运用优化的毛细管等电聚焦电泳方法评价叶酸受体α单抗电荷不均一性

目的:用优化的cIEF方法分析叶酸受体α单抗(McAb)及其参照品的异构体分布,根据等电点差异,将样品带电荷的异构体分离开来,更好地评价叶酸受体α单抗电荷不均一性。方法:毛细管等电聚焦电泳条件:中性涂层的毛细管50μm×30cm(有效长度为20cm);阳极电泳缓冲液200mmo·lL-1磷酸,阴极电泳缓冲液300 mmo·lL-1氢氧化钠;聚焦电压25 kV,迁移电压30 kV,温度20℃检测波长280 nm。结果:用优化后的方法使样品的电荷异构体能够有效分离,电泳图谱与参照品电泳图谱一致。样品主峰峰面积百分比为60.59%,等电点为7.78,次主峰面积百分比为24.38%,等电点为7.59,主峰与次主峰分离度为1.185(USP)。连续5针进样,主峰面积百分比的RSD为7.05%,等电点的RSD为0.31%;次主峰面积的百分比RSD为5.99%,等电点的RSD为0.69%;待测样品在12 h内稳定。结论:采用优化的cIEF方法分析叶酸受体α单抗的电荷不均一性,具有更高的分离度,更好的重现性,为质量控制提供了更准确的手段。     

分析单克隆抗体电荷异质性的成像毛细管等电聚焦电泳技术平台方法的建立

目的  采用成像毛细管等电聚焦电泳技术（imaged capillary isoelectric focusing, iCIEF）建立分析单克隆抗体（单抗）电荷异质性的平台方法,并用不同亚型单抗（IgG1、IgG2、IgG4）确认该平台方法的适用性。方法  优化该平台方法的部分参数,包括两性电解质和阴极稳定剂体积、聚焦时间和尿素浓度。采用3种亚型单抗（IgG1、IgG2、IgG4）对该平台方法的专属性、精密度、线性、准确度和耐用性进行验证。结果   对样品（单抗）的处理条件为：3 mol/L尿素-0.5%甲基纤维素溶液70 μl、两性电解质（pH3~10）4 μl、阴极稳定剂（500 mmol/L 精氨酸）2 μl、等电点 6.14和9.99 Marker 各2 μl,最终完成0.2 mg/ml单抗（样品）的制备。检测参数：预聚焦1 500 V、1 min,聚焦3 000 V、8 min。该平台方法的专属性良好,制剂缓冲液对检测无干扰。重复检测6份平行样品以及不同分析员于不同时间检测12份样品各成分含量的相对标准偏差均符合规定的要求。单抗（样品）终浓度为0.1～0.3 mg/ml时,主要和酸性成分的线性决定系数（R2）≥0.99,碱性成分的线性R2≥0.98。该平台方法检测样品各成分的准确度为92～105%。耐用性实验设计结果表明,两性电解质（pH3~10）体积和毛细管批次对该平台方法有显著影响。结论  建立的iCIEF平台方法分离度较高,精密度、准确度和耐用性良好,为单抗制品的电荷异质性表征和质量控制提供了更有效的工具。     

