 ⁇ M.
  • the trace elements also include uridine.
  • the concentration of uridine is 1-4mM.
  • the antibody is a fully human antibody targeting RANKL.
  • the trace elements added to the medium are 2.5 ⁇ M manganese ions, 20 ⁇ M copper ions, 20 ⁇ M ferric ions and 1 mM uridine.
  • the trace elements added to the medium are 100 ⁇ M manganese ions, 320 ⁇ M copper ions, 160 ⁇ M ferric ions and 4 mM uridine.
  • the host cells are preferably eukaryotic cells, such as but not limited to CHO, COS, RSF, SP2/0, NSO, HEK-293, PER-C6 cells and the like.
  • the host cells are CHO cells, including CHO-K1, CHO-DXB11 and CHO-DG44.
  • CHO-K1 is the closest to wild-type CHO, while DXB11 and DG44 have eliminated the endogenous dihydrofolate reductase (dihydrofolate reductase, DHFR) gene through random mutation to establish a gene amplification screening system for DHFR metabolic defects.
  • DHFR dihydrofolate reductase
  • CHOK1SV that has been adapted to serum-free suspension culture (Porter AJ, Dickson AJ, Racher AJ. Biotechnology progress, 2010, 26(5): 1446-1454.) and CHO-S cells, etc.
  • the host cells are CHO-K1 cells. In some more preferred embodiments, the host cell is a CHO-K1 cell in which the GS gene has been knocked out. It can preferably express the fully human antibody targeting RANKL in some embodiments of the present invention, and can obtain a product with good activity and good stability.
  • the present invention significantly increases the expression of antibodies by adding one or more trace elements, especially the metal elements manganese ion, copper ion, ferric ion and uridine, to the serum-free medium, and the dosage and matching of various trace elements It has been optimized to the best for the fully human antibody targeting RANKL of the present invention. Compared with the commercially available serum-free medium of the prior art, the cost increase is negligible, but the expression level of the antibody is greatly increased, and has significant market promotion value.
  • trace elements especially the metal elements manganese ion, copper ion, ferric ion and uridine
  • Figure 1 is a schematic diagram of the effect of different concentrations of uridine on cell density and antibody expression.
  • Figure 2 is a schematic diagram of the effect of different concentrations of manganese ions on cell density and antibody expression.
  • Figure 3 is a schematic diagram of the effect of different concentrations of copper ions on cell density and antibody expression.
  • Figure 4 is a schematic diagram of the effect of different concentrations of ferric ions on cell density and antibody expression.
  • Fig. 5 is a schematic diagram of the effect of different concentrations of complex metal ions on cell density.
  • Figure 6 is a schematic diagram of the effect of different concentrations of complex metal ions on the expression of antibodies.
  • Figure 7 is a schematic diagram of the effect of combined use of uridine and a single metal ion on cell density and antibody expression.
  • Figure 8 is a schematic diagram of the effect of combined use of compound trace elements on cell density.
  • Figure 9 is a schematic diagram of the influence of combined use of compound trace elements on the expression of antibodies.
  • the fusion proteins of the present invention all use CHO-K1 as the host cell, and the fed-batch culture is the large-scale culture method.
  • the basal medium CHOM-B03 is purchased from Shanghai Maitai Junao Biotechnology Co., Ltd. .
  • the culture equipment is a 50L bioreactor, suspension culture, the initial temperature is 37°C, the temperature is lowered to 34°C until the fourth day, the stirring speed is 100 ⁇ 150rpm, the microbubbles are kept deep aeration, the pH is controlled at 6.8 ⁇ 7.2, and the DO is controlled at About 40%.
  • Example 1 The effect of adding different concentrations of uridine in the medium on the expression of antibodies
  • the purpose of this example is to test the effect of adding different concentrations of uridine to the basal medium CHOM-B03 on the cell density and antibody expression.
  • the dosage of uridine is shown in Table 1.
  • Example 2 The effect of adding different concentrations of manganese ions in the medium on the expression of antibodies
  • the purpose of this example is to test the effect of adding different concentrations of manganese ions in the basal medium CHOM-B03 on cell density and antibody expression.
  • the amount of manganese ion is shown in Table 2.
