Characterization of a Monoclonal Antibody Cell Culture Production Process Using a Quality by Design Approach

The goal of quality by design (QbD) in cell culture manufacturing is to develop manufacturing processes which deliver products with consistent critical quality attributes (CQAs). QbD approaches can lead to better process understanding through the use of process parameter risk ranking and statistical design of experiments (DOE). The QbD process starts with an analysis of process parameter risk with respect to CQAs and key performance indicators (KPIs). Initial DOE study designs and their factor test ranges are based on the outcomes of the process parameter risk ranking exercises. Initial DOE studies screen factors for significant influences on CQAs as well as characterize responses for process KPIs. In the case study provided here, multifactor process characterization studies using a scale-down model resulted in significant variation in charge heterogeneity of a monoclonal antibody (MAb) as measured by ion-exchange chromatography (IEC). Iterative DOE studies, using both screening and response surface designs, were used to narrow the operating parameter ranges so that charge heterogeneity could be controlled to an acceptable level. The data from the DOE studies were used to predict worst-case conditions, which were then verified by testing at those conditions. Using the approach described here, multivariate process parameter ranges were identified that yield acceptable CQA levels and that still provide operational flexibility for manufacturing.     

基于DOE设计和氨基酸补加策略提高CHO细胞表达抗CD20单克隆抗体*

中国仓鼠卵巢细胞(CHO)流加培养生产单克隆抗体是目前主流培养方式,其中环境参数(pH和温度)和营养成分均影响细胞生长、碳氮源代谢和外源蛋白表达,是培养过程中关键的控制参数。采用实验设计(design of experiment,DOE)方法研究培养参数(温度、pH)对CHO细胞生长和抗CD20抗体表达的影响,建立营养限制型氨基酸流加策略,实现抗CD20抗体的高表达。结果表明,温度是影响蛋白质表达的显著因素,35℃有助于提高细胞密度和目标抗CD20抗体表达,而pH对抗CD20表达影响不显著,且温度和pH无交互作用,经DOE预测分析最佳培养条件是温度35℃和pH7.0。在该最佳培养条件下,在培养后期酪氨酸和半胱氨酸的浓度都低于0.1mmol/L。在培养的第2天通过补加1.5mmol/L酪氨酸和1mmol/L半胱氨酸避免营养限制,抗CD20抗体表达水平提高了24.1%,且对蛋白糖型无影响。     

基于DOE设计和氨基酸补加策略提高CHO细胞表达抗CD20单克隆抗体*

中国仓鼠卵巢细胞(CHO)流加培养生产单克隆抗体是目前主流培养方式,其中环境参数(pH和温度)和营养成分均影响细胞生长、碳氮源代谢和外源蛋白表达,是培养过程中关键的控制参数。采用实验设计(design of experiment,DOE)方法研究培养参数(温度、pH)对CHO细胞生长和抗CD20抗体表达的影响,建立营养限制型氨基酸流加策略,实现抗CD20抗体的高表达。结果表明,温度是影响蛋白质表达的显著因素,35℃有助于提高细胞密度和目标抗CD20抗体表达,而pH对抗CD20表达影响不显著,且温度和pH无交互作用,经DOE预测分析最佳培养条件是温度35℃和pH7.0。在该最佳培养条件下,在培养后期酪氨酸和半胱氨酸的浓度都低于0.1mmol/L。在培养的第2天通过补加1.5mmol/L酪氨酸和1mmol/L半胱氨酸避免营养限制,抗CD20抗体表达水平提高了24.1%,且对蛋白糖型无影响。     

氨基酸生产和海洋生物的氨基酸资源开发

氨基酸在医药、食品、饲料等领域有着极为重要和广泛的用途,世界上氨基酸总需求量以５～１０％递增,市场竞争十分激烈。生物资源提取、化学合成、生物合成和综合法是生产氨基酸的４种技术,目前的发展趋势为生物合成和综合法,特别是将现代生物工程技术应用于氨基酸生产。另外,氨基酸生产领域另一个新的倾向是海洋生物氨基酸资源的开发和应用,尤其是海洋生物所产生的特殊氨基酸、肽及其衍生物的开发,同时,综合利用海产品加工后的废弃物来生产氨基酸也受到重视。     

