摘要
人血清白蛋白是血浆中含量最大的一种蛋白,含量达到40-50g/L,对维持体内渗透压具有重要的作用,此外它还有运输脂类、激素、药物等重要功能。白蛋白已被广泛应用于烧伤、肝腹水和低白蛋白症等的临床治疗,另外白蛋白还被用于药物的赋形剂,稳定剂,以及细胞培养的添加剂量。由于极其广泛的应用,人源血清白蛋白已不能满足现在的市场需求。实验室利用水稻种子这一新型的表达体系表达重组人血清白蛋白,并实现经济有效的大规模生产。临床应用中,通常人血清白蛋白的使用剂量为每人每次10-20g,因此,在利用水稻种子生产重组人血清白蛋白时,急需对其宿主杂质的安全性进行相关评估,并对其进行有效的质量控制。宿主杂质中,宿主蛋白和宿主DNA是最主要的两个宿主杂质,也是重组蛋白药质量控制中最为关注的要素。欧洲药品审评署(EMEA)明确指出,所有生物制品都需要对这两种宿主杂质进行详细的说明以确保最终产品的临床应用安全性。本论文的主要结果如下：
1.残留宿主DNA定量方法的建立：试验通过定量PCR的方法,以水稻5S核糖体基因为内参扩增序列,建立水稻宿主残留DNA定量方法。实验比较了两个常用的定量PCR体系,SYBRGreen Ⅰ荧光染料和Taqman探针法,在没有明显差异的情况下,基于成本考虑,选择SYBRGreen Ⅰ荧光染料为最终的定量体系。通过引物的选择,引物浓度的优化,最终建立的定量方法具有高度的线性相关性,相关系数R2达到0.9991,扩增效率接近100%,检测区间为2×104pg到0.2pg每个反应体系,最低定量限度为0.2pg每个反应体系。qPCR具有很好的重复性与特异性,回收实验的回收率在80%到122.2%之间,且标准样品的选择不会对结果有明显的影响。对一个重组人血清白蛋白典型样品的定量分析,最终样品的宿主残留DNA为每剂量(10g)含3.5ng,少于法规每剂量10ng的要求。
2.残留宿主蛋白定量方法的建立：利用水稻种子抗原制备抗体建立夹心ELISA定量检测方法,制备的抗体具有很好的覆盖性且不对目的蛋白白蛋白有交叉反应,实验比较了分别以HRP标记抗体为检测抗体和Biotin标记抗体为检测抗体的灵敏度,最后选择灵敏度较高的Biotin标记抗体为检测抗体的反应体系。实验主要通过包被抗体和检测抗体的浓度优化,建立的ELISA定量方法具有良好的非线性相关性,相关系数R2达到0.99,定量区间从25ng/ml到1.25ng/ml之间,最低定量限为1.25ng/ml。ELISA具有很好的重复性及精确度,定量结果的cv值小于11.5%,添加回收试验中宿主蛋白的回收率在86%到110.6%之间,样品的最终定量纯度达到99.9998%。
3.残留宿主蛋白的鉴定研究：为了避免目的产物人血清白蛋白对残留宿主蛋白鉴定的干扰,实验设计抗体免疫层析的方法去除目的蛋白-重组人血清白蛋白,并达到浓缩残留宿主蛋白的效果。实验通过人源血清白蛋白免疫兔子制备抗人血清白蛋白多克隆抗体,用制备的人血清白蛋白抗体与CNBr-activatived SepharoseTM4Fast Flow进行偶联,获得能够吸附约100mg人血清白蛋白的抗体免疫层析柱。利用该层析柱分离目的蛋白(重组人血清白蛋白)和残留宿主蛋白。实验建立的方法对99.8%样品的宿主蛋白回收率高于90%。收集的残留宿主蛋白经过label free NanoLC-ESI-MS/MS鉴定分析,结果显示,几乎所有的残留宿主蛋白都能被有效鉴定。
4.残留宿主蛋白反复给药的毒性研究：实验收集99.9%纯度产品中的白蛋白宿主蛋白,以1倍(0.2mg/kg)、5倍(1mg/kg)、25倍(5mg/kg)的剂量进行28天的反复静脉注射大鼠的毒性试验。整个试验过程中,大鼠没有出现样品相关的致命性毒性引起的死亡现象,主要的毒性表现为贫血症状以及脾功能亢进,初步推测与低渗性溶血或免疫性溶血有关。
Human serum albumin (HSA) is the most abundant protein in plasma, the level of which reaches40-50g/L. It plays an important role in maintaining osmotic pressure in the body, as well as transporting lipids, hormones and medicine molecules. In clinic, this protein has been widely used to treat burns, liver ascites, low albumin disease etc. Besides, HSA can also be applied as pharmaceutical excipient, stabilizer and additive in cell culture. Due to the extensive application, currently, HSA has been not able to meet the demand of market. Our lab developed a new rice-seed expression system to produce recombinant HSA (rHSA), and large-scale production of rice-derived rHSA in a cost-effective way has been realized. Generally, HSA is used clinically at a high dosage of10-20g per person, hence, it is urgent to assess the safety of the impurities from host cells when rice-derived rHSA was produced, and the effective quality control is necessary. Host cell proteins (HCPs) and host cell DNA is the two main impurities from host cells and considered the most important factors in recombinant protein drug quality control. EMEA make it clear that these two kinds of impurities should be characterized distinctly to ensure the safety of the products in clinical use. The main results of this dissertation are as follows:
