The similarity between a proposed biosimilar product and the reference product can be affected by many factors. This study is designed to examine whether any subtle difference in the distribution of the charge variants of an Avastin biosimilar can affect its in vitro potency and in vivo PK. Here, the acidic, basic and main peak fractions of a biosimilar product were isolated using high-performance cation-exchange chromatography and were subjected to various studies to compare their in vitro properties and in vivo PK profile. A serial of analytical methods, including size exclusion chromatography (SEC), imaged capillary isoelectric focusing (icIEF) capillary zone electrophoresis (CZE) and cation-exchange chromatography (CEX-HPLC) were also used to characterize the isolated charge variants. The kinetics constant was measured using a Biacore X100 system. The study indicates the biosimilar product has a high similarity with avastin in physicochemical properties. The potency in vitro and PK profile in rat of charge variants and biosimilar product are consistent with avastin.

A stable HMG-CoA reductase (HMGR) reaction in vitro was developed by a sensitive, selective and precise liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The optimized enzyme reaction condition contained 1.5 μg of HMGR, 20 nM of NADPH with 50 min of reaction time. The method was validated by several intra- and inter-day assays. The production transitions of m/z 147.0/59.1 and m/z 154.0/59.1 were used to detect and quantify mevalonolactone (MVAL) and MVAL-D7, respectively. The accuracy and precision of the method were evaluated over the concentration range of 0.005-1.000 μg/mL for MVAL and 0.010-0.500 μg/mL for lovastatin acid in three validation batch runs. The lower limit of quantitation was found to be 0.005 μg/mL for MVAL and 0.010 μg/mL for lovastatin acid. Intra-day and inter-day precision ranged from 0.95% to 2.39% and 2.26% to 3.38% for MVAL, 1.46% to 3.89% and 0.57% to 5.10% for lovastatin acid, respectively. The results showed that the active ingredients in Xuezhikang capsules were 12.2 and 14.5 mg/g, respectively. This assay method could be successfully applied to the quality control study of Xuezhikang capsule for the first time.