Latest ArticlesMembrane proteins are the main undertakers of biofilm function, and also the most important target group for innovative drug discovery and research. About 60% of drugs targets are membrane proteins. Due to the obvious aggregation and denaturation tendency of membrane proteins in aqueous solution, it is difficult to simulate the membrane like environment to maintain the correct conformation of membrane proteins in vitro, which results in the slower-growing research on the structure and function of membrane proteins and related ligand drugs than that of water-soluble proteins. Membrane protein stabilization technology is the premise of establishing high specificity, high sensitivity and high throughput drug screening methods for membrane protein ligands, which is of great significance. In this paper, some techniques for stable separation and purification of membrane proteins are reviewed, including detergents, artificial membranes, polymers, lentiviral particles and so on, as well as their specific applications in drug screening.
Oligonucleotides have attracted the widespread attention in disease diagnosis and gene therapy. At present, the nucleic acid drugs are at the forefront of biomedical and pharmaceutical research. The bioanalysis of therapeutic oligonucleotides has been slow, however, due to the requirements for pharmacokinetic/toxicokinetic and pharmacodynamic studies in pharmaceutical development. Conventionally, the hybridization-enzyme linked immunosorbent assay (hybridization-ELISA) is widely used in the bioanalysis of therapeutic oligonucleotides. Recentlly, many technologies such as real-time quantitative PCR (qPCR) and high performance liquid chromatography (HPLC)-based technologies have also showed a broad application prospects in the bioanalysis of therapeutic oligonucleotides. However, each technology has its own advantages and limitations. This review summarizes the currently used techniques in the bioanalysis of oligonucleotide therapeutics and reviews the challenges of regulated bioanalysis.
The paper aims to establish the method to determine the monosaccharide composition and monosaccharide ratio in propylene glycol alginate sodium sulphate (PSS). Samples were hydrolyzed with trifluoroacetic acid, neutralized with sodium hydroxide solution after the reaction conditions for sample pretreatment were optimized via orthogonal analysis. A high performance anion exchange chromatograghy (HPAEC) coupled with pulsed amperometric detector (PAD) was performed on a CarboPac®PA20, using 200 mmol·L-1 sodium hydroxide solution and 1 mol·L-1 sodium acetate solution as mobile phase. The established HPAEC-PAD method was validated by testing the linear relationship, precision and accuracy, and showed exclusive, sensitive, rapid and wide use. The monosaccharide composition of PSS from different manufacture can be accurately determined with great significance for the structural identification of PSS.
Deamidation is one of the most common degradation impurities in protein and peptide drugs. The deamidation of glutamine and asparagine in the protein sequence can lead to changes in the chemical and biological properties of the protein. However, the rutine trypsin-based pretreatment process can significantly increase the artificial deamidation impurities during the digestion process, resulting in high determination level. In this study, after optimizing the conditions of Glu-C enzymatic hydrolysis, we obtained the best enzymatic conditions under acidic condition and the artificial deamidation impurities significantly reduced in digestion process, identified the deamidation site (N48). Through the methodological investigation and comparison of the measurement results of different methods, the specificity, reproducibility and accuracy of the method are verified. The method established in this research has laid a solid foundation for the accurate determination of deamidation impurities in cobratide and its similar protein peptide biochemical drugs.
In recent years, the biopharmaceutical industry has grown rapidly, and the market size of monoclonal antibody drugs has increased significantly. Accurate structural characterization and quality control are the supporting technologies for the development of monoclonal antibody drugs. As a significant post-translational modification of antibody drugs, glycosylation has an important influence on its efficacy, stability, and immunogenicity. The existing literature usually uses liquid chromatography-mass spectrometry to perform major glycosylation modifications of monoclonal antibody drugs. Characterization, there are few studies on low-abundance glycosylation, but the characterization and control of low-abundance glycosylation cannot be ignored. In this study, we have established a qualitative and quantitative analysis technology for N-glycans based on RapiFluor-MS reagent-labeled monoclonal antibody drugs. This method has a short sample processing time and high sensitivity. It can not only characterize the main glycoforms of three monoclonal antibody drugs (adalimumab, bevacizumab, and trastuzumab) but also can quantify low-abundance N-glycans. The results of the study showed that the main glycoforms specified in the Pharmacopoeia could be detected in different batches of monoclonal antibody drugs, but the content of N-glycans in different batches of samples is not identical. After that, we analyzed the N-glycans connection sites and glycoforms at the intact glycopeptide level, further enriching the N-glycans structure information of the monoclonal antibody. The qualitative and quantitative analysis technology of N-glycans based on RapiFluor-MS reagent-labeled monoclonal antibody drugs can realize the in-depth characterization and control of glycosylation modification of monoclonal antibody drugs.
IMM0306 is a recombinant human signal regulatory protein α-anti-CD20 mouse chimeric antibody fusion protein, intended to treat refractory or recurrent CD20 positive B-cell non-Hodgkin lymphoma (B-NHL) in clinical. In this study, an ELISA method was established to evaluate the pharmacokinetics of IMM0306 in human serum. The experiment was approved by the Ethics Committee of the Cancer Hospital of the Chinese Academy of Medical Sciences (No. CTR20192612). Recombinant human CD47 protein was coated with the plate overnight, blocking the plate with 5% skin milk for 2 h. After washing, 100 μL per well standard and unknown samples were added, and incubated for 1.5 h. After washing, the detection antibody Anti-IMM0306-Biotin was added and incubated for 1 h, and then HRP-labeled streptavidin was added for 1 h, and the color was detected. The optimal concentration of coating reagent was 2 μg·mL-1 by ELISA method, and the optimal dilution of anti-IMM0306-biotin and SA-HRP were 1:500 and 1:5 000, respectively. The lower limit of quantitation was 4 ng·mL-1, and the standard curve range was 4-100 ng·mL-1. The verification results of the method meets the corresponding acceptance criteria, and can be used in IMM0306 clinical pharmacokinetic studies.