抗CD19单抗的质量控制研究

目的：研究并建立针对抗白细胞分化抗原19(Cluster of Differentiation 19,CD19)单抗的关键质量属性质控方法。方法：运用肽图对抗CD19的单抗进行鉴别;采用还原/非还原十二烷基磺酸钠毛细管电泳(Capillary Electrophoresis-sodium Dodecyl sulfonate,CE-SDS)和分子排阻色谱(Size ExclusionHigh Performance Liquidchromatography,SEC-HPLC)进行单抗纯度的控制;运用成像毛细管等电聚焦电泳(Imaging Capillary Isoelectricfocusing Electrophoresis,iCIEF)测定电荷异质性;运用高效液相色谱法(High Performance Liquid Chromatography, HPLC)分析抗CD19单抗的糖基化,采用基于报告基因的抗体依赖的细胞介导的细胞毒效应(Antibody-Dependent Cell-mediated Cytotoxicity,ADCC) 测定生物学活性。其他各项指标均应符合现行版《中华人民共和国药典》以及其他相关要求。结果： 肽图检测抗CD19单抗具有相应的特征图谱,能够用于鉴别;还原CE-SDS的轻链与重链峰面积之和百分比为(98.77±0.05)%;非还原CE-SDS主峰峰面积百分比为(96.19±0.05)%,片段百分比为 (3.81±0.05)%;SEC-HPLC单体的峰面积百分比为(99.42±0.001)%,聚合物峰面积百分比为 (0.54±0.001)%;iCIEF分析的主要亚型的峰面积百分比为(66.41±0.38)%,酸性亚型的峰面积百分比为(13.69±0.16)%,碱性亚型的峰面积百分比为(19.90±0.46)%;在糖型分析中,占比最高的三个糖型分别为G0、G0-GN和M5,G0的含量为(64.64±0.61)%,G0-GN的含量为(9.75±0.42)%, M5含量为(6.22±0.35)%,生物学活性的半数最大效应浓度(Concentration for 50% of Maximal Effect, EC₅₀)值为(10.09±1.40)ng·mL^-1。结论：针对抗CD19单抗的关键质量属性,研究并建立了相应的质量控制方法,以确保其安全、有效和质量可控,为该类单抗产品的质量控制方法和策略提供参考依据。     

阿柏西普与雷珠单抗治疗新生血管性AMD的临床疗效对比分析

目的 对比研究两种不同药物阿柏西普和雷珠单抗对新生血管性年龄相关性黄斑变性（age-related macular degeneration,AMD）的临床疗效。方法 在2018年1月至2021年12月期间,收集徐州市第三人民医院眼科确诊为新生血管性年龄相关性黄斑变性合并黄斑水肿的患者56例56眼,随机分为两组,其中一组行玻璃体腔注射阿柏西普治疗,其中一组行玻璃体腔注射雷珠单抗治疗,分别于术后1个月、2个月、3个月、6个月随诊复查,观察两组间最佳矫正视力（BCVA）、黄斑区视网膜厚度（CSFT）及注药针数,并观察两组间6个月内的有无不良反应或严重并发症发生。结果 术前阿柏西普组和雷珠单抗组的最佳矫正视力（best corrected visual acuity,BCVA）、黄斑区视网膜厚度（central subfield thickness,CSFT）差异无统计学差异（均P> 0.05）,术后1个月、2个月、3个月、6个月,两组的BCVA与术前相比明显提高,CSFT明显降低,比较有统计学差异（P <0.05）,术后1个月、2个月、3个月两组间的BCVA及CSFT比较无统计...     

康柏西普与阿柏西普对新生血管性老年性黄斑变性的作用

目的：对比康柏西普和阿柏西普对新生血管性老年性黄斑变性（nAMD）患者的视力及不良反应的影响。方法：选取2020年6月至2023年2月江阴市中医院行玻璃体腔内药物注射治疗的nAMD患者109例（109眼）,根据玻璃体腔注射药物的不同将患者分为康柏西普组（57眼）与阿柏西普组（52眼）,随访3个月以上,对比分析两组治疗前后的最佳矫正视力（BCVA）、黄斑中心凹视网膜厚度（CMT）、脉络膜新生血管（CNV）渗漏情况以及不良反应等指标。结果：治疗后1个月、3个月的BCVA、CMT的数值均降低[康柏西普组：（0.57±0.15）、（0.46±0.12）Log MAR,(256.27±24.58)、（237.58±25.67）μm;阿柏西普组：（0.55±0.13）、（0.44±0.13）Log MAR,(247.94±26.27)、（232.49±27.29）μm],差异具有统计学意义（均P <0.05）。两组各时间点BCVA、CMT比较,差异均无统计学意义（均P> 0.05）,康柏西普组与阿柏西普组CNV渗漏治疗总有效率（85.96%vs 88.46%）及不良反应发生率（19.2...     

