Surface Activity of a Monoclonal Antibody

The development of high concentration antibody formulations presents a major challenge for the formulation scientist, as physical characteristics and stability behavior change compared to low concentration protein formulations. The aim of this study was to investigate the potential correlation between surface activity and shaking stress stability of a model antibody-polysorbate 20 formulation. The surface activities of pure antibody and polysorbate 20 were compared, followed by a study on the influence of a model antibody on the apparent critical micelle concentration (CMC) of polysorbate 20 over a protein concentration range from 10 to 150 mg/mL. In a shaking stress experiment, the stability of 10, 75, and 150 mg/mL antibody formulations was investigated containing different concentrations of polysorbate 20, both below and above the CMC. The antibody increased significantly the apparent CMC of antibody-polysorbate 20 mixtures in comparison to the protein-free buffer. However, the concentration of polysorbate required for stabilization of the model antibody in a shaking stress experiment did not show dependence on the CMC. A polysorbate 20 level of 0.005% was found sufficient to stabilize both at low and high antibody concentration against antibody aggregation and precipitation. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4525–4533, 2009     

Fragmentation of a Monoclonal Antibody by Peroxotungstate

Purpose

Tungsten and tungsten oxide leachates found in glass pre-filled syringes were identified to initiate protein precipitation and aggregation. Here, we tested the possibility of tungsten and tungsten oxide to induce the chemical degradation of proteins via reaction with hydrogen peroxide, a possible impurity present in protein formulations, to yield peroxotungstate.

Methods

A monoclonal antibody (mAb) was incubated with various concentrations of peroxotungstate and the reaction mixtures analyzed by SDS-PAGE and mass spectrometry.

Results

Exposure of a mAb to 1.07–1070 ppm peroxotungstate (based on tungsten content) at temperatures of 4°C and 22°C (pH 5–7) induced protein fragmentation. The extent of fragmentation increased with higher temperatures, lower pH and higher peroxotungstate concentrations. The mAb fragments were identified to contain different combinations of heavy chains (H) and light chains (L). Analogous mAb fragments were generated when the protein was exposed to H₂O₂ and orthotungstate at levels as low as 5 ppm. In addition, extracts from tungsten pins used to manufacture glass pre-filled syringes, in combination with H₂O₂ caused comparable fragmentation of the mAb. Mass spectrometric identification of the fragments suggests fragment generation by oxidative disulfide bond cleavage between the heavy and light chains, confirmed by mass spectrometry data on product formation. The mechanism of oxidative fragmentation was separately confirmed with insulin.

Conclusion

Fragmentation of the mAb by peroxotungstate is proposed to occur through inter-chain disulfide bond oxidation to form thiosulfinate (CyS(═O)SCy) and thiosulfonate [CyS(═O)₂SCy], followed by hydrolysis.

     

单克隆抗体药物进展

单克隆抗体药物特异性高、结构与性质均一稳定,其制备技术日益完善,临床应用越来越广泛,已有18种产品上市并用于人类疾病的诊断和治疗,100多种单克隆抗体药物进入临床研究阶段;单克隆抗体药物研究在生物技术药物中占有突出位置。文章综述了其国内外研究开发情况。     

新型抗B淋巴细胞CD22单克隆抗体Epratuzumab

CD22抗原是表达于B细胞的一种跨膜唾液酸糖蛋白,对B细胞的生长、发育及功能的维持具有重要作用.CD22抗原还可高表达于异常活化的B细胞及B细胞来源的恶性肿瘤细胞表面.Epratuzumab是人源化抗CD22单克隆抗体,与CD22结合,通过诱导靶细胞调亡、启动抗体依赖性细胞介导的细胞毒性以及抑制靶细胞的增殖等机制,来治疗自身免疫病及恶性B细胞肿瘤.因为具有高效、低毒、半衰期长等特点,Epratuzumab将在未来的临床治疗中发挥重要作用.     