1. The establishment of residual host cell DNA quantative method:,A quantitative real-time polymerase chain reaction (qPCR) assay, based on the5S ribosomal RNA (rRNA) genes, was used to develop a quantitative method of rice residual DNAs. The two common qPCR assays, S YBR Greean I and TaqMan, were compared. In the case of no significant differences existing between the two reaction systems, SYBR Green I was chosen as the residual DNA quantitative method because of its cost-effectivity. Through the choice of appropriate primers and the optimization of primer concentration, the established residual DNA quantitative method exhibited a high degree of linear relationship with a R2of0.9991, when the template concentration was in the range of2×104pg to0.2pg per reaction system. The amplification efficiency was close to100%and the detective limit lowed to0.2pgper reaction system. The established qPCR assay had a good repeatability and specificity, showing a high recovery of80%to122.2%in the recovery experiments, which was also not affected by the choice of standard samples. The amount of residual DNA in a typical rHSA product was quantified to be3.5ng per dosage (10g) by this method,lower than the requirements of10ng per dosage by regulations.
2. The establishment of residual HCPs quantitative method:An enzyme-linked immune sorbent (ELISA) assay was developed using antibodies prepared by rice seed antigens. The produced antibody gave high antibody coverage and did not cross react with the target protein, HSA. The sensitivity of two detecting antibodies coupled with HRP or Biotin respectively was compared, and the results demonstrated that the antibody with Biotin gave higher sensitivity, which was chosen to be the detecting antibody in the ELISA method. Following the optimization of concentrations of capture and detecting antibodies, the established ELISA assay had a good non-linear relationship with a R2of0.99. The quantitative range was from25ng/ml to1.25ng/ml, and the minimum quantitative limit was1.25ng/mL. The ELISA method also had a robust repeatability and accuracy. The coefficient of variation (CV) of quantitative results was lower than11.5%, and the recovery of spiked standard HCPs in the recovery experiments ranged from86%to110.6%. By quantifying the residual HCPs using the established method, the purity of an rHSA product could be calculated to be99.9998%.
3. The identification of residual HCPs:To avoid the interference from rHSA in HCPs identification, rHSA wasremoved and the HCPs were concentrated by an immune chromatography method. Anti-human serum albumin polyclonal antibodywas produced in rabbits through human serum albumin as antigen. The immune chromatography with a capacity of100mg HSA was achieved by coupling anti-HSA antibodies with CNBr-activatived SepharoseTM4Fast Flow, which was used to seperate rHSA from HCPs. The recovery of HCPs from rHSA with a purity of99.8%was higher than90%in this seperation technique. The collected HCPs were concentrated and subsequently characterized by label free NanoLC-ESI-MS/MS, showing that at least seven-orders of magnitude identification range was achieved. Almost all impurities were identified effectively by this method.
4. The toxicity research of residual HCPs by repeated administration:The collected HCPs from products with the purity of99.9%were intravenously injected into mice at a clinical dosage of1-fold (0.2mg),5-fold (1mg) and25-fold (5mg) for28-day toxisity test. During the whole testing, mice did not die from the deadly toxicity caused by samples. The major toxic symptoms included anemia and hypersplenism, which were preliminarily speculated to be related to low permeability hemolysis or immuno hemolytic disease..
引文
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