Evaluation of Hydrogen Exchange Mass Spectrometry as a Stability-Indicating Method for Formulation Excipient Screening for an IgG4 Monoclonal Antibody

Antibodies are molecules that exhibit diverse conformational changes on different timescales, and there is ongoing interest to better understand the relationship between antibody conformational dynamics and storage stability. Physical stability data for an IgG4 monoclonal antibody (mAb-D) were gathered through traditional forced degradation (temperature and stirring stresses) and accelerated stability studies, in the presence of different additives and solution conditions, as measured by differential scanning calorimetry, size exclusion chromatography, and microflow imaging. The results were correlated with hydrogen exchange mass spectrometry (HX-MS) data gathered for mAb-D in the same formulations. Certain parameters of the HX-MS data, including hydrogen exchange in specific peptide segments in the C_H2 domain, were found to correlate with stabilization and destabilization of additives on mAb-D during thermal stress. No such correlations between mAb physical stability and HX-MS readouts were observed under agitation stress. These results demonstrate that HX-MS can be set up as a streamlined methodology (using minimal material and focusing on key peptide segments at key time points) to screen excipients for their ability to physically stabilize mAbs. However, useful correlations between HX-MS and either accelerated or real-time stability studies will be dependent on a particular mAb's degradation pathway(s) and the type of stresses used.     

The Selection of a Pharmaceutical Salt—The Effect of the Acidity of the Counterion on Its Solubility and Potential Biopharmaceutical Performance

A roadmap for the selection of a pharmaceutical salt form for a development candidate is presented. The free base of the candidate did not have sufficient chemical stability for development. The initially selected salt form turned out to be undevelopable because it was unstable during scale-up synthesis and storage. The rationale for the new solid form screening and the criteria for selection are discussed. Before the final selection, the pH solubility profiles of the 2 new salts, a benzoate and a besylate, were compared. Atypical solubility behavior was observed for the benzoate salt in hydrochloric acid with and without normal saline. A scheme is proposed illustrating how the pKas of the counterion and active pharmaceutical ingredient, the medium composition, and final pH affect the solubility and solution equilibria of the 2 selected salt forms. This scheme also includes the equilibria between solution and solid phases in different pH ranges. The pharmaceutical importance of this research is that it sheds light on how the acidity of the counterion can affect the solubility of the selected salt form in the gastric environment. With a well-designed formulation strategy, this property potentially can be translated to optimal biopharmaceutical performance of the drug product.     

Impact of Glycosylation on the Local Backbone Flexibility of Well-Defined IgG1-Fc Glycoforms Using Hydrogen Exchange-Mass Spectrometry

We have used hydrogen exchange–mass spectrometry to characterize local backbone flexibility of 4 well-defined IgG1-Fc glycoforms expressed and purified from Pichia pastoris, 2 of which were prepared using subsequent in vitro enzymatic treatments. Progressively decreasing the size of the N-linked N297 oligosaccharide from high mannose (Man8-Man12), to Man5, to GlcNAc, to nonglycosylated N297Q resulted in progressive increases in backbone flexibility. Comparison of these results with recently published physicochemical stability and Fcγ receptor binding data with the same set of glycoproteins provide improved insights into correlations between glycan structure and these pharmaceutical properties. Flexibility significantly increased upon glycan truncation in 2 potential aggregation-prone regions. In addition, a correlation was established between increased local backbone flexibility and increased deamidation at asparagine 315. Interestingly, the opposite trend was observed for oxidation of tryptophan 277 where faster oxidation correlated with decreased local backbone flexibility. Finally, a trend of increasing C'E glycopeptide loop flexibility with decreasing glycan size was observed that correlates with their FcγRIIIa receptor binding properties. These well-defined IgG1-Fc glycoforms serve as a useful model system to identify physicochemical stability and local backbone flexibility data sets potentially discriminating between various IgG glycoforms for potential applicability to future comparability or biosimilarity assessments.     

Coformulation of Broadly Neutralizing Antibodies 3BNC117 and PGT121: Analytical Challenges During Preformulation Characterization and Storage Stability Studies

In this study, we investigated analytical challenges associated with the formulation of 2 anti-HIV broadly neutralizing antibodies (bnAbs), 3BNC117 and PGT121, both separately at 100 mg/mL and together at 50 mg/mL each. The bnAb formulations were characterized for relative solubility and conformational stability followed by accelerated and real-time stability studies. Although the bnAbs were stable during 4°C storage, incubation at 40°C differentiated their stability profiles. Specific concentration-dependent aggregation rates at 30°C and 40°C were measured by size exclusion chromatography for the individual bnAbs with the mixture showing intermediate behavior. Interestingly, although the relative ratio of the 2 bnAbs remained constant at 4°C, the ratio of 3BNC117 to PGT121 increased in the dimer that formed during storage at 40°C. A mass spectrometry-based multiattribute method, identified and quantified differences in modifications of the Fab regions for each bnAb within the mixture including clipping, oxidation, deamidation, and isomerization sites. Each bnAb showed slight differences in the levels and sites of lysine residue glycations. Together, these data demonstrate the ability to differentiate degradation products from individual antibodies within the bnAb mixture, and that degradation rates are influenced not only by the individual bnAb concentrations but also by the mixture concentration.     