OAS1蛋白单克隆抗体的制备及其表征

目的:用OAS1蛋白免疫小鼠,获得OAS1特异性单克隆抗体,为OAS1的含量测定提供基础。方法:通过全基因合成的方法获得目的基因序列,转化大肠杆菌BL21细胞诱导His-OAS1及OAS1蛋白表达,纯化后用作抗原免疫小鼠,取脾融合,筛选稳定分泌抗体的阳性细胞株,制备并纯化单抗,通过SDS-PAGE,ELISA,Western blot等方法进行检测。结果:体外高效表达OAS1蛋白,并成功制备特异性单克隆抗体,效价在5×10^-11mol/L以上,亲和常数为3.37×10⁸ L·mol^-1。结论:获得高亲和力OAS1单克隆抗体,为其含量的检测奠定了基础。     

工程原核生物和酵母菌中生产单克隆抗体和抗体片段研究进展 *

抗体药物和抗体片段药物在药物市场占据了重要的地位,主要通过哺乳动物细胞系统进行生产,操作复杂并且成本高。为了能够克服哺乳动物细胞系统生产抗体药物的弊端,越来越多的抗体及抗体片段在原核细胞及酵母菌中生产,但是产率往往不高并且没有糖基化。从基因转录和翻译的优化、分子伴侣的共表达和抑制蛋白水解降解等方面概述了在原核生物表达系统及酵母菌中提高单克隆抗体和抗体片段产量的研究进展,为未来利用原核生物和酵母菌实现工业化生产单克隆抗体及抗体片段奠定基础。     

氨基酸分析仪测定饲料添加剂——叶酸的方法

用６ｍｏｌ／Ｌ盐酸于１１０℃条件下水解饲料添加剂——叶酸,使之游离出谷氨酸,用氢氧化钠中和调节ｐＨ到２,氨基酸分析仪测定谷氨酸含量,经与标准叶酸水解样品比较,计算出叶酸的纯度。该方法重现性好,变异系数ＣＶ＝０．０８％,平均回收率为９８．３４％,浓度与峰面积呈线性相关,相关系数ｒ＝０．９９８７,可随氨基酸分析同时进行,不需改变任何分析条件。     

补料优化降低抗体生产过程中宿主细胞蛋白残留的研究

目的:通过上游工艺中补料培养基优化以降低单克隆抗体生产中的宿主细胞蛋白(HCP)水平。方法:本文在3 L反应器的工艺开发过程中考察了不同的商品化补料培养基和细胞接种密度对HCP水平的影响,筛选出最优条件后,进行了补料工艺的优化和金属离子的添加试验,最后将优化后的工艺放大至200 L中试规模。结果:在小试阶段发现Cellvento 4Feed可以显著降低HCP,同时CuSO4可以进一步促进HCP降低的水平,最终将工艺放大至200 L中试进行生产并取得了相似的结果,验证了工艺的稳定性和可放大性,中试规模的HCP水平相比最初的工艺降低了65%左右。结论:补料培养基优化可以有效降低细胞对HCP的比生产速率,使收获液中整体的HCP水平显著下降。     

单克隆抗体制备的细胞工程学研究进展

单克隆抗体是近年来发展最快、最成功的大分子药物之一,哺乳动物细胞作为单抗大规模制备最适宜的宿主,在工业生产中仍然存在成本高、产率低等缺点。近年来,抗细胞凋亡、控制细胞周期、优化代谢过程等细胞工程学方面的研究极大地推动了抗体表达及翻译后修饰技术的发展。以下对近年来单克隆抗体制备在细胞工程学方面取得的进展作一综述,并探讨该领域未来可能的研究方向。     

Therapeutic Targeting of Lewisy and Lewisb with a Novel Monoclonal Antibody 692/29

Background

Several monoclonal antibodies (mAbs) recognising Lewis^y, such as BR96, have reached the clinic but have failed to show good anti-tumour responses with an acceptable level of toxicity. No Lewis^b mAbs have been trialled in patients. In this study we compare the specificity of three mAbs; BR96 (Lewis^y), 2-25 LE (Lewis^b) and 692/29 that recognises a unique facet of both Lewis^y and Lewis^b. We then assessed the in vivo therapeutic effect of 692/29 using xenograft models.

