Identification and Characterization of a Monoclonal Antibody Variant Species with a Clipping in the Complementarity Determining Region Isolated by Size Exclusion Chromatography Under Native Conditions

The content of monoclonal antibody (mAb) fragments in pharmaceutical mAb products is a critical quality attribute and should be controlled for safety. Peptide bonds in the hinge region of mAbs are susceptible to hydrolysis, generating Fc-Fab fragments, which are associated with lower efficacy than the intact antibody. Fc-Fab fragments can be separated from intact antibody molecules under native conditions by size exclusion chromatography (SEC). Although several fragments generated by a clip in the complementarity determining region (CDR) have been reported, their efficacies have not been analyzed. This is because these fragments could not be separated from intact antibodies under native conditions owing to their similar molecular sizes. Here, we report that bevacizumab variant with clipping in the CDR, with the resulting fragments remaining intact in the variant, can be isolated under native conditions by selecting an adequate SEC column.     

柱前衍生化高效液相色谱法分离分析奥美拉唑对映体

目的 建立柱前衍生化高效液相色谱法分离奥美拉唑对映体.方法 以(S)-(+)-樟脑磺酰氯为柱前手性衍生化试剂,衍生物在Diamonsil C18色谱柱上,以10 mmol·L-1醋酸铵(pH 7.5)-异丙醇(75:25,v/v)为流动相分离,流速为0.5 mL· min-1.结果 奥美拉唑衍生物可达到基线分离,分离度为1.72.结论 采用柱前衍生化高效液相色谱法,在普通C18色谱柱上分离奥分析美拉唑衍生物,为此类手性药物的分离分析提供一个新方法.     

Development of a size exclusion chromatography method for the determination of molar mass for poloxamers

An aqueous size exclusion chromatography (SEC) method for determination of the molar mass of poloxamers 188 and 407 has been developed as an alternative to the pharmacopoeia methods. During the development work two different columns and several different eluent compositions were investigated. With a PL-aquagel-OH column, non-exclusion behaviour was obtained. A TSKgel column gave good separation of both poloxamers. The best separation was obtained with an eluent consisting of sodium chloride (0.01 M)-methanol (90:10, v/v) on the TSKgel column. The method was shown to be linear within the elution time of the two poloxamers and to have acceptable precision. The results from the SEC method was compared to results obtained using SEC with online multi angle light scattering detection (MALS) and to results obtained with matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS).     

RP-HPLC柱前衍生化法测定白舒非血药浓度

目的建立测定人血浆中白舒非浓度的柱前衍生高效液相色谱法。方法以1,5-戊二醇二甲磺酸酯为内标,血浆样品经二乙基二硫代氨基甲酸钠衍生化处理后,以乙腈-0.7%冰醋酸水溶液(69∶31)为流动相,流速1.0mL·min-1,采用XterraP18色谱柱分离,在280nm波长下进行检测。结果白舒非线性范围为0.20～4.00mg·L-1,以加权最小二乘法计算得到回归方程为Y=2.56X+16.7(r=0.9993,n=7),提取回收率81.01%～82.72%,方法回收率96.50%～97.90%,日内、日间精密度(RSD)均小于10%。最低血浆检测质量浓度为0.080mg·L-1。结论柱前衍生高效液相色谱法准确、灵敏,适用于白舒非血浆浓度的测定及其药代动力学研究。     

HPLC法测定奥赛博司原料药中有关物质的含量

目的:建立以高效液相色谱法测定奥赛博司原料药中有关物质含量的方法。方法:色谱柱为SunFireTMRP C18,流动相为磷酸盐缓冲溶液(pH3.05)-乙腈(8∶2),流速为1mL.min-1,检测波长为220nm,进样量为20μL,柱温为室温。结果:3批原料药经破坏试验后奥赛博司主峰与其它杂质峰均分离较好,有关物质含量均小于1.19%。结论:本方法简便、灵敏度高,可用于奥赛博司有关物质的含量检测。     

