Salt-Induced Aggregation of a Monoclonal Human Immunoglobulin G1

Physical stability is critical for any therapeutic protein's efficacy and economic viability. No reliable theory exists to predict stability de novo, and modeling aggregation is challenging as this phenomenon can involve orientation effects, unfolding, and the rearrangement of noncovalent bonds inter- and intramolecularly in a complex sequence of poorly understood events. Despite this complexity, the simple observation of protein concentration-dependent diffusivity in stable, low ionic-strength solutions can provide valuable information about a protein's propensity to aggregate at higher salt concentrations and over longer times. We recently verified this notion using two model proteins, and others have shown that this strategy may be applicable to antibodies as well. Here, we expand our previous study to a monoclonal human immunoglobulin G1 antibody and discuss both merits and limitations of stability assessments based on the diffusional virial coefficient k_D. We find this parameter to be a good predictor of relative protein stability in solutions of different chaotropic salts, and a telling heuristic for the effect of kosmotropes. Both temperature and glycosylation are seen to have a strong influence on k_D, and we examine how these factors affect stability assessments. Protein unfolding is monitored with a fluorescence assay to assist in interpreting the observed aggregation rates. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:377–386, 2013     

Origin of the Isoelectric Heterogeneity of Monoclonal Immunoglobulin h1B4

The origin of the microheterogeneity of a highly purified antiinflammatory humanized monoclonal antibody prepared in mammalian cell culture has been investigated. This antibody is an IgG directed toward human CD 18 (a subunit of leukocyte integrins). When the IgG preparation is subjected to isoelectric focusing, it is found to contain four major species with pI values ranging from 6 to 7. Although the relative amounts of each form differ and some species are present only in small quantities, each has been isolated by a combination of high-resolution anion-exchange chromatography and isoelectric focusing. Comparative studies reveal no detectable differences in overall secondary (far UV circular dichroism) or tertiary (intrinsic fluorescence) structure, molecular weight (laser-desorption mass spectroscopy), or antigen binding activity. When each of the isolated species is incubated under conditions which favor deamidation, it is converted to forms of lower pI which appear to correspond to naturally observed species. While the isolated light chain is relatively homogeneous, the heavy chain exhibits a pattern of isoelectric focusing bands similar to that of the intact immunoglobulin. These results suggest that in this case, charge microheterogeneity is due to the sequential deamidation of the immunoglobulin heavy chain.     

郑州市3～5岁儿童龋患率及危险因素分析

目的了解儿童龋患率现状,分析其危险因素,为儿童龋齿的综合防治提供科学依据。方法对郑州市二七区7所幼儿园3~5岁儿童进行口腔检查及问卷调查。Logistic回归分析龋齿危险因素。结果共调查1 492名儿童,龋患率为50.13%,男48.55%,女52.31%,两性间龋患率差异无统计学意义(χ2=0.68,P=0.4101)。各年龄组间龋患率差异有统计学意义(χ2=21.39,P≤0.0001)。结论对双亲进行口腔健康知识教育,使儿童养成良好的口腔卫生习惯,预防龋齿的发生。     

Gamma scintigraphy: an evolving technology in pharmaceutical formulation development-Part 1

Gamma scintigraphy provides a noninvasive method to `see' the in vivo fate of a pharmaceutical dosage form. The power of the technique is realized fully by correlating deposition and transit of the delivery system with resulting systemic drug levels. Since their inception 25 years ago, these methods have evolved into the preferred technology for evaluation of the in vivo behavior of drug delivery systems. In part one of a two part review, the authors describe general scintigraphic methods and provide illustrations that exemplify the use of gamma scintigraphy for the in vivo evaluation of oral formulations. Part 2 of this article will appear in the July issue of PSTT.     

某市从业人员健康体检肥胖症的患病率及相关疾病分析

目的调查广东省惠州市从业人员肥胖症的患病率及相关疾病患病率。方法偶遇抽样调查惠州市12632名从业人员,内容包括问卷调查、测量身高、体质量、血压,检查空腹血糖、血脂、肝脏B超。结果肥胖症的总体患病率为12.80%,在年龄段为18²4岁以及总体的人群中,男女肥胖症患病率无明显差异;在2524岁以及总体的人群中,男女肥胖症患病率无明显差异;在25³4、5534、55⁶5岁的年龄段中女性患病率高于男性,在3565岁的年龄段中女性患病率高于男性,在35⁴4、4544、45⁵4岁的年龄段中男性患病率高于女性;在肥胖症者中,高血压、高脂血症、脂肪肝、高血糖的患病率均明显高于非肥胖症者,差异均有统计学意义。结论肥胖症的发病率随着年龄的增长而上升,在不同的年龄段存在着性别差异,肥胖者中慢性病的患病率增加。     