  • Example 3 The effect of adding different concentrations of copper ions in the medium on the expression of antibodies
  • the purpose of this example is to test the effect of adding different concentrations of copper ions in the basal medium CHOM-B03 on cell density and antibody expression.
  • the dosage of copper ions is shown in Table 3.
  • Example 4 The effect of adding different concentrations of ferric ions in the medium on the expression of antibodies
  • the purpose of this example is to test the effect of adding different concentrations of ferric ions to the basal medium CHOM-B03 on cell density and antibody expression.
  • the dosage of ferric ion is shown in Table 4.
  • the purpose of this example is to test the effect of adding pairwise metal elements in the basal medium CHOM-B03 on cell density and antibody expression.
  • the matching and dosage of manganese ions, copper ions and ferric ions are shown in Table 5.
  • Example 6 The effect of adding complex trace elements in the medium on the expression of antibodies
  • the purpose of this example is to test the effect of adding different concentrations of urea and metal elements in the basal medium CHOM-B03 to form a complex trace element, on cell density and antibody expression.
  • the collocation and dosage of urea, manganese ions, copper ions and ferric ions are shown in Table 6.
  • the antibody expression level reached 4.612 g/L, the highest in all examples of the present invention, an increase of more than 150%, and the effect of increasing the antibody expression level was very good obvious.
  • the expression level of the antibody in the medium supplemented with 2.5 ⁇ M manganese ions, 20 ⁇ M copper ions, 20 ⁇ M ferric ions, and 1 mM uridine reached 4.598 g/L, an increase of over 150%, and the expression level was also very good.
  • the most suitable combination of adding trace elements is 100 ⁇ M manganese ions, 320 ⁇ M copper ions, 160 ⁇ M ferric ions and 4 mM uridine, or 2.5 ⁇ M ⁇ M manganese ion, 20 ⁇ M copper ion, 20 ⁇ M ferric ion and 1 mM uridine. Under these two combined conditions, the antibody expression was more than 4g/L, and the degree of improvement was significant.