毛细管区带电泳方法评价曲妥珠单抗类似物的电荷异质性北大核心CSCD

目的:建立毛细管区带电泳(CZE)方法用于评价曲妥珠单抗类似物的电荷异质性。方法:实验中选用甲基纤维素(MC)和尿素作为缓冲液添加剂以改善曲妥珠单抗类似物电荷变体间的分离;0.1 mol·L-1盐酸溶液与0.1 mol·L-1氢氧化钠溶液前处理冲洗以有效减少单抗吸附。本文考察了运行缓冲液pH及浓度、分离电压、温度、添加剂对主峰与酸、碱变体峰分离度的影响。优化电泳条件为:5 kPa进样10 s,分离温度为15℃,分离电压为20 kV,运行缓冲液为600 mmol·L-1ε-氨基己酸(EACA)pH 5.4,包含0.1%MC和4 mol·L-1尿素溶液。结果:在优化的实验条件下,曲妥珠单抗类似物与其电荷变体能够得到较好分离,并且主成分的相对百分含量与迁移时间的日内精密度均小于2%。结论:本方法操作简单,分离度良好,成本较低,可以满足曲妥珠单抗类似物电荷异质性的分析要求。     

重组抗人表皮生长因子受体2单克隆抗体的电荷异质体分析

目的  探讨重组抗人表皮生长因子受体2（human epidermal growth factor receptor 2,Her2）单克隆抗体（单抗）的酸碱异质体对其生物活性和亲和力的影响。方法  分别用过氧化氢、肽-N-糖苷酶F、羧肽酶B对抗Her2单抗进行氧化、脱糖、酶切处理。用阳离子交换高效液相色谱法和毛细管等电聚焦电泳分析抗Her2单抗的电荷异质体,使用微量差示扫描荧光法分析抗Her2单抗的热稳定性,用细胞增殖抑制法和表面等离激元共振技术分析抗Her2单抗的亲和力。结果  脱糖处理抗Her2单抗的酸性电荷异质体增加了5.58%,与免疫球蛋白G Fc受体1（high affinity immunoglobulin gamma Fc receptor Ⅰ,FcGR1A）的亲和力明显减弱,亲和力常数为9.032×10^-8 mol/L。氧化和酶切处理抗Her2单抗的热稳定性降低,去折叠温度分别为63.6和59.5 ℃,均低于未处理抗Her2单抗（66.7 ℃）。结论  3种处理均会使抗Her2单抗产生电荷异质性,脱糖处理抗Her2单抗与FcGR1A的亲和力减弱,氧化和酶切处理抗Her2单抗的热稳定性降低。     

重组激素类药物质量分析专栏

<正>[编者按]重组激素类药物是最早用于临床治疗的生物技术药物，是目前全球销售额仅次于单抗的第二大类治疗性蛋白药物。在临床上对诸如糖尿病、生殖类疾病、骨质疏松症等重大慢性病、退行性疾病的治疗具有不可替代的作用，对于国家医保、公众大健康等民生工程具有重大意义。由于重组激素类药物设计基础主要来自于人和动物体内天然存在的蛋白和多肽活性分子，经过100多年的研究，人类对其分子类别、药理和药效的认知十分完善，基于新靶点、新分子实体的源头创新药物开发现阶段难度很大。国际上的研发热点主要集中在有重大临床价值的长效化，蛋白质工程改构，复方制剂产品，诸如甘精胰岛素、赖脯胰岛素、德谷胰岛素、利拉鲁肽、度拉鲁肽等“重磅炸弹”级的生物药。我国人口基数大、老龄化问题日渐凸显，临床上对于重组激素类药物的需求仍处于快速增长阶段。上市产品个数、生产企业数量，近几年有显著增加，某些当前仍依赖进口的临床亟需品种也陆续进入上市申报阶段。     