维生素A在分泌型免疫球蛋白A生成中的作用

维生素A(VA)是最早被认识并应用于临床的维生素,VA有维持上皮完整性、提高免疫力和抗感染的作用,常应用于防治小儿呼吸道及消化道等感染性疾病.分泌型免疫球蛋白A(SIgA)广泛存在于呼吸道及消化道等外分泌液中,是人体粘膜免疫的主要抗体,许多感染性疾病的发生与SIgA结构、含量及活性的改变有关.研究表明VA缺乏时,SIgA水平下降,粘膜局部特异性免疫功能降低,对病原微生物的易感性增高.     

克拉霉素分散片的相对生物利用度

目的 :比较国产克拉霉素分散片与进口克拉霉素片 (克拉仙 )的相对生物利用度。方法 :采用微生物法测定8名健康男性志愿者随机单剂量口服两种片剂500mg后 ,药物在体内的经时过程。结果 :药 -时曲线符合一级吸收二室模型 ,国产分散片与进口片AUC分别为 (18 58±5 46) μg/(h·ml)和 (19 05±5 75) μg/(h·ml) ;Cmax 分别为 (2 88±0 74) μg/ml和 (2 74±0 65) μg/ml;Tmax 分别为 (1 28±0 41)h和 (1 47±0 51)h ,分散片的相对生物利用度为 (98 49±16 00) %。结论 :国产克拉霉素分散片与进口克拉霉素普通片生物等效。     

MTT比色法与相对增殖度法对输液用包装材料的细胞毒性检测结果的比较

目的 比较四唑盐(MTT)比色法和相对增殖度法对4种输液用包装材料细胞毒性检查结果的相关性,评价2种方法在检测药包材体外细胞毒性作用上的优缺点。为建立快速有效的药包材检测新标准提供有力依据。方法 L-929细胞分别采用MTT法和细胞增殖度法对4种包装材料的浸提液进行细胞毒性研究,比较材料对细胞的毒性等级,算出细胞的相对增殖度,分析比较2种方法相关性。结果 在同样实验条件下,4种药品包材表现出对L-929细胞不同的细胞毒性。与细胞增殖度法比较,MTT法与细胞增殖法检测结果具有很好的相关性r=0.966(P<0.05)。结论 与相对增殖度法相比,MTT比色法由于实际检测所需的细胞量相对较少、试验步骤相对简便、检测周期短,因此具有一定的优越性,值得推荐作为细胞毒性检测方法。     

骨化醇类化合物的质谱裂解规律

以10个骨化醇类化合物为研究对象,采用电喷雾串联质谱及高分辨质谱元素组成进行分析,探讨该类化合物的质谱裂解规律。C-17位侧链基团的裂解差异、各环上断裂的位点以及结构中双键位置对质谱裂解的影响反映了该类化合物的结构特征。该研究可用于骨化醇类化合物的结构鉴定,为该类药物未知杂质的快速定性提供参考。     

初始剂量丙种球蛋白无反应性川崎病的治疗进展

虽然静脉用丙种球蛋白(IVIG)可以有效的治疗川崎病(KD),但仍有10%~20%的KD患儿对初始剂量IVIG治疗无反应,使他们合并冠状动脉病变(CAL)的风险增加.对初始剂量丙种球蛋白无反应性川崎病的治疗方案仍有争议.其他疗法主要包括再次输注IVIG、糖皮质激素药物、TNF-α抑制剂、钙调磷酸酶抑制剂、他汀类药物、MTX治疗、血浆置换等.本文综述初始剂量IVIG无反应性KD的治疗进展.     

Gamma irradiation of Sennae Folium

The effect of different doses of gamma irradiation on senna leaves was studied. No changes in sennoside content and composition were observed after irradiation with 25 kGy. It was demonstrated that a minimum dose of 10 kGy was necessary to obtain a product of good microbiological quality. Cold maceration of senna leaves has to be discouraged, because of unavoidable microbial growth. Hot extraction is the method of choice for tea preparation. The rationale of gamma irradiation is discussed.     

