Development and validation of a quantitative cell-based bioassay for comparing the pharmacokinetic profiles of two recombinant erythropoietic proteins in serum

An in vitro cell-based bioassay was developed and validated to assess the pharmacokinetic profiles of two novel therapeutic recombinant proteins (EP1 and EP2) with erythropoiesis stimulating properties in Sprague-Dawley rats. While immunoassays are the standard choice for evaluating the pharmacokinetic parameters of drugs, no immunoassay was available for EP2, necessitating the need for a quantitative bioassay capable of measuring both EP1 and EP2 separately so that appropriate comparisons could be made. The bioassay described here utilizes a sub clone of the murine 32D cell line transfected with the gene encoding for the human erythopoietin (HuEPO) receptor. Erythropoietin (EPO), EP1 and EP2 exert their proliferative effect on the cell line by signaling through the HuEPO receptor. The proliferation induced by the erythropoietic proteins was measured by [methyl-(3)H]thymidine incorporation into the cellular DNA. The assay was conducted in 96-well microtiter plates and had relatively high throughput. The Guidelines of the International Conference on Harmonization (ICH) were followed for the validation of the different assay parameters including robustness, linearity, accuracy, precision, limit of quantitation (LOQ) and specificity. The robustness of the bioassay is demonstrated by the lack of an effect of age of the 32D cell culture on the performance of the EP2 bioassay. The bioassay demonstrated good linearity, yielding a coefficient of determination of 0.99 or higher for both EP1 and EP2. The assay showed reproducible dose-response curves for EP1 in the range of 0.039-2.5 ng/mL and for EP2 in the range of 0.125-8 ng/mL. The accuracy estimates ranged between 98% and 108% for EP1 and between 90% and 110% for EP2 in the reproducible range mentioned above. Intermediate precision (within-plate R.S.D.) in the same range was within 26% and 17% for the EP1 and EP2 bioassays, respectively. The validated bioassays for EP1 and EP2 were utilized to quantitatively analyze serum samples from a pharmacokinetic study conducted to compare the profiles of the two compounds in Sprague-Dawley rats.     

A signal-to-noise crossover dose as the point of departure for health risk assessment

Background: The U.S. National Toxicology Program (NTP) cancer bioassay database provides an opportunity to compare both existing and new approaches to determining points of departure (PoDs) for establishing reference doses (RfDs).Objectives: The aims of this study were a) to investigate the risk associated with the traditional PoD used in human health risk assessment [the no observed adverse effect level (NOAEL)]; b) to present a new approach based on the signal-to-noise crossover dose (SNCD); and c) to compare the SNCD and SNCD-based RfD with PoDs and RfDs based on the NOAEL and benchmark dose (BMD) approaches.Methods: The complete NTP database was used as the basis for these analyses, which were performed using the Hill model. We determined NOAELs and estimated corresponding extra risks. Lower 95% confidence bounds on the BMD (BMDLs) corresponding to extra risks of 1%, 5%, and 10% (BMDL₀₁, BMDL₀₅, and BMDL₁₀, respectively) were also estimated. We introduce the SNCD as a new PoD, defined as the dose where the additional risk is equal to the “background noise” (the difference between the upper and lower bounds of the two-sided 90% confidence interval on absolute risk) or a specified fraction thereof.Results: The median risk at the NOAEL was approximately 10%, and the default uncertainty factor (UF = 100) was considered most applicable to the BMDL₁₀. Therefore, we chose a target risk of 1/1,000 (0.1/100) to derive an SNCD-based RfD by linear extrapolation. At the median, this approach provided the same RfD as the BMDL₁₀ divided by the default UF.Conclusions: Under a standard BMD approach, the BMDL₁₀ is considered to be the most appropriate PoD. The SNCD approach, which is based on the lowest dose at which the signal can be reliably detected, warrants further development as a PoD for human health risk assessment.     

Investigation of prion removal/inactivation from chromatographic gel

BACKGROUND AND OBJECTIVES: Concerns about the potential for prions to be retained on chromatography gels during the manufacture of plasma products prompted development of an investigational strategy for detecting infectious prions bound to gels. The objective was to firstly examine methods of implanting gels intracerebrally (IC) in mice, then to examine prion cleaning from a scaled-down version of the DEAE Sepharose column used in a production process to fractionate immunoglobulins and albumin from human plasma. MATERIALS AND METHODS: The study consisted of two parts: (i) the pathophysiological impact by IC inoculation of ground gel beads was compared to whole gel beads; (ii) the feedstreams to two DEAE Sepharose columns were spiked with scrapie ME7. One column was subjected to the protein loading and elution portions of the chromatography cycle. The other column was subjected to the full cycle of protein loading and elution, followed by regeneration with 0.5 m NaCl, 1 m NaOH and solvent/detergent washes. The gels were unpacked and bioassayed by IC implantation in mice to quantify infectivity. RESULTS: IC inoculation of ground gel beads resulted in unacceptably high pathological impact in the mice whereas whole gel bead inoculation resulted in a reduced affect. Accordingly, the whole bead model system was used to assess prion removal/inactivation from chromatography gels at the pre- and postcleaning stage of the chromatography cycle. Infectious prions were detected on the DEAE Sepharose prior to the cleaning step; however, the gel cleaning cycle reduced infectivity by a log reduction factor (LRF) of > or = 2.75, thus reducing infectivity by bioassay to below detectable limits. CONCLUSIONS: A model system for assessment of prion inactivation/removal from chromatography gels has been established. Spiked prion infectivity does bind to DEAE Sepharose gel; however, the cleaning cycle removed infectivity to levels below that detectable by bioassay.     

全蝎蛋白药效组分的生物鉴定法研究

目的 :研究全蝎蛋白药效组分的提取方法,摸索全蝎蛋白药效组分SDS-PAGE的最佳电泳条件,测定全蝎蛋白药效组分电泳图谱条带的大致分子质量,确定其SDS-PAGE的表达特征,从而建立全蝎蛋白药效组分的电泳鉴别法。 方法 :采用水提与盐析法获得全蝎蛋白药效组分,考马斯亮蓝法测定蛋白药效组分的蛋白质含量,SDS-PAGE法和Alpha Ease FC 4.0.0软件测定全蝎蛋白药效组分的蛋白质相对分子质量。 结果 :全蝎蛋白药效组分的蛋白质质量分数为50.37%;得到较好的全蝎蛋白药效组分的10条电泳谱带,相对分子质量分别为94.162 0,66.400 0,40.357 0,27.541 0,22.518 0,19.520 0,12.766 0,11.190 0,9.665 3,8.534 2 KDa。 结论 :这10条谱带是全蝎蛋白药效组分的特征谱带,可用于全蝎蛋白药效组分的鉴别。     

琼脂扩散法生物检定的理论方程式 Ⅰ.杯碟法

本文根据二维扩散方程式并考虑到琼脂相与水相间的平衡关系,推导得杯碟法生物检定的理论方程式。利用放线酮作为试样,在不同瞬间、不同径向距离处取样,测得放线酮在琼脂中的表观扩散系数为0.013cm~2/h(25℃),并计算得浓度随半径和时间变化的理论曲线,与实验结果吻合较好。结合临界浓度和临界时间概念,方程式能对生物检定的实际应用有指导意义。