Determination of Immunological Characterization and Verification of Recombinant Streptococcal Protein G | Chapter 2 | New Visions in Biological Science Vol.10

The purpose of this study was to identify the immunological characterisation and verification of recombinant streptococcal protein G (rSPG), which is essential since it has a higher specificity for immunoglobulin (Ig) than staphylococcal protein A. However, the high expense of commercial recombinant (r) SPG has stymied further study into rSPG's applicability. The immunological characterization of purified rSPG was compared to commercial SPG using western blot analysis in this study, which was accomplished through effective high cell density fermentation of genetically modified Escherichia coli in earlier work. Purified rSPG had a significantly better IgG-binding capacity than commercial SPG, according to the findings of the current investigation. Purified rSPG cross-linked with Q Sepharose® Fast Flow also had a high affinity for IgG in mouse serum. The purified rSPG was detected using Nanoflow Liquid Chromatography-Mass Spectrometry (MS)/MS spectrum in order to achieve relatively pure and accurate rSPG. The two peptide segments of purified rSPG were found to belong to the Streptococcus sp. GX7805 protein G listed in the National Center for Biotechnology Information database. The approach reported in this paper provides a novel practical method for verifying rSPG in a relatively pure form, which can be used to purify IgG or perform immunolabeling operations.

 

Author(s) Details:

Hucheng Zhang,
Department of Biology Engineering, Beijing Polytechnic, Beijing-100176, P.R., China.


Weifeng Liang,
Beijing Buchang Pharm Biopharmaceutical R&D Center, Beijing-100080, P.R., China.


Haitao Fan,
Department of Biology Engineering, Beijing Polytechnic, Beijing-100176, P.R., China.

 

Jun Yang,
Department of Biology Engineering, Beijing Polytechnic, Beijing-100176, P.R., China.


Guowei Yang,
Department of Biology Engineering, Beijing Polytechnic, Beijing-100176, P.R., China.


Xiaojie Wang,
Department of Biology Engineering, Beijing Polytechnic, Beijing-100176, P.R., China.

Liang Chen,
Department of Biology Engineering, Beijing Polytechnic, Beijing-100176, P.R., China.


Tao Liang,
Department of Life Sciences, Tianjin Normal University, Tianjin-300384, P.R. ,China.


Please see the link here: https://stm.bookpi.org/NVBS-V10/article/view/5981

Development of Validated LC-MS/MS Method for Pharmacokinetic and Bioequivalence Studies of Azelastine in Korean Healthy Volunteers | Chapter 5 | Current Topics in Medicine and Medical Research Vol. 1

The pharmacokinetic (PK) and bioequivalence (BE) studies of two formulations of azelastine (AZ) was carried out in 18 healthy male Korean volunteers according to a randomized crossover-design and were performed by a new validated liquid chromatography coupled to tandem mass spectrometry (LCMS/MS) method for quantitative analysis of plasma AZ. Subjects were given single dose of 2 tablets of 1 mg AZ of each formulation with 240 ml of water to subjects on 2 treatment days separated by one week washout period. After dosing, serial blood samples were collected for a period of 96 hours. AZ and the clomipramine (IS) were separated using a mobile phase of acetonitrile-5 mM NH4AC (8：2, v/v, pH=6.4) with flow rate of 0.25 ml/min over Luna C18 column. Analysis required 0.2 ml of plasma and involved a solid extraction with an Oasis HLB cartridge by on-line clean-up extraction automation system and then directly injected into HPLC with an API 3000 MS system by multiple reactions monitoring (MRM) mode. Several PK parameters (including AUC0-t, AUC0-infinity, Cmax, Tmax, T1/2 and Ke) were determined from the plasma concentration of AZ of both formulations. The AUC and Cmax was tested for BE after log-transformation of the data. The ionization was optimized using ESI (+) and selectivity was achieved by MS-MS analysis, m/z 112.0 and m/z 86.1 for AZ and IS, respectively. The calibration curves with r2＞0.999 were linear over a working range of 20∼2000 pg/ml. No endogenous compounds were found to interfere with the analysis. The inter- and intra-day accuracy was in the ranges of 89.9∼109.3% and precision of inter- and intra-day expressed as relative standard deviation were 2.73∼8.79 %. No significant difference was found based on ANOVA; 90% confidance intervals (91.77∼101.94% for AUC0-t; 90.44∼ 106.27% for AUC0-infinity; 92.22∼108.57% for Cmax) for the test and reference drugs were found within FDA guideline of 80∼125%. Based on these statistical considerations, it was concluded that test drug was bioequivalent to the reference drug and therefore, may be interchangeably in the management of allergic or bronchial asthma and prophylaxis of mucositis due to cancer chemoradiotherapy. This validated method was found to be good performance on PK and BE studies for AZ. (Cancer Prev Res 12, 84-91, 2007). 

Author (s)  Details

Professor Ju-Seop Kang,
Department of Pharmacology and Clinical Pharmacology Laboratory, College of Medicine, Hanyang University, Seoul 133-791, Korea.

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