诺和诺德旗下德谷胰岛素/利拉鲁肽复方药物赢得EU批准

诺和诺德公司研究的一款名为Xultophy的复方药物,已赢得欧盟的批准,这是首创的一天使用一次的2型糖尿病复方药物。这款复方药物由德谷胰岛素与利拉鲁肽配对组成,它被预填入一个单一的注射笔中使用,该公司预计将于明年上半年在整个欧洲推出。 周期为52周的试验数据在6月份的美国糖尿病协会会议上得到发布,使用该复方药物的患者有6.4%的血糖基线值,优于利拉鲁肽用药患者的7.1%,及德谷胰岛素用药患者的6.9%。     

一种新型聚乙二醇化重组人生长激素的制备及性质考察

目的制备一种新型聚乙二醇化重组人生长激素(PEG-(rhGH)2),并对其理化性质及药理作用进行研究。方法以分子质量为2×104u的聚乙二醇二丙醛(PEG-(ALD)2)对重组人生长激素(rhGH)进行修饰,采用Sephacryl S-200凝胶过滤色谱、Q Sepharose FF离子交换色谱对修饰产物进行分离纯化,SDS-PAGE鉴定各组分,通过SEC-HPLC、IEF、MALDI-TOF-MS等方法对目标产物进行分析,并采用去脑垂体大鼠模型研究其体内生物活性与长效作用。结果获得了一种新型聚乙二醇化重组人生长激素(PEG-(rhGH)2)。其纯度质量分数达到97%以上,等电点为5.27,分子质量为6.6×104u。与未修饰的rhGH相比,PEG-(rhGH)2具有高效、长效的促进机体生长的药理作用。结论新型聚乙二醇化重组人生长激素PEG-(rhGH)2是一种具有开发前景的长效蛋白质药物,值得进一步深入研究。     

Properties of a cleaved two-chain form of recombinant human growth hormone

Escherichia coli cells transformed with plasmids engineered for the expression of recombinant human growth hormone as a secreted product also produced a proteolytically cleaved form of rhGH. This variant is isolated at a high resolution anion exchange chromatography stage during the manufacturing process. The higher isoelectric point of this form is demonstrated by isoelectric focusing and chromatofocusing and the two-chain nature by tryptic mapping, N- and C-terminal sequence analyses, and sodium dodecyl sulfate polyacrylamide gel electrophoresis. These data indicate that the single site of cleavage is between Thr-142 and Tyr-143, in contrast to the two-chain variant isolated from human pituitary glands, which has a clip after residue Phe-139. The recombinant two-chain form was further characterized by reversed-phase high performance liquid chromatography at both acidic and basic pHs. The assay utilizing bicarbonate-containing mobile phases was determined to be the most efficient and sensitive method. The bioactivity of this two-chain form was measured by the in vivo rat weight gain assay and by the in vitro Nb2 cell bioassay. Its immunological similarity to intact one-chain rhGH was demonstrated with an enzyme-linked immunosorbent assay.     

Injection force and dose accuracy of FlexTouch for the delivery of a new basal insulin

Objective: The aim of the paper is to determine the dose accuracy and injection force of FlexTouch (FT) filled with insulin degludec 100 U/ml, insulin degludec 200 U/ml and insulin degludec/insulin aspart 100 U/ml, SoloStar (SS) filled with insulin glargine 100 U/ml and KwikPen (KP) filled with insulin lispro mix 75/25 100 U/ml.

Methods: Dose accuracy was measured at minimum, midpoint and maximum doses (FT 1, 2, 40, 80 and 160 U; SS 1, 40 and 80 U; KP 1, 30 and 60 U). Injection force was measured during the injection of the maximum dose.

Results: All doses delivered from FT were within ISO limits (ISO 11608-1:2012) for degludec 100 U/ml, degludec 200 U/ml and degludec/aspart 100 U/ml, and the pens delivered insulin accurately and consistently at all doses tested. Similarly, all tested doses from KP filled with insulin lispro mix 75/25 100 U/ml were within ISO limits, while some doses from SS filled with insulin glargine 100 U/ml were outside ISO limits. FT had a significantly lower injection force than SS and KP (p < 0.05).