人源化抗HER2单克隆抗体Pertuzumab

人表皮生长因子受体2（HER2）为受体酪氨酸激酶家族成员,是多种肿瘤细胞的重要生长调节因子,其过度表达对肿瘤的发生和恶性转化有促进作用,而HER2单体无活性,必须经配体结合后与自身或HER家族其他成员形成二聚体才能被激活。因此,HER2已成为肿瘤治疗研究中的一个潜在有效的靶标。目前临床上开发使用的HER2特异性靶向药物主要有两类,即人源化抗体和小分子酪氨酸激酶抑制剂。     

Freezing-Induced Perturbation of Tertiary Structure of a Monoclonal Antibody

We studied the effects of pH and solution additives on freezing-induced perturbations in the tertiary structure of a monoclonal antibody (mAb) by intrinsic tryptophan fluorescence spectroscopy. In general, freezing caused perturbations in the tertiary structure of the mAb, which were reversible or irreversible depending on the pH or excipients present in the formulation. Protein aggregation occurred in freeze–thawed samples in which perturbations of the tertiary structure were observed, but the levels of protein aggregates formed were not proportional to the degree of structural perturbation. Protein aggregation also occurred in freeze–thawed samples without obvious structural perturbations, most likely because of freeze concentration of protein and salts, and thus reduced protein colloidal stability. Therefore, freezing-induced protein aggregation may or may not first involve the perturbation of its native structure, followed by the assembly processes to form aggregates. Depending on the solution conditions, either step can be rate limiting. Finally, this study demonstrates the potential of fluorescence spectroscopy as a valuable tool for screening therapeutic protein formulations subjected to freeze–thaw stress.     

Aggregation Stability of a Monoclonal Antibody During Downstream Processing

Purpose  

To study the effect of several operative parameters, particularly pH and salt concentration, on the stability and aggregation kinetics of IgG solutions under the conditions typically encountered in downstream processing.     

Fragmentation of a Recombinant Monoclonal Antibody at Various pH

PURPOSE: To determine the relative importance of direct hydrolysis and beta-elimination, two common mechanisms of antibody hinge region fragmentation, and the impact of the conserved N-linked oligosaccharides in affecting antibody fragmentation under various pH. METHODS: A recombinant monoclonal antibody was incubated in buffers of various pH at 40 degrees C for 5 weeks. The level of fragmentation was measured using size-exclusion-chromatography (SEC). The specific sites of fragmentation were determined by analyzing SEC fractions using liquid chromatography mass spectrometry (LC-MS). RESULTS: Direct hydrolysis was accelerated by acidic and basic pH, while beta-elimination contributed to hinge region fragmentation at pH 7 and above. In addition, a shift of the major peptide bond hydrolysis sites in the hinge region towards the C-terminal direction with the decrease of sample pH from 9 to 5 was observed. At pH 4, the major cleavage site shifted outside the hinge region and was localized in the CH2 domain. Oligosaccharides did not affect hinge region fragmentation in the pH range of 5-9, however, at pH 4 oligosaccharides slowed down fragmentation in the CH2 domain. CONCLUSIONS: Antibody fragmentation level, sites and mechanisms were affected by pH. Oligosaccharides only affected the rate of fragmentation at pH 4.     

Monoclonal Antibody Therapies against Anthrax

Anthrax is a highly lethal infectious disease caused by the spore-forming bacterium Bacillus anthracis. It not only causes natural infection in humans but also poses a great threat as an emerging bioterror agent. The lethality of anthrax is primarily attributed to the two major virulence factors: toxins and capsule. An extensive effort has been made to generate therapeutically useful monoclonal antibodies to each of the virulence components: protective antigen (PA), lethal factor (LF) and edema factor (EF), and the capsule of B. anthracis. This review summarizes the current status of anti-anthrax mAb development and argues for the potential therapeutic advantage of a cocktail of mAbs that recognize different epitopes or different virulence factors.     

抗呼吸道合胞体病毒感染的人源单克隆抗体——Motavizumab

呼吸道合胞体病毒（RSV）是诱发婴幼儿及儿童病毒性细支气管炎和肺炎的主要病原体,为有包膜RNA病毒,主要人侵呼吸道上皮细胞。病毒包膜F糖蛋白介导包膜与宿主细胞膜的融合,被感染细胞随之也发生融合,最终形成合胞体。不同RSV株的F蛋白序列类似,故而F蛋白可作为治疗靶标。RSV感染性疾病的症状直接由病毒性细胞病变以及对感染的宿主反应所引起。     

抗神经生长因子单克隆抗体Tanezumab

目前在对疼痛和骨关节炎之类的炎症的治疗中多联合使用非甾体类解热镇痛抗炎药（NSAIDs）与硫酸吗啡等阿片类药物,但这些药物可能对某些病人无效或能引起复杂的不良反应,如胃肠道溃疡和呼吸抑制,因而亟待开发更为安全有效的治疗药物。神经生长因子（NGF）是一种在超敏反应的发生和异常性疼痛的产生中具有关键作用的递质。大量临床前研究表明：阻止NGF与其受体的相互作用,     