Failure Mode Identification of Insulin Drug Products – Impact of Relevant Stress Conditions on the Quality of the Drug

Fast-acting insulin drug products (DPs) are carried and administered by diabetic patients to maintain their blood glucose level throughout the day, exposing the DPs to stress conditions. Apidra, Novolog, and Humalog insulin DPs were tested under various stress conditions. Dynamic light scattering (DLS), and size exclusion chromatography (SEC) were used to monitor the stability and aggregation. Thermal stress alone did not influence the stability. However, 24 hr exposure to vigorous mechanical stress shifted the DLS size peaks of Novolog and Humalog from 5 ± 1 nm to > 50.9 ± 25.6 nm, and the SEC native protein peak areas decreased 52% for Novolog and 18.4% for Humalog. Combined stress accelerated protein aggregation more drastically. Novolog and Humalog size shifted (>75 nm) after 3 hr and the peak area decreased > 97.9% after 6 hr exposure, indicating that high temperature accelerated the aggregation triggered by agitation. Soluble aggregates were captured by DLS early on compared to SEC. Apidra was comparably stable indicating DP formulation plays a critical role in stability. Our study provides a greater understanding of potential failure modes patients and care givers may encounter while handling insulin DPs.     

发酵虫草菌粉指纹图谱研究

目的通过对发酵虫草菌粉指纹图谱的深入研究,为发酵虫草菌粉类产品的质量提高提供思路。方法液质联用确定发酵虫草菌粉类产品(百令胶囊)指纹图谱中6个主色谱峰的化学成分;采用中药色谱指纹图谱相似度评价系统,比较天然虫草和不同厂家发酵虫草菌粉指纹图谱相似度及不同干燥方式对指纹图谱的影响。结果百令胶囊指纹图谱中的6个主色谱峰成分为尿苷5-单磷酸、鸟苷酸、5’-腺嘌呤核苷酸、尿苷、鸟苷、腺苷。发酵虫草菌粉的指纹图谱与天然虫草均存在差异,其中百令胶囊的指纹图谱与天然虫草最接近。干燥方式对指纹图谱影响较大,采用沸腾干燥方式对应的指纹图谱主色谱峰的面积比最接近天然虫草晒干方式。结论指纹图谱可准确反映发酵虫草菌粉质量及质量控制方式的有效性。     

Erythrocyte Pyrimidine 5′-Nucleotidase and Deoxynucleotidase Isozymes: Metallosensitivity and Kinetics

ABSTRACT

Erythrocytes from a subject with classical pyrimidine 5′-nucleotidase (P5N) deficiency (PND) possessed typically low nucleotidase activity with uridine 5′-monophosphate (UMP) and cytidine 5′-monophosphate (CMP) as the substrates with subnormal dephosphorylation of 2′-deoxy thymidine 5′-monophosphate (dTMP) and 2′-deoxyuridine 5′-monophosphate (dUMP) and intermediate activity with 2′-deoxycytidine 5′-monophosphate (dCMP). Contrary to previously reported data, there was enzyme activity with all five substrates. The metallosensitivities of the P5N isozymes using UMP, dUMP, dCMP and dTMP as the substrates were defined. Nucleotidase (P5N) is slightly more sensitive than 2′-deoxy-pyrimidine 5′-nucleotidase (dP5N) to inhibition by lead, copper and mercury. Chromium stimulated more enzyme activity with dCMP than other substrates. Apparent Michaelis constants (K_m) were calculated for UMP, CMP, dUMP, dCMP, and dTMP for PND and control subjects. Optimal activity was at pH 7.0–7.8 when using UMP and CMP as the substrates but at pH 5.5–6.5 with dUMP, dCMP and dTMP as the substrates. The K_m, pH optima and metallosensitivities were, however, consistent with electrophoretic evidence for two and probably three isozymes: P5N with maximal affinity for UMP and CMP; dP5N with maximal affinity for dUMP and dTMP; and a closely related dCMPase. The relative activity with representative substrates can be used to distinguish homozygotes and heterozygotes for P5N deficiency and subjects with heavy metal exposure.     