Methodology/Principal Findings

Using a glycan array, each mAb was shown to display a different binding pattern with only 692/29 binding to both Lewis^y and Lewis^b. 692/29 was able to kill tumour cells over-expressing Lewis^y/b directly, as well as by antibody and complement mediated cytotoxicity (ADCC/CDC), but failed to kill cells expressing low levels of these haptens. In contrast, BR96, directly killed cells expressing either high or low levels of Lewis^y perhaps explaining its toxicity in patients. 2-25 LE failed to cause any direct killing but did mediate ADCC/CDC. Both 692/29 and BR96 bound to >80% of a panel of over 400 colorectal tumours whereas 2-25 LE showed lower reactivity (52%). 692/29 demonstrated more restricted normal tissue reactivity than both BR96 and 2-25 LE. 692/29 anti-Lewis^y/b mAb also showed good in vivo killing in xenograft models.

Conclusions/Significance

MAbs targeting both Lewis^y and Lewis^b may have a therapeutic advantage over mAbs targeting just one hapten. 692/29 has a more restricted normal tissue distribution and a higher antigen threshold for killing which should reduce its toxicity compared to a Lewis^y specific mAb. 692/29 has an ability to directly kill tumours whereas the anti-Lewis^b mAb does not. This suggests that Lewis^y but not Lewis^b are functional glycans. 692/29 showed good anti-tumour responses in vivo and is a strong therapeutic candidate.     

Effect of Polysorbate 80 Quality on Photostability of a Monoclonal Antibody

Polysorbate 80 is one of the key components of protein formulations. It primarily inhibits interfacial damage of the protein molecule due to mechanical stress during shipping and handling. However, polysorbate 80 also affects the formulation photostability. Exposure to light of polysorbate 80 aqueous solution results in peroxide generation, which in turn may result in oxidation of the susceptible amino acid residues in the protein molecule. The purpose of this study was to determine if the photostability of our proprietary IgG(1) monoclonal antibody formulation containing polysorbate 80 is affected by the quality (grade/vendor) of polysorbate 80. Following four types of polysorbate 80 were tested: (1) Polysorbate 80 Super-Refined, Mallinckrodt Baker, (2) Polysorbate 80 NF, Mallinckrodt Baker, (3) Polysorbate 80 NF, EMD Chemicals, and (4) Ultra-pure Polysorbate 80 (HX), NOF Corporation. The samples were exposed to light as per ICH guidelines Q1B. The results of the study show that photostability of the antibody formulation is indeed affected by the quality of polysorbate 80. This study underscores the importance of carefully choosing the quality of polysorbate 80 to ensure the robustness of formulation.     

Investigating the Degradation Behaviors of a Therapeutic Monoclonal Antibody Associated with pH and Buffer Species

This study aimed in understanding the degradation behaviors of an IgG 1 subtype therapeutic monoclonal antibody A (mAb-A) associated with pH and buffer species. The information obtained in this study can augment conventional, stability-based screening paradigms by providing the direction necessary for efficient experimental design. Differential scanning calorimetry (DSC) was used for studying conformational stability. Dynamic light scattering (DLS) was utilized to generate B ₂₂*, a modified second virial coefficient for the character of protein-protein interaction. Size-exclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC) were employed to separate degradation products. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used for determining the molecular size and liquid chromatography mass spectrometry (LC-MS) were used for identifying the sequence of the separated fragments. The results showed that both pH and buffer species played the roles in controlling the degradation behaviors of mAb-A, but the pH was more significant. In particular, pH 4.5 induced additional thermal transition peaks occurring at a low temperature compared with pH 6.5. A continual temperature-stress study illustrated that the additional thermal transition peaks related to the least stable structure and a greater fragmentation. Although mAb-A showed the comparable conformational structures and an identical amount of aggregates at time zero between the different types of buffer species at pH 6.5, the aggregation formation rate showed a buffer species-dependent discrepancy over a temperature-stress period. It was found that the levels of aggregations associated with the magnitudes of protein-protein interaction forces.     