Inhibition of Organic Anion Transporting Polypeptide 1B1 and 1B3 by Betulinic Acid: Effects of Preincubation and Albumin in the Media

Betulinic acid (BA), a plant-derived pentacyclic triterpenoid, may interact with the members of the organic anion transporting polypeptide 1B subfamily. Here, we investigated the interactions of BA and its analogs with OATP1B1/3 and rat Oatp1b2 in vitro and in vivo. BA inhibited the activity of OATP1B1/3 and rat Oatp1b2 in vitro. Systemic exposure of atorvastatin was substantially altered with the intravenous co-administration of BA (20 mg/kg). Preincubation (incubation with inhibitors, followed by washout) with BA led to a sustained inhibition of OATP1B3, which recovered rapidly in the media containing 10% fetal bovine serum. The addition of albumin to the media decreased intracellular concentrations of BA and expedited the recovery of OATP1B3 activity following preincubation. For asunaprevir and cyclosporin A (previously known to inhibit OATP1B3 upon preincubation), the addition of albumin to the media shortened recovery time with asunaprevir, but not with cyclosporin A. Overall, our results showed that BA inhibits OATP1B transporters in vitro and may incur hepatic transporter-mediated drug interactions in vivo. Our results identify BA as another OATP1B3 inhibitor with preincubation effect and suggest that the preincubation effect and its duration is impacted by altered equilibrium of inhibitors between intracellular and extracellular space (e.g., albumin in the media).     

基于液相色谱-质谱技术的乳腺癌转移相关 代谢标志物的筛选

摘要：目的 应用代谢组学技术筛选与乳腺癌转移相关的代谢标志物。方法 收集100例乳腺癌患者和50例 健康志愿者的血清标本,采用高效液相色谱-轨道离子阱质谱联用（HPLC-LTQ Orbitrap XL MS）代谢组学研究平台分 析乳腺癌未转移患者、乳腺癌转移患者和健康人群血清标本的代谢轮廓,并通过模式识别方法结合非参数检验对数 据进行分析。结果 由乳腺癌未转移组、乳腺癌转移组和健康对照组的代谢轮廓构建的正交偏最小二乘判别分析 （OPLS-DA）模型具有很好的判别能力（R2=95.2%,Q2=86.7%）,可以鉴别出用于区分乳腺癌转移与否的8个代谢标志 物,包括溶血磷脂酸［18∶1（9Z）/0∶0］、溶血磷脂酰胆碱（18∶0）、溶血磷脂酰胆碱［20∶3（5Z,8Z,11Z）］、胆碱、磷酸二羟 丙酮（18∶0e）、2R,3S-番石榴酸、芥酸、L-氢化乳清酸。结论 利用代谢组学方法获得的血清代谢轮廓可以用来构建 区分模型和寻找乳腺癌转移相关的代谢标志物,为乳腺癌的早期诊治、预后评估和药物治疗靶点的选择提供支持和 依据。     

2型糖尿病患者体质量指数与甲状旁腺素、 25羟维生素D的相关性研究

摘要：目的 探讨 2 型糖尿病（T2DM）患者体质量指数（BMI）与甲状旁腺素（PTH）、25 羟维生素 D［25（OH）D］的 相关性。方法 收集首都医科大学附属北京朝阳医院内分泌科住院治疗的 2 型糖尿病患者 110 例,BMI 在 18.5～ 23.9 kg/m2患者作为正常体质量组（n=24）,BMI 在 24.0～27.9 kg/m2患者作为超重组（n=47）,BMI≥28.0 kg/m2患者作为 肥胖组（n=39）。记录患者年龄、性别、吸烟史、BMI、腹围和臀围。采集清晨空腹外周血,通过全自动生物化学分析仪 检测总胆固醇（TC）、高密度脂蛋白胆固醇（HDL-C）、低密度脂蛋白胆固醇（LDL-C）、三酰甘油（TG）、血清白蛋白 （ALB）、尿素氮（BUN）、血肌酐（Cr）、血清钙（Ca2+）、血清磷（P）、游离脂肪酸（FFA）、空腹血糖（FPG）和空腹胰岛素 （FINS）。高效液相色谱法测定糖化血红蛋白（HbA1c）。采用乳胶增强免疫透射比浊法测定超敏 C 反应蛋白（hs CRP）。采用免疫化学发光法测定 PTH、25（OH）D。计算稳态模型胰岛素抵抗指数（HOMA-IR）。利用简化肾脏病膳 食改善公式（MDRD）估算肾小球滤过率（eGFR）。比较 3 组间 PTH、25（OH）D 及各指标的差异,多重线性逐步回归 分析 BMI 与 PTH、25（OH）D 等指标的相关性。结果 与正常体质量组比较,超重组的腹围、臀围、FINS 明显升高 （P＜0.05）;肥胖组的腹围、臀围、PTH、FINS、HOMA-IR 明显升高（P＜0.05）。与超重组比较,肥胖组的腹围、臀围、 hs-CRP、PTH 明显升高（P＜0.05）。多元线性逐步回归分析结果提示,BMI 与臀围（β=0.293）、腹围（β=0.060）、FINS （β=0.026）、PTH（β=0.019）呈正相关（P＜0.05）。结论 T2DM 患者 BMI 与 PTH 密切相关,与 25（OH）D 无关。     