rhIL-1ra蛋白质的HPLC法测定

建立了分子排阻色谱-高效液相色谱法测定重组人白介素-1受体拮抗剂(rhIL-1ra)蛋白。使用凝胶色谱柱,流动相为0.01 mol/L枸橼酸缓冲液-0.1 mol/L氯化钠溶液,检测波长280 nm。rhIL-1ra蛋白在0.018～2.4 mg/mm浓度范围内线性关系良好。回收率为99.1%,RSD为1.09%。     

RP-HPLC测定天然与人工冬虫夏草中游离麦角甾醇的含量

目的：比较天然与人工冬虫牙草中游离麦角甾醇的含量,探讨麦角甾醇作为冬虫夏草质控指标的可行性。方法：采用HP1100高效液相色谱仪,Allsphere C18色谱柱,甲醇－水（95∶5）为流动相,检测波长为275nm,流速1ml/min。结果：不同来源的天然冬虫夏草游离麦角甾醇含量差异显著,但均明显高于人工虫草菌丝。结论：麦象甾醇可作为冬虫夏草质控指标之一。     

Development and validation of a liquid chromatographic method for the determination of branched-chain amino acids in new dosage forms

A reversed-phase liquid chromatographic method (RP-LC) is proposed and validated for the analysis of branched-chain amino acids (l-leucine, l-isoleucine and l-valine) in new pharmaceutical formulations. The pre-column derivatization reaction of these amino acids with 2,4-dinitrofluorobenzene (DNFB) has been investigated considering the matrix effect. The compound reacts at 60 degrees C for 10 min at pH 9 with the amino function, in presence of cetyltrimethylammonium bromide (CTAB), to give adducts that have been separated on a RP amide C16 column and detected at lambda=360 nm. Linear responses were observed for each derivative. The intra-day precision (R.S.D.) was 相似文献   

湘葛一号根HPLC指纹图谱及5种异黄酮含量的同时测定

目的采用高效液相色谱法建立湘葛一号根指纹图谱,并同时测定5种异黄酮的含量。方法色谱条件:采用AT·LICHROM C18色谱柱(250mm×4.6mm,5μm);以甲醇-水溶液为流动相进行梯度洗脱;流速为1.0mL·min~(-1);检测波长为250nm;柱温为30℃。对不同采收期的10批样品的图谱进行测定,运用中国药典委员会的"中药色谱指纹图谱相似度评价软件2004AB"进行评价,并对5种异黄酮成分进行含量测定。结果建立了湘葛一号根HPLC指纹图谱,各色谱峰分离度良好,确定了7个共有峰,并采用对照品指认了其中5个色谱峰。含量测定条件通过方法学验证,葛根素、大豆苷、染料木苷、大豆苷元、染料木素的线性范围分别为5.00~50.00,1.00~10.00,1.82~18.20,1.00~10.00和2.00~20.00μg·mL~(-1),线性关系良好(r>0.999),平均加样回收率为98.8%~101.8%,5种异黄酮含量测定结果可初步确定适宜采收期为12月份。结论该方法所建立的湘葛一号根指纹图谱特征性强、方法简单、结果可靠,结合主要有效成分含量测定可更好地用于其质量控制,对其鉴定也具有参考价值。     

HPLC-fluorescence determination of amino acids in pharmaceuticals after pre-column derivatization with phanquinone

4,7-Phenanthroline-5,6-dione (phanquinone) was used as a fluorogenic labeling reagent in pre-column derivatization for the quality control of amino acids in pharmaceuticals. The amino acid adducts were efficiently separated by C12 RP high-performance liquid chromatography (HPLC) using a ternary mixture of triethylamine (TEA) phosphate buffer (pH 2.5, 0.05 M)-methanol- tetrahydrofuran (THF) as mobile phase by varying composition gradient elution and detected fluorometrically. The results obtained by the proposed method were compared statistically, by means of the Student's t-test and the variance ratio F-test, with those obtained by a rapid reference method, which involved o-phthaldialdehyde (OPA) as pre-column reagent; no significant difference was found. The stronger derivatization conditions (60 degrees C, pH 8, 60 min) required for the method with phanquinone are compensated by the major stability of derivatives and by the absence of fluorescent degradation products.     