Conclusions: FT filled with insulin degludec and insulin degludec/insulin aspart, delivered insulin accurately and consistently within ISO limitations at all doses tested; similarly, KP delivered insulin within ISO limitations at all doses tested and SS delivered most doses within ISO limitations. The significantly lower injection force of FT compared to SS and KP is an important feature that has the potential to make the injection process easier for people with diabetes.     

Fixed-ratio combination therapy with GLP-1 receptor agonist liraglutide and insulin degludec in people with type 2 diabetes

Introduction: A fixed combination of basal insulin degludec and glucagon-like peptide-1 receptor agonist (GLP-1RA) liraglutide (IDegLira; 50 units degludec/1.8 mg liraglutide) has been developed as a once daily injection for the treatment of type 2 diabetes (T2D). In the phase 3a trial programme ‘Dual action of liraglutide and insulin degludec in type 2 diabetes’ (DUAL?), five trials of 26 weeks duration and one trial of 32 weeks duration have evaluated the efficacy and safety of IDegLira compared with administration of insulin degludec, insulin glargine, liraglutide alone or placebo.

Areas covered: Combination therapy with IDegLira reduces HbA1c more than monotherapy with a GLP-1RA (liraglutide) or insulin (degludec or glargine). Combination therapy leads also to weight loss, or a stable body weight, with no increase in hypoglycaemia. Rates of adverse events did not differ between treatment groups; however, gastrointestinal side effects were fewer with IDegLira compared with liraglutide treatment alone. A limitation of the DUAL? development programme is that patients receiving basal insulin doses in excess of 50 units were excluded from the studies.

Expert commentary: In conclusion, IDegLira combines the clinical advantages of basal insulin and GLP-1RA treatment, and is a treatment strategy that could improve the management of patients with T2D.     

Pharmacokinetic evaluation of fixed-ratio combination of insulin degludec and liraglutide in the treatment of type 2 diabetes

Introduction: Type 2 diabetes is a complex disease requiring individualized and often multi-faceted treatment plans. Balancing glycemic control with adverse medication side effects can be challenging. Combination therapy of basal insulin and GLP-1 receptor agonist therapy appears to provide a balance between glycemic efficacy, hypoglycemia and weight gain.

Areas covered: Available pharmacokinetic data, clinical trials and abstracts regarding fixed-ratio combination of insulin degludec and liraglutide were reviewed. Literature was searched from PubMed and available abstracts using the search term IDegLira up to June 2016.

Expert opinion: Fixed-ratio combination of insulin degludec and liraglutide is associated with sustained glucose-lowering effects as assessed by HbA1c reduction. Overall, hypoglycemia is reduced and weight loss is observed compared to basal insulin alone. There is no significant increase in gastrointestinal side-effects, including nausea compared to non-glucagon-like peptide-1 receptor agonists. Pen injector delivery and dose-step titration allow for individualized therapy intensification. Combined treatment for type 2 diabetes with degludec and liraglutide therapy provides complementary therapeutic efficacy in the treatment of individuals with type 2 diabetes.     

Patient-reported outcomes from a randomized,crossover trial comparing a pen injector with insulin degludec versus a pen injector with insulin glargine U100 in patients with type 2 diabetes

Objective: Type 2 diabetes (T2D) is associated with insulin resistance and deteriorated glycemic control that can be restored with insulin injections. Choice of insulin pen injector may affect complexity, adherence, efficacy of treatment and health-related quality of life. We describe detailed patient-reported outcomes (PROs) on treatment impact and preference comparing insulin degludec (degludec) using FlexTouch¹ versus insulin glargine U100 (glargine U100) with SoloStar² pen injector.