Effect of residual doxycycline concentrations on resistance selection and transfer in porcine commensal Escherichia coli

Pig feed may contain various levels of antimicrobial residues due to cross-contamination. A previous study showed that a 3% carry-over level of doxycycline (DOX) in the feed results in porcine faecal concentrations of approximately 4?mg/L.The aim of this study was to determine the effect of residual DOX concentrations (1 and 4?mg/L) in vitro on selection of DOX–resistant porcine commensal Escherichia coli and transfer of their resistance plasmids.Three different DOX–resistant porcine commensal E. coli strains and their plasmids were characterised. These strains were each brought in competition with a susceptible strain in a medium containing 0, 1 and 4?mg/L DOX. Resistant bacteria, susceptible bacteria and transconjugants were enumerated after 24?h and 48?h.The tet(A)–carrying plasmids showed genetic backbones that are also present among human E. coli isolates. Ratios of resistant to susceptible bacteria were significantly higher at 1 and 4?mg/L DOX compared with the blank control, but there was no significant difference between 1 and 4?mg/L. Plasmid transfer frequencies were affected by 1 or 4?mg/L DOX in the medium for only one of the resistance plasmids.In conclusion, DOX concentrations of 1 and 4?mg/L can select for resistant E. coli in vitro. Further research is needed to determine the effect of these concentrations in the complex environment of the porcine intestinal microbiota.     

琥珀酸美托洛尔缓释片在健康中国人体空腹和高脂餐后单次给药的药代动力学研究

目的评价单次口服琥珀酸美托洛尔缓释片在中国健康受试者体内的药代动力学特征和安全性。方法 7例中国健康志愿者空腹单次口服琥珀酸美托洛尔缓释片47. 5 mg,8例中国健康志愿者在高脂餐后单次口服琥珀酸美托洛尔缓释片47. 5 mg,用液相色谱-串联质谱联用法测定给药后不同时间美托洛尔的血药浓度,并用Win Nonlin 6. 4软件计算主要药代动力学参数。结果本研究未观察到药物相关的不良事件和严重不良事件。空腹单次口服琥珀酸美托洛尔缓释片后的药代动力学参数如下:C_max为(14. 63±8. 93) ng·m L^-1,t_max为(9. 43±2. 76) h,t1/2为(9. 36±5. 76) h,AUC_0-t为(343. 63±262. 07) ng·m L^-1·h,AUC_inf为(359. 65±259. 29) ng·m L^-1·h,清除率为(188. 58±109. 10) L·h^-1,表观分布容积为(3031. 36±4029. 78) L。高脂餐后单次口服琥珀酸美托洛尔缓释片后的药代动力学参数如下:C_max为(19. 56±14. 89) ng·m L^-1,t_max(6. 00±1. 07) h,t1/2为(6. 27±1. 37) h,AUC_0-t为(391. 07±307. 06) ng·m L^-1·h,AUC_inf为(395. 84±311. 42) ng·m L^-1·h,清除率为(225. 97±201. 53) L·h^-1,表观分布容积为(2022. 20±1842. 51) L。结论高脂餐后给予琥珀酸美托洛尔缓释片,t_max较空腹给药提前,暴露量未见明显差异。     

分子排阻色谱法测定头孢克洛胶囊中高分子杂质的含量

目的 建立分子排阻色谱法测定头孢克洛胶囊中的高分子杂质。方法 采用球状亲水硅胶柱(TSK-GEL?G2500PWXL色谱柱,7.8mm×300mm, 7μm),流动相为磷酸盐缓冲液(pH7.0)[0.02mol/L磷酸氢二钠溶液和0.02mol/L磷酸二氢钠溶液(61:39)]-乙腈(95:5)为流动相,流速为0.8mL/min,检测波长为265nm,以头孢克洛对照品外标法计算高分子杂质的含量。结果 头孢克洛在0.532~21.280μg/mL的浓度范围内,面积与浓度呈良好的线性关系(r=0.9999);最小检出浓度为0.161μg/mL;高分子杂质与头孢克洛峰能有效分离,并先于主峰流出,方法专属性良好。结论 该方法适于测定头孢克洛胶囊中高分子杂质,灵敏度高,重复性好,操作简便。