Abstract

提供了一种高效表达融合蛋白的方法，通过针对特定融合蛋白优化培养基用量、pH、渗透压等工艺条件，并将其搭配组合优化至最优，显著提高了抗体的表达量，具有显著的经济意义和广泛的推广价值。

Description

一种提高宿主细胞中抗体表达量的方法 技术领域
本发明属于生物技术领域，具体地，本发明提供了一种提高宿主细胞中抗体表达量的方法，属于抗体表达技术领域。
 背景技术
核因子κ-B配体受体致活剂（Receptor activator of nuclear factor kappa-B ligand，RANKL）是肿瘤坏死因子超家族的一员，为破骨细胞分化激活的关键因子，具有诱导破骨细胞生产活化，抑制破骨细胞凋亡的作用，可用于骨质疏松的治疗。
RANKL/RANK/OGP系统是RANKL参与骨重建的典型途径，成骨细胞表达的RANKL与破骨前体细胞的RANK相结合，RANK与下游肿瘤坏死因子相关受体6（TRAF6）相结合可启动破骨细胞生产基因的转录，将破骨前体细胞诱导为成熟的破骨细胞。
骨保护素(OPG)在上世纪90年代由Amgen科学家发现，其是ＲANK的一种可溶形式，是ＲANKL的诱导受体，可与成骨细胞上的RANKL结合，从而阻断RANKL/RANK的结合削弱整个系统的效应。
在基于该通路的药物开发中，考虑到全长的OPG在体内容易与肝素相结合表现出较差的药代动力学特征，科学家们尝试将OPG与lgG1抗体的Fc区融合改善其药代动力学活性，但融合蛋白在临床中存在安全性问题，因此科学家最终靶向RANKL抗体的开发。
另外，研究显示RANKL/RANK通路可通过活化破骨细胞促进骨转移,通过抑制RANKL可阻止肿瘤转移引起的骨质溶解和肿瘤生长，RANKL抗体在肿瘤治疗中也有应用价值。
2010年6月，Amgen靶向RANKL的单克隆抗体Denosumab（商用名Prolia）获得FDA批准上市用于治疗女性更年期后的骨质疏松症，这是世界首款也是迄今唯一一款用于骨质疏松的单克隆抗体。在同年11月，Denosumab又收获预防实体瘤骨转移适应症，商品名Xgeva。之后，Denosumab又收获骨巨细胞瘤、恶性高钙血症、多发性骨髓瘤骨骼相关事件等适应症。2017年，Prolia和Xgeva分别为Amgen贡献了19.68亿美元和15.75亿美元。
骨质疏松症是一种内分泌、营养、免疫、遗传多种复合因素引起的代谢性疾病，在老年人、绝经后妇女和恶性肿瘤患者中发生率较高。根据美国国家骨质疏松基金2015年度报告，大约有5400万的美国成年人目前有患骨质疏松的风险。我国大规模流行病学调查研究显示，国内骨质疏松症总患病率为12.4%，总人数已超过1.6亿。全球骨质疏松市场容量在100亿左右，虽然远低于肿瘤市场规模，但该市场竞争程度也不像肿瘤市场那样白热化，曾诞生过数个重磅炸弹药物。目前全球治疗骨质疏松治疗药物主要有抑制骨吸收（双磷酸盐、选择性雌激素受体调节剂、降钙素、RANKL抑制剂单抗）和增加骨形成两种（甲状旁腺素）等。
Denosumab是首款用于骨质疏松的单抗药物，为骨质疏松市场带来了新的血液，也给传统的小分子药物带来了挑战。Denosumab之后，另外两款用于骨质疏松的单克隆抗体Romosozumab、Blosozumab正处于临床后期，这两款药物均是针对骨硬化蛋白 (sclerostin)。不过在2017年7月，FDA拒绝而Romosozumab的上市申请，骨硬化蛋白类抗体药物能否延续Denosumab的辉煌还未可知。
 技术问题
现有的靶向RANKL的抗体，虽然种类繁多，竞争激烈，但在大规模工业化生产过程中，仍然存在性状不稳定、或表达量不高的问题。
 技术解决方案
本发明提供一种提高宿主细胞中抗体表达量的方法，通过在无血清培养基中添加一种或多种微量元素显著提高抗体的表达量。
本发明提供了一种提高宿主细胞中抗体表达量的方法，其特征在于，在无血清培养基中添加一种或多种微量元素。
在本发明的一些实施例中，所述宿主细胞为CHO细胞。
在本发明的一些实施例中，所述微量元素包括金属元素。
在一些优选的实施例中，所述锰离子的浓度为2.5~100μM，所述铜离子的浓度为20~320μM，所述三价铁离子的浓度为20~160μM。
在本发明的一些实施例中，所述微量元素还包括尿苷。
在一些优选的实施例中，所述尿苷的浓度为1-4mM。
在本发明的一些实施例中，所述抗体为靶向RANKL的全人源抗体。
在一些优选的实施例中，培养基中添加的微量元素为2.5μM锰离子、20μM铜离子、20μM三价铁离子和1mM尿苷。