治疗性单克隆抗体药物的现状及发展趋势

治疗性单克隆抗体药物经历了三十多年的发展,已经成为生物医药的最重要组成部分之一.在疾病治疗上具有广阔的应用前景,成功用于治疗肿瘤、自身免疫性疾病、感染性疾病和移植排斥反应等多种疾病.截至2012年已有29种治疗性单克隆抗体药物通过FDA审批并上市销售.治疗性单抗的安全性和有效性很大程度上由其作用的靶点决定,上市和在研的单抗药物有些靶向相同的靶点,有些有自己独特的作用靶点,新的作用靶点也在不断出现.以治疗性单抗的作用靶点为切入点,对目前上市销售和研发中的此类药物进行了简要总结.详述了肿瘤坏死因子α、白细胞分化抗原20、表皮生长因子受体及血管内皮生长因子等4种靶点的特点及相关单克隆抗体药物的情况,并对我国单抗药物的现状进行了分析,提出未来发展对策.     

不同产地天麻氨基酸的含量测定

氨基酸用途广泛,人们对氨基酸质量和品种的要求也越来越高.我国氨基酸从无到有,到现在的大市场份额,经历了五十多年的发展历程,其中包括谷氨酸、赖氨酸、苏氨酸和蛋氨酸在内的大品种氨基酸已经发展成为全球的主要供应品种,部分小品种氨基酸已形成自己的优势.绿色制造和现代生物技术的应用将成为氨基酸行业发展的主流.本文重点论述了我国氨基酸品种的现状、水平以及未来发展方向.     

猪链球菌2型次黄嘌呤核苷酸脱氢酶单克隆抗体的制备及其识别表位分析

用纯化的硫氧还蛋白-IMPDH融合蛋白免疫BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合,对杂交瘤细胞及时筛选,阳性孔经4次有限稀释法克隆,成功获得1A8、1F2、2D2和2D12共4株能稳定传代并分泌抗IMPDH的单克隆抗体(McAb)的杂交瘤细胞株.4株腹水型单克隆抗体间接ELISA效价分别为100x211、100x211、100x210和100x28,经Western-blot分析表明,4株单抗与硫氧还蛋白-IMPDH融合蛋白均具有特异性反应,并且通过4种IMPDH全基因分片段缺失表达的融合蛋白,分析了4株单抗所识别抗原决定簇的差异性,发现1A8、1F2,2D2识别表位的编码基因集中在IMPDH基因片段的627 bp～790 bp之间,2D12识别表位的编码基因则集中在IMPDH基因片段的411 bp～790 bp之间.猪链球菌2型中IMPDH单克隆抗体的获得及相应表位分析为研究IMPDH蛋白的生物学活性及免疫学活性奠定了基础.     

Characterization of a Monoclonal Antibody Specific for Lipoteichoic Acid from Various Gram-Positive Bacteria

A hybrid cell line, 3G6, producing monoclonal antibody (mAb) against the polyglycerophosphate (PGP) backbone of lipoteichoic acids has been derived by the polyethylene glycol-induced fusion of mouse myeloma cells and spleen cells from mice immunized with partially purified glucosyltransferase from culture supernatant of Streptococcus mutans strain 6715. Immunodiffusion tests and ELISA revealed that the antibody reacted with purified PGP from group A Streptococcus pyogenes strain Sv as well as crude phenol-water and saline extracts of various gram-positive bacteria except for a few species such as biotype B S. sanguis, Micrococcus sp., and Actinomyces viscosus. Whole cells of serotype b S. mutans and Staphylococcus epidermidis were agglutinated upon addition of 3G6 mAb, while those of most other species were not significantly affected by this procedure. A hapten inhibition study showed that glycerophosphate was only a potent inhibitor of passive hemagglutination reactions between LTA coated sheep erythrocytes and 3G6 mAb.     

治疗性单抗与抗体产业关键技术

抗体技术历经动物血清多克隆抗体、杂交瘤单克隆抗体,以及重组基因工程抗体等不同发展时期,尤其是后者使得治疗性抗体的生产进入产业化阶段.在已上市的抗体药物中,人源化抗体、全人源抗体由于免疫原性小,临床药效好,目前已经成为抗体药物的主流.随着抗体药物在癌症、免疫调节等治疗领域的广泛应用.抗体产业已经成为国际制药行业的主要组成部分.我国的抗体产业由于品种不足、技术落后,尚处于起步阶段,其行业发展受限于诸多技术瓶颈,如:工程细胞系构建与筛选、大规模培养工艺开发,单抗的纯化与质控等,上述产业化关键技术的突破可加快我国抗体产业的发展进程.