单克隆抗体纯化研究进展

综述了单克隆抗体的纯化技术,讨论了各单元操作的关键问题和解决方法,并重点介绍了模拟移动床色谱、双水相萃取、膜色谱等操作简单、能连续生产、低成本的纯化技术。     

完全人源化抗TNF-α单克隆抗体Golimumab

强生Centocor公司和先灵葆雅公司联合开发的完全人源化抗TNF-α单克隆抗体golimumab应用了Medarex公司的HuMAb-Mouse^TM技术，为一种每月1次皮下注射产品，最近已在其全球首个市场——加拿大以Simponi商品名获准上市，其适应证包括与甲氨蝶呤联用治疗中度至重度类风湿性关节炎、单独或与甲氨蝶呤联用治疗活动性银屑病性关节炎和用于治疗活动性关节强硬性脊椎炎。“Golimumab每月1次皮下给药，为风湿病医生及其患者提供了一种重要且方便的新的治疗选择。”     

抗CD80单克隆抗体Galiximab

Galiximab（IDEC-144）是一种猴源性的、抗CD80（B7—1）IgG1的单克隆抗体，包含短尾猴IgG，的可变区和人IgG1的恒定区。本品由美国BiogenIdec公司研制，并在研制中使用了一项名为PRIMATIZED的抗体技术以降低免疫原性。     

治疗高嗜酸性粒细胞综合征的药物——美泊利单抗

人源化单克隆抗体美泊利单抗（SB-240563）曾用于治疗由变应性嗜酸性粒细胞增多引起的一些症状，如变应性哮喘和特应性皮炎，但临床疗效并不好。不过该药在治疗嗜酸性粒细胞异常增多方面显示了更大潜力，其用于高嗜酸性粒细胞综合征（HES）的治疗已进人Ⅲ期临床研究。     

Adsorption Behavior of a Surfactant and a Monoclonal Antibody to Sterilizing-Grade Filters

Formulations of therapeutic proteins usually contain a surfactant such as polysorbate 80 to protect them against interfacial stresses. Since surfactants may interact with surfaces, the aim of the present work was to study the adsorption behavior of low concentrations of polysorbate 80 and of a monoclonal antibody during sterile filtration. Lab-scale tests were performed to study the adsorption behavior of a monoclonal antibody to different filter materials (PVDF, PES, CA, and Nylon) from different suppliers. Subsequently, protein and polysorbate 80 adsorption were tested in manufacturing scale experiments. It was found that the extent of protein adsorption differed with filter materials, but also with different suppliers. Prominently, Nylon filters showed the highest degree of protein adsorption. In manufacturing-scale filtration experiments, significant adsorption of polysorbate 80 to sterilizing-grade filters was found. Thus, the adsorption of both protein and polysorbate to filters should be taken into consideration in the formulation and manufacturing process and assessed on a case-by-case basis depending on the manufacturing process set-up.     

Identification and Characterization of Polysorbate-Degrading Enzymes in a Monoclonal Antibody Formulation

Degradation of polysorbate (PS) by hydrolytically active host cell proteins (HCPs) in drug products may impair the protein-stabilizing properties of PS and lead to the formation of particles due to the accumulation of poorly soluble free fatty acids upon long-term storage. The identification of the causative enzymes is challenging due to their low-abundance even when using state-of-the-art instrumentation and workflows. To overcome these challenges, we developed a rigorous enrichment strategy for HCPs, utilizing both Protein A and anti-HCP affinity chromatography, which facilitated the in-depth characterization of the HCP population in a monoclonal antibody formulation prone to PS hydrolysis. Based on the HCPs identified by liquid chromatography coupled to tandem mass spectrometry, a number of enzymes annotated as hydrolases were recombinantly expressed and characterized in terms of polysorbate degradation. Among the selected candidates, Lipoprotein Lipase, Lysosomal Acid Lipase (LIPA) and Palmitoyl-Protein Thioesterase 1 (PPT1) exhibited notable activity towards PS. To our knowledge, this is the first report to identify LIPA and PPT1 as residual HCPs that can contribute to PS degradation in a biological product.