多巴对映异构体的柱前衍生化HPLC法手性拆分

目的：建立多巴对映异构体的柱前衍生化拆分分析方法。方法：采用柱前衍生化反相高效液相色谱法,以氯甲酸乙酯为酰化剂,L-丙氨酸-β-萘胺（L-Ala-β-NA）为柱前衍生化试剂,在室温反应20 min 后,依利特 Hypersil ODS2（4.6 mm ×250 mm,5μm）为色谱柱;乙腈-0.05%三氟乙酸为流动相,梯度洗脱;流速为1.0 mL·min-1;柱温为25℃;检测波长为280 nm。结果：多巴对映体衍生物获得了基线分离,并确认了R（-）-和S（+）-两个衍生物的色谱峰。结论：该方法经济可行,操作较简便,为多巴的药理学研究和光学异构体纯度测定提供了参考。     

LC assay for salmon calcitonin in aerosol formulations using fluorescence derivatization and size exclusion chromatography.

A highly sensitive LC procedure was developed that utilizes fluorescence derivatization and detection coupled with size exclusion chromatography for the analysis of salmon calcitonin in salmon calcitonin aerosols. The LC procedure uses fluorescamine derivatization to label the primary amino groups of the peptide. The derivatization procedure is completely automated by an autosampler capable of pre-column mixing. Size exclusion chromatography is performed using a Supelco G2000 SWXL column. The method can be used to assay the amount of salmon calcitonin delivered per actuation of an aerosol unit. The procedure is simple, accurate, and precise and can detect as little as 2 ng ml-1 concentrations of salmon calcitonin.     

梯度洗脱高效液相色谱法检测注射用辅酶A有关物质

目的建立注射用辅酶A有关物质的测定法。方法采用Hypersil ODS色谱柱,以磷酸盐缓冲液(pH7.0)与乙腈为流动相梯度洗脱;检测波长259 nm,柱温25℃,流速1.0 mL/min。结果在此条件下,辅酶A与有关物质得到有效分离,辅酶A、5′-磷酸腺苷、氧化型辅酶A和3′-脱磷辅酶A的检测限分别为0.01 u,0.15 ng,50 ng和100 ng。结论此法专属、灵敏、准确,适用于药品的质量控制。     

A method for determination of the plasma levels of modafinil enantiomers, (+/-)-modafinic acid and modafinil sulphone by direct human plasma injection and bidimensional achiral-chiral chromatography

Coupled-column separation using restricted access media as the first dimension in order to exclude macromolecules and retain micromolecules has been successfully used for a number of biological fluids. This paper describes the first method developed and validated for the analysis in a single run of the enantiomers of modafinil and its two major metabolites. The method was developed using a bidimensional HPLC system by coupling a restricted access medium (RAM) bovine serum albumin (BSA) column (1.0 cm × 0.46 cm i.d.) to an amylose tris[(S)-1-phenylethylcarbamate] chiral column. The method was fully validated and showed good linearity, precision, accuracy, sensitivity and selectivity, allowing it to be used for pharmacokinetic studies. The quality of the performance of both columns was maintained with over 280 plasma injections of 100 μl.     