Middle-Down Fragmentation for the Identification and Quantitation of Site-Specific Methionine Oxidation in an IgG1 Molecule

A middle-down LC/MS approach, for the rapid quantitation and characterization of site-specific methionine oxidation in a recombinant monoclonal IgG1 molecule, is described. An IgG1 antibody was digested with endoprotease LysC under limited proteolytic conditions to produce two major components; an antigen binding fragment (Fab) and a crystallizable fraction (Fc). These fractions were then reduced to produce three major species; light chain (LC), Fc/2 which is the C terminal region of the heavy chain (HC) and the N-terminal heavy chain region (Fd). These three fragments were separated by reversed-phase HPLC using a diphenyl column. The diphenyl column resolved site-specific methionine oxidation in all three subunits. Middle- down N-terminal sequencing with a LCT premier mass spectrometer was used to identify the sites of oxidation in the LC. Sites of oxidation in the Fc/2 were identified using middle-down collision-induced dissociation (CID) on a Qtof premier. This method allowed for the rapid quantitation and identification of oxidation on each methionine residue in an IgG1 molecule.     

Fragmentation of 3-oximino-4-chromanone.

Heating either the methanesulfonate ester of 3-oximino-4-chromanone or 3-oximino-4-chromanone and an alternative acylating agent such as p-toluensulfonyl chloride or acetic anhydride in the presence of aqueous base afforded two major fragments: salicyclic acid and 2-carboxyphenoxyacetonitrile. These compounds are derived from two separate cleavage pathways involving the acylated oxime. In one pathway, fragmentation appears to be assisted by the ether ring oxygen; in the other, it is assisted by the alpha-carbonyl group of the oxime ester.     

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.

     

Relative pharmacological potency in mice of optical isomers of delta 1-tetrahydrocannabinol

The tritium-labelled unnatural enantiomorph of Δ¹-tetrahydrocannabinol(Δ¹-THC) was synthesized. The ³H-(+)-Δ¹-THC had a specific activity of 1.3 Ci/mmole and an optical purity of ca. 97%. The equipotent molar ratio for (+) and (?)-Δ¹-tetrahydrocannabinols was determined in mice by an established behavioural bioassay. The (+)-Δ¹-THC was found to be significantly less potent than the laevorotatory isomer, the mean potency ratio being 13 (95 per cent confidence limits: 7 and 24). Brain levels of (+)-Δ¹-THC and its metabolites were measured in mice 20 min after intravenous injection of ³H-(+)-Δ¹-THC (2 mg.kg) and were compared with the corresponding levels of (?)-Δ¹-THC and its metabolites. With the exception of the concentrations of one metabolite, no statistically significant differences were observed between the mean levels of enantiomorphs of the cannabinoids in the brain. In the case of the single metabolite (which was tentatively assigned the structure of 7-hydroxy-Δ¹-THC) the brain level of the dextrorotatory isomer was 1.8-times higher than that of the laevorotatory isomer, a difference which was statistically significant. On incubation in vitro with an enriched mouse liver homogenate, (+)-Δ¹-THC was partially metabolized to more polar compounds; the principal metabolite was shown to be (+)-7-hydroxy-Δ¹-THC. It was concluded that the differences in the psychopharmacological potencies in vivo of the optical isomers of Δ¹-THC are determined within the central nervous system and are not due to gross differences in metabolism or body distribution.     

总体率的估计方法

概述已有的各种率的估计方法以及各方法的优缺点,并对方法的改进提出进一步的设想,以期为率的估计方法的研究和应用提供参考。     

Relative potencies of histamine H1-agonists on guinea-pig tracheal smooth muscle

The relative potencies of a series of histamine H₁-agonists in causing contraction of spirally cut strips of guinea-pig trachea, measured in the presence of cimetidine, indomethacin, methylatropine and propranolol, were similar to those reported on other guinea-pig tissues. However, whereas 2-methylhistamine and N^α,N^α-dimethylhistamine are apparently partial agonists in potentiating the adenosine-stimulated accumulation of cyclic AMP in slices of guinea-pig cerebral cortex, on tracheal spirals they are full agonists producing the same maximum contraction as histamine.     

γ能谱的蒙特卡罗模拟

采用蒙特卡罗方法及通用程序M CNP 4C模拟了核物理实验中的γ能谱测量,阐述了γ能谱模拟思想及方法。结果表明,蒙特卡罗模拟得到的能谱与实验测量得到的能谱具有完全相同的特征。