Mass Spectrometry Characterization Of Insulin Analogues

Diabetes has become one of the major chronic diseases threatening human life and health in the 21 st century,and it has seriously affected human health and quality of life.Currently,insulin therapy has been shown an important means to control hyperglycemia.Among them,insulin analogues are significantly better than animal or recombinant human insulin in simulating normal physiological insulin secretion and reducing the risk of hypoglycemia due to their stable structure and strong binding ability to receptors.However,insulin analogues are recombinant genetically engineered products.Compared with traditional small-molecule drugs,they have large molecular weights,complex structures,and large batch-to-batch differences.A series of analytical techniques are required to characterize the physical and chemical properties of insulin analogues.High resolution mass spectrometry has the advantages of high quality accuracy and wide dynamic range.The primary structure information measured by mass spectrometry can provide qualitative and quantitative results of protein molecular weight,primary amino acid sequence and most post-translational modifications(PTMs).In addition,mass spectrometry is often used to study and characterize higher-order structures of proteins in structural biology.This study selected insulin aspart and insulin degludec as the research objects,and discussed the application of mass spectrometry in the quality control of insulin analogues,and conducts in-depth research from the aspects of primary structure,higher-order structure,and determination of chiral impurities.The main contents of this thesis are as follows:1.The primary amino acid sequence of protein drugs determines its high-level structure and biological functions.The "top-down" high-resolution tandem mass spectrometry method was used for the first time to comprehensively analyze the amino acid sequence and the non-protein modification sequence of insulin aspart and insulin degludec.In this study,we optimized the reduction conditions of insulin degludec and the collision conditions of tandem mass spectrometry.The results indicated that insulin degludec can be fully reduced to two independent peptide chains when 50 mmol/L tris(2-carboxyethyl)phosphine and 6 mol/L guanidine hydrochloride are used in combination for 40 min at 45 °C.The reduction products were separated by Accucore C18 column and analyzed by orbitrap high resolution tandem mass spectrometry.When the higher energy collision induced dissociation is optimized,the most abundant fragment information can be obtained,which not only meets the basic requirements of 100 % peptide coverage in biotechnology drug evaluation,but also provides more comprehensive sequence analysis for the problem samples.This method avoids the costly enzymatic hydrolysis step in the traditional sequence analysis,limits the risk of introducing artifactual modification to obtain better sequence information,thereby,greatly saves the cost,and significantly improves the work efficiency,and provides a new solution for sequence analysis of insulin analogs.2.Disulfide bonds belong to the primary structure of proteins,and play a significant role in stabilizing the spatial structure of protein drugs and maintaining their activity.Insulin analogues will undergo disulfide bond mismatches during the synthesis process,so the characterization of disulfide bonds is very important for the quality control of insulin analogues.Insulin contains disulfide bonds in the chain,which will significantly inhibit the mass fragmentation of the peptide chain,especially the fragmentation of the peptide chain loop formed by the disulfide bonds in the chain.Conventional CID and HCD are incapable of this.In this thesis,we use source collision-induced dissociation(SID)technique,which can effectively improve the fragmentation of peptide chains in the loop to obtain key mass spectrometry fragments that prove the disulfide bond connection.For the first time,enzymatic digestion-mass spectrometry and SID-mass spectrometry were used to analyze the disulfide bonds of insulin aspart,which proved the existence of three disulfide bonds of insulin aspart: intra-chain disulfide bond(A:C6=A:C11),inter-chain disulfide bond(A:C7=B:C7)and inter-chain disulfide bond(A:C20=B:C19).This method has the advantages of fast analysis speed and simple operation process.The method of disulfide bond analysis established in this experiment can be extended to disulfide bond analysis of other insulin analogens.3.Hydrogen-deuterium exchange(HDX)is a well-known technology for studying protein higher order structure(HOS),which is based on the labile hydrogen atoms in proteins can be exchanged with deuterium in solution.Avoiding the exchange of hydrogen and deuterium atoms,improving the accuracy and reproducibility of HDX experiments,and rapid detection are the bottlenecks of the widespread application of HDX-MS technology.Therefore,we developed a matrix assisted laser description ionization time-of-flight mass spectrometry(MALDI-TOF MS)based on solid-phase microextraction sample target and HDX to study the insulin analog HOS method,which effectively solved the above problems.In addition,the conformational differences of insulin analogues HOS were compared in a high-throughput manner to achieve rapid and batch analysis of insulin analogues,and the effects of pharmaceutical excipients and storage conditions on the dynamics and conformation of insulin analogues were studied.The results showed that phenol has a positive effect on the maintenance of hexamers in insulin aspart injection and insulin degludec injection;excessive storage temperature will affect the HOS of insulin in the injection,resulting in changes in its efficacy,especially for the effect of rapid-acting insulin,such as insulin aspart.4.Peptide drugs are prone to form racemic peptides during the synthesis process.Racemic peptides are peptide chains formed by one or more amino acid residues with unintended chiral configuration in the peptide chain.The presence of racemic peptides may cause significant differences in stability and biological activity.Insulin analogues are synthesized using genetic engineering techniques.First,biosynthetic proinsulin,and then cleave C peptide with protease to obtain insulin.Due to the restriction of the restriction site and the need for subsequent chemical modification,artificial introduction of amino acids is required,which will lead to the production of racemic peptides.We established a liquid chromatography-high resolution mass spectrometry(LC-HRMS)method for detecting chiral amino acid impurities,and optimized the reaction conditions for hydrolysis of racemic peptides to amino acids and chiral amino acids and derivatization reagents,which significantly improved the detection limit and linear range of L/D-threonine and glutamic acid.The limit of quantification of threonine is 2.2 nmol/L,and the limit of quantification of glutamic acid is 2.5 nmol/L.Compared with the existing tandem mass spectrometry method for the detection of chiral amino acid impurities,this method is basically the same for the detection of L and D type standard curves and quantification limits of the same amino acid.Therefore,the chromatographic peak area normalization method can be used to directly obtain the relative content ratio of the L/D-amino acid based on the LC-HRMS chromatographic peak area ratio of the L/D-amino acid,thereby saving the work of preparing a standard curve.