Quantitation of poloxamers in pharmaceutical formulations using size exclusion chromatography and colorimetric methods

Poloxamers have been used as functional excipients in pharmaceutical products. They function as surfactants, emulsifying agents, solubilizing agents, dispersing agents, and in vivo absorbance enhancer. Despite their wide range of applications, limited analytical techniques have been reported in literature for characterizing poloxamers and few are targeted to quantify poloxamer contents in formulations with desired sensitivity and accuracy. In this paper, two distinct analytical methods for quantifying low levels of poloxamers in pharmaceutical formulations have been developed and optimized: a colorimetric method and a size exclusion chromatography method. The colorimetric method is based on the formation of a colored complex between poloxamers and cobalt(II) thiocyanate in aqueous medium, which has a maximum UV absorbance at 624 nm. The feasibility of this method is product specific. In this report, adequate specificity and sensitivity was demonstrated for only one of the several products tested. The size exclusion chromatography (SEC) method utilizes size exclusion columns with THF as mobile phase and refractive index detection. The SEC method provides a limit of quantitation (LOQ) of 0.005 mg/mL (0.0005%, w/w) and at least three orders of magnitudes of linear range. We applied the SEC method to pharmaceutical products containing 0.3–10% poloxamer 188 or poloxamer 407, such as Avapro, Neurontin, Sudafed and other developmental formulations. The results obtained with the SEC method agreed very well with literature and theoretical values with 97–102% recovery. The SEC method was proven to be widely applicable, accurate, precise and simple to use.     

Comparison of free solution capillary electrophoresis and size exclusion chromatography for quantitating non-covalent aggregation of an acylated peptide

There are few methods available for the rapid and precise quantitation of non-covalent aggregation. The very methods used to measure the aggregation can easily disrupt the weak forces holding an aggregate together. This paper describes the novel application of free solution capillary electrophoresis (CE) for the quantitation of a biologically inactive non-covalent aggregate of C8GLIP (Des-amino-histidine-7-arginine-26 N(epsilon)-octanoyl-lysine-34-human glucagon-like insulinotropic peptide), an acylated peptide. The CE results are compared to a more traditional approach using size exclusion chromatography (SEC). Under the conditions explored in this paper, SEC showed a significantly slower apparent rate of aggregation than CE. This is due to the disruption of the aggregate during the SEC process. The cause of the disruption is complex and is potentially related to the separation process itself, on-column dilution effects, and/or interactions of the aggregate with the column packing or SEC components. Analysis times and dilution are greatly reduced by CE, and, because there is no potentially interactive stationary phase and because both the protein and the walls of the capillary are negatively charged, potential disaggregation due to surface interactions is reduced. Thus, CE is shown to be superior to SEC for this peptide in that disruption of the aggregate is minimized.     

黄牛木的化学成分

目的研究黄牛木的化学成分,为开发利用黄牛木植物资源提供依据。方法运用大孔吸附树脂、硅胶柱色谱、LH-20葡聚糖凝胶柱色谱、ODS柱色谱及制备液相等方法进行分离,根据化合物的理化性质及光谱数据鉴定化合物的结构。结果从黄牛木体积分数为65%乙醇提取物的正丁醇部分分离得到8个化合物,分别鉴定为(-)-南烛木树脂酚-3α-O-β-D-葡萄糖苷(-)-lyo-niresinol-3α-O-β-D-glucopyranoside(1)、(-)-5′-甲氧基异落叶松脂醇-9-O-β-D-葡萄糖苷((-)-5′-methoxyisolariciresinol-3α-O-β-D-glucopyranoside,2)、(+)-异落叶松脂醇-9-O-β-D-葡萄糖苷(isolariciresinol-9-O-β-D-glucopyranoside,3)、2,6-二甲氧基-4-羟基苯酚-1-短线问题-O-β-D-吡喃葡萄糖(2,6-dimethoxy-4-hydroxyphenol-1-O-β-D-glucopyranoside,4)、儿茶素(catechin,5)、表儿茶素(epicatechin,6)、原矢车菊素B-2(procyanidinB-2,7)、2a,3a-环氧-5,7,3′,4′-四羟基黄烷-(4b→8)-表儿茶素(2a,3a-epoxy-5,7,3′,4′-tetrahydroxyflavan-(4b→8)-epicatechin,8)。结论化合物1-4、8为首次从该属植物中分离得到。