在一些优选的实施例中，培养基中添加的微量元素为100μM锰离子、320μM铜离子、160μM三价铁离子和4mM尿苷。
在本发明的一些实施例中，所述的宿主细胞优选的是真核细胞，例如但不限于CHO、COS、RSF、SP2/0、NSO、HEK-293、PER-C6细胞等。
在一些优选的实施例中，所述宿主细胞为CHO细胞，包括CHO-K1，CHO-DXB11和CHO-DG44。其中，CHO-K1最接近野生型CHO，而DXB11和DG44均经过随机突变消除了内源性二氢叶酸还原酶（dihydrofolate reductase，DHFR）基因而建立DHFR代谢缺陷的基因扩增筛选系统。还有已经适应无血清悬浮培养的CHOK1SV（Porter AJ, Dickson AJ, Racher AJ. Biotechnology progress, 2010, 26(5): 1446-1454.）和CHO-S细胞等。
在一些优选的实施例中，所述宿主细胞为CHO-K1细胞。在一些更优选的实施例中，所述宿主细胞为敲除了GS基因的CHO-K1细胞。其可较佳地表达本发明一些实施例中靶向RANKL的全人源抗体，可获得活性良好，稳定性良好的产物。
 有益效果
本发明通过在无血清培养基中添加一种或多种微量元素，尤其是金属元素锰离子、铜离子、三价铁离子以及尿苷，显著提高抗体的表达量，各种微量元素用量及搭配已针对本发明靶向RANKL的全人源抗体优化至最优。与现有技术的市售无血清培养基相比，成本增加可忽略不计，但大幅提高了抗体的表达量，具有显著的市场推广价值。
 附图说明
图1是不同浓度的尿苷对细胞密度和抗体表达量的影响示意图。
图2 是不同浓度的锰离子对细胞密度和抗体表达量的影响示意图。
图3是不同浓度的铜离子对细胞密度和抗体表达量的影响示意图。
图4是不同浓度的三价铁离子对细胞密度和抗体表达量的影响示意图。
图5是不同浓度的复合金属离子对细胞密度的影响示意图。
图6不同浓度的复合金属离子对抗体表达量的影响示意图。
图7尿苷和单一金属离子联合使用对细胞密度和抗体表达量的影响示意图。
图8复合微量元素联合使用对细胞密度影响示意图。
图9复合微量元素联合使用对抗体表达量的影响示意图。
 本发明的最佳实施方式
下面结合附图进一步阐述本发明提高宿主细胞中抗体表达量的方法的技术方案。
在下述实施例中，本发明所述融合蛋白皆以CHO-K1作为宿主细胞，以分批补料培养为大规模培养方式，基础培养基CHOM-B03购自上海迈泰君奥生物技术有限公司。培养设备为50L生物反应器，悬浮培养，初始温度为37℃，培养至第4天降温至34℃，搅拌转速为100~150rpm，保持微泡深层通气，pH控制在6.8~7.2，DO控制在40%左右。
实施例 1 、培养基中添加不同浓度的尿苷对抗体表达量的影响
本实施例的目的在于试验在基础培养基CHOM-B03中添加不同浓度的尿苷，对细胞密度和抗体表达量的影响。尿苷的用量如表1所示。
表1.
Figure 310070dest_path_image001
基础培养基中添加不同浓度的尿苷对细胞密度和抗体表达量的影响如图1所示。
由图1可知，与不添加尿苷的基础培养基相比，添加了不同浓度尿苷的培养基中，宿主细胞密度和抗体表达量都有不同程度的提高，抗体表达量由不添加尿苷的1.788g/L提高到超过3.192 g/L，增幅约80%。
实施例 2 、培养基中添加不同浓度的锰离子对抗体表达量的影响
本实施例的目的在于试验在基础培养基CHOM-B03中添加不同浓度的锰离子，对细胞密度和抗体表达量的影响。锰离子的用量如表2所示。
表2.
Figure 7637dest_path_image002
基础培养基中添加不同浓度的锰离子对细胞密度和抗体表达量的影响如图2所示。
由图2可知，与不添加锰离子的基础培养基相比，添加了不同浓度锰离子的培养基中，宿主细胞密度和抗体表达量都有不同程度的提高，抗体表达量由不添加锰离子的1.788g/L提高到最高3.375 g/L，增幅约90 %。
实施例 3 、培养基中添加不同浓度的铜离子对抗体表达量的影响
本实施例的目的在于试验在基础培养基CHOM-B03中添加不同浓度的铜离子，对细胞密度和抗体表达量的影响。铜离子的用量如表3所示。
表3.
Figure 508413dest_path_image003
基础培养基中添加不同浓度的铜离子对细胞密度和抗体表达量的影响如图3所示。
由图3可知，与不添加铜离子的基础培养基相比，添加了不同浓度铜离子的培养基中，宿主细胞密度和抗体表达量都有不同程度的提高，抗体表达量由不添加铜离子的1.788g/L提高到最高3.346g/L，增幅约90%。
实施例 4 、培养基中添加不同浓度的三价铁离子对抗体表达量的影响
本实施例的目的在于试验在基础培养基CHOM-B03中添加不同浓度的三价铁离子，对细胞密度和抗体表达量的影响。三价铁离子的用量如表4所示。
表4.