FGF21-164 is a fusion protein obtained by structural modification and coupling of endogenous FGF21. It is a candidate drug used in the treatment of glucose and lipid metabolic disorders caused by obesity. In this study, the candidate peptide mass spectrometry information of the protein hydrolyzed by trypsin was predicted by Skyline software and verified by high resolution mass spectrometry. The specific surrogate peptide (YLYTDDAQQTEAHLEIR) with the best mass response was selected after optimizing ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Under ESI positive ion mode, the parent ion m/z 689.3 with 3 charge and the product ion m/z 738.4 with single charge can be monitored. After dilution by PBS, the serum samples were denatured under 60℃ and alkylated to reduce the matrix effect, then incubated with trypsin at 37℃ for 2 h, to obtain the surrogate peptide. The chromatographic separation was carried out on an EclipsePlus C18 column (2.1 mm×50 mm, 1.8 μm) using aqueous solution containing 0.1% formic acid (phase A) and acetonitrile solution containing 0.1% formic acid (phase B). Finally, the concentration of FGF21-164 fusion protein in mouse serum was quantitatively analyzed by external standard method by monitoring the above ion pairs using triple quadrupole mass spectrometer. This method showed a good linearity in the range of 2.50-500 μg·mL-1 (r=0.998 8), and was successfully applied to the pharmacokinetic study of FGF21-164 fusion protein in mice. This experiment was approved by the Experimental Animal Ethics Committee of Shanghai Institute of Materia Medica, Chinese Academy of Sciences (batch number: 20180004040450). Compared with the endogenous FGF21, the t1/2 of FGF21-164 fusion protein was prolonged from 0.5 h to 2.6 h, which is expected to prolong the therapeutic efficacy of this protein.
GeXIVA[1,2] is a new type of conotoxin recently discovered in the transcriptome of Conus generalis and it is expected to be used clinically as a new type of analgesic. This study established and verified a sandwich enzyme-linked immunosorbent assay method for the marine drug GeXIVA[1,2] in the plasma of rats and Beagle dogs. The mouse monoclonal antibody 4B2 and biotin-labeled rabbit polyclonal antibody 2# were developed. The checkerboard method was used to optimize the antibody pairing concentration, minimum dilution ratio, incubation temperature, and incubation time to establish an antibody sandwich ELISA detection method. Verify the established testing methods. The established ELISA method has a quantitative range of 1.25-80 ng·mL-1 in rat and Beagle plasma. The precision, accuracy, selectivity, specificity, stability, dilution linearity, and hook effect all meet the requirements for biological sample analysis. All the procedures for the animal experiments were approved by the Animal Ethics Committee of the Institute (Permit Number: IACUC-DWZX-2020-698). This method can support the preclinical pharmacokinetic study of the marine drug GeXIVA.
Compared with human insulin, insulin lispro shows a faster hypoglycemic effect and a higher peak plasma concentration, which can better control postprandial hyperglycemia. In this study, we used a solid phase extraction pretreatment method and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify insulin lispro in rat plasma. Bovine insulin was used as an internal standard. Plasma samples were separated on an ACQUITY UPLC Peptide CSH C18 column (2.1 mm × 50 mm, 1.7 μm) after solid phase extraction. Positive electrospray ionization was performed using multiple reaction monitoring (MRM) with transitions of m/z 1 162.5→217.2 for insulin lispro and m/z 1 157.5→136.0 for insulin bovine (internal standard). The method validation results showed that the linear range was 0.1 ng·mL-1-100 ng·mL-1; intra- and inter-day accuracy and precision met the acceptance criteria for biological sample analysis. The recovery of insulin lispro ranged from 63.1% to 68.1%. The method was applied in a pharmacokinetic study of insulin lispro following a single-dose subcutaneous administration to rats. Animal experiments were approved by the Experimental Animal Ethics Committee of Shanghai Institute of Materia Medica, Chinese Academy of Sciences.
A fast and sensitive UPLC-MS/MS method was established for the direct quantification of insulin aspart in human plasma. The plasma samples were extracted by solid phase extraction (SPE), an ESI ion source was used and operated in the positive ion mode with multiple reaction monitoring (MRM). Bovine insulin was chosen as internal standard and the chromatographic separation of insulin aspart was performed on Waters ACQUITY UPLC CSH C18 column (50 mm×2.1 mm, 1.7 μm). A mixture of acetic acid aqueous solution and acetonitrile with acetic acid at a flow rate of 0.6 mL·min-1 in gradient elution mode was employed as mobile phase. We found that the method was validated over the range of 0.200-10.0 ng·mL-1 for insulin aspart and showed excellent linearity. The intra-and inter-assay accuracy and precision were below 14.5% and the recovery was 36.7%-41.7% over the three concentration levels evaluated. The UPLC-MS/MS method was selective, accurate, sensitive and robust, and the method was successfully applied in supporting the pharmacokinetic research of two insulin aspart injections (Test Product and NovoRapid?) in heathy male subjects. This clinical trial was approved according to the Ethics Committee of West China Hospital, Sichuan University (2017 Clinical Trial (Western Medicine) Approval (148)).
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