Methods: In this randomized, multicenter (USA), open-label, crossover, treat-to-target study (NCT01570751), patients with T2D using high-dose insulin (≥81?U/day from vials) were randomized (n?=?145) 1:1 to 16?weeks of degludec U200 (3?mL FlexTouch) followed by 16?weeks of glargine U100 (3?mL SoloStar) or vice versa. PRO questionnaires assessed treatment impact and patient preference of pen injectors.

Results: Significantly more patients (p?<?.01) considered FlexTouch “extremely easy” for learning (62.5 vs. 43.0%), maintaining (63.2 vs. 42.2%) and adjusting the dose (63.2 vs. 44.4%), and significantly more were “very” or “extremely confident” in using the device (60.3 vs. 36.3%) and in its accuracy (50.7 vs. 30.4%) versus SoloStar. Significantly more were “not at all bothered” by device discomfort (74.3 vs. 54.1%), whereas device size (83.8 vs. 80.0%) or public use (69.9 vs. 60.7%) were numerically in favor of FlexTouch. Significantly more patients preferred degludec treatment with FlexTouch (59 vs. 22%), preferred to continue (67 vs. 15%) and recommend (67 vs. 14%) use of FlexTouch compared with SoloStar with glargine U100.

Conclusions: In this randomized, crossover trial, lower treatment impact and higher patient preference were reported for FlexTouch versus SoloStar pen injectors.     

The Stability of Recombinant Human Growth Hormone in Poly(lactic-co-glycolic acid) (PLGA) Microspheres

Purpose. The development of a sustained release formulation for recombinant human growth hormone (rhGH) as well as other proteins requires that the protein be stable at physiological conditions during its in vivo lifetime. Poly(lactic-co-glycolic acid) (PLGA) microspheres may provide an excellent sustained release formulation for proteins, if protein stability can be maintained. Methods. rhGH was encapsulated in PLGA microspheres using a double emulsion process. Protein released from the microspheres was assessed by several chromatrographic assays, circular dichroism, and a cell-based bioassay. The rates of aggregation, oxidation, diketopiperazine formation, and deamidation were then determined for rhGH released from PLGA microspheres and rhGH in solution (control) during incubation in isotonic buffer, pH 7.4 and 37°C. Results. rhGH PLGA formulations were produced with a low initial burst (<20%) and a continuous release of rhGH for 30 days. rhGH was released initially from PLGA microspheres in its native form as measured by several assays. In isotonic buffer, pH 7.4 and 37°C, the rates of rhGH oxidation, diketopiperazine formation, and deamidation in the PLGA microspheres were equivalent to the rhGH in solution, but aggregation (dimer formation) occured at a slightly faster rate for protein released from the PLGA microspheres. This difference in aggregation rate was likely due to the high protein concentration used in the encapsulation process. The rhGH released was biologically active throughout the incubation at these conditions which are equivalent to physiological ionic strength and pH. Conclusions. rhGH was successfully encapsulated and released in its fully bioactive form from PLGA microspheres over 30 days. The chemical degradation rates of rhGH were not affected by the PLGA microspheres, indicating that the internal environment of the microspheres was similar to the bulk solution. After administration, the microspheres should become fully hydrated in the subcutaneous space and should experience similar isotonic conditions and pH. Therefore, if a protein formulation provides stability in isotonic buffer, pH 7.4 and 37°C, it should allow for a safe and efficacious sustained release dosage form in PLGA microspheres.     

我国已上市治疗用重组蛋白多肽类产品分析

目的：找出我国治疗用重组蛋白多肽类产品存在的不足,明确未来努力方向。方法：基于我国已上市治疗用重组蛋白多肽类产品批准数据进行统计分析和对比分析。结果：对我国已上市的重组蛋白多肽类产品的基本情况、技术代次、治疗领域等进行了回顾梳理。结论：我国重组蛋白多肽类药物在新一代产品的开发上,由于企业创新不足及技术落后,与国际先进水平的差距近年来在进一步加大。