Figure 418469dest_path_image004
基础培养基中添加不同浓度的三价铁离子对细胞密度和抗体表达量的影响如图4所示。
由图4可知，与不添加三价铁离子的基础培养基相比，添加了不同浓度三价铁离子的培养基中，宿主细胞密度和抗体表达量都有不同程度的提高，抗体表达量由不添加三价铁离子的1.788g/L提高到最高3.412g/L，增幅超过90%。
实施例 5 、培养基中添加复合金属元素对抗体表达量的影响
本实施例的目的在于试验在基础培养基CHOM-B03中添加两两搭配的金属元素，对细胞密度和抗体表达量的影响。锰离子、铜离子和三价铁离子的搭配和用量如表5所示。
表5.
Figure 959739dest_path_image005
基础培养基中添加复合金属元素的搭配组合对细胞密度和抗体表达量的影响如图5和图6所示。
由图5、图6可知，与不添加任何微量元素的基础培养基相比，添加了不同浓度两两搭配的复合金属元素的培养基中，宿主细胞密度和抗体表达量都有不同程度的提高，抗体表达量由不添加复合金属元素的1.788g/L提高到最高3.855g/L，增幅超过110%。
实施例 6 、培养基中添加复合微量元素对抗体表达量的影响
本实施例的目的在于试验在基础培养基CHOM-B03中添加不同浓度的尿素和金属元素的搭配形成的复合微量元素，对细胞密度和抗体表达量的影响。尿素、锰离子、铜离子和三价铁离子的搭配和用量如表6所示。
Figure 719885dest_path_image006
基础培养基中添加尿苷和单一金属离子的搭配组合对细胞密度和抗体表达量的影响如图7所示。
基础培养基中添加尿苷和复合金属离子，或三种复合金属离子的搭配组合对细胞密度和抗体表达量的影响如图8和图9所示。
由图7-9可知，与不添加任何微量元素的基础培养基相比，添加了不同浓度的尿素和复合金属元素，或多种金属元素搭配形成的复合微量元素的培养基中，宿主细胞密度和抗体表达量都有显著的提高，抗体表达量由不添加复合金属元素的1.788g/L提高到最高超过4g/L。其中添加100μM锰离子、320μM铜离子、160μM三价铁离子和4mM尿苷培养基中的抗体表达量达到本发明所有实施例最高的4.612 g/L，增幅超过150%，提升抗体表达量效果非常明显。综合成本因素来看，添加2.5μM锰离子、20μM铜离子、20μM三价铁离子、1mM尿苷培养基中的抗体表达量达到4.598 g/L，增幅也超150%，表达量也非常优秀。
总结：经过反复摸索与优化，最终确定本发明所述融合蛋白的基础培养基中，添加微量元素最合适的组合为100μM锰离子、320μM铜离子、160μM三价铁离子和4mM尿苷，或2.5μM锰离子、20μM铜离子、20μM三价铁离子和1mM尿苷。这两种组合条件下，抗体表达量均超过4g/L，提升程度显著。
 

Claims (10)

  1. 一种提高宿主细胞中抗体表达量的方法，其特征在于，在无血清基础培养基中添加一种或多种微量元素。
  2. 如权利要求1所述的方法，其特征在于，所述宿主细胞为CHO细胞。
  3. 如权利要求1所述的方法，其特征在于，所述微量元素包括金属元素。
  4. 如权利要求3所述的方法，其特征在于，所述金属元素为锰离子、铜离子、三价铁离子中的一种或多种。
  5. 如权利要求4所述的方法，其特征在于，所述锰离子的浓度为2.5~100μM，所述铜离子的浓度为20~320μM，所述三价铁离子的浓度为20~160μM。
  6. 如权利要求3所述的方法，其特征在于，所述微量元素还包括尿苷。
  7. 如权利要求6所述的方法，其特征在于，所述尿苷的浓度为1-4mM。
  8. 如权利要求2所述的方法，其特征在于，所述抗体为靶向RANKL的全人源抗体。
  9. 如权利要求8所述的方法，其特征在于，所述微量元素为2.5μM锰离子、20μM铜离子、20μM三价铁离子和1mM尿苷。
  10. 如权利要求8所述的方法，其特征在于，所述微量元素为100μM锰离子、320μM铜离子、160μM三价铁离子和4mM尿苷。
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