Analysis Of Protein Therapeutics By Non-Reducing Sodium Dodecyl Sulfate-Capillary Gel Electrophoresis

Objective:The analysis of protein-based therapeutics is an important part of drug research,which is of great significance to the development of new drugs.Among them,nonreducing sodium dodecyl sulfate capillary gel electrophoresis(CE-SDS)is a powerful tool to analyze the size isomers of protein therapeutics.On the one hand,this paper aims to study the influence of alkylation reagents such as iodoacetamide(IAM)and N-ethyl maleimide(NEM)on the non-reducing analysis results of recombinant protein X,and to explore the formation mechanism of specific related impurities.At the same time,a variety of polypeptides and monoclonal antibodies with different molecular weights are studied to expand the research objects,so as to enrich the industry’s understanding of the analysis and characterization of related products as well as the quality control aspects and matters need to pay attention.On the other hand,the use of CE-SDS method for analysis of site specificity glycation of protein X,as to study the effect of reducing sugar that used in cell culture(such as glucose and galactose and so on).We hope to provide research cases and technical support for the quality control in the production process of biological drugs by using relevant sample and analysis technology.Methods:In the first part,we detected abnormal shoulder peak in the results of non-reducing sodium dodecyl sulphate-capillary gel electrophoresis(CE-SDS),and studied the presence or absence of alkylation reagents like iodoacetamide(IAM)and N-ethyl maleimide(NEM),sample incubation time,sample incubation temperature and the presence or absence of SDS and so on.Then,the degraded samples were analyzed by reverse phase-high performance liquid chromatography(RP-HPLC)from the perspective of hydrophilicity and hydrophobicity,and the molecular weight of the samples was analyzed by MALDI-TOF-MS.The molecular weight of the degraded protein was further analyzed and characterized by liquid chromatography coupled with mass spectrometry(LC-MS)and the degradation sites were analyzed by peptide mapping.The advanced structure of the samples before and after degradation was analyzed by farultraviolet circular dichroism(Far-UV CD)and intrinsic fluorescence spectroscopy.In addition,non-reducing CE-SDS and RP-HPLC techniques were mainly used to analyze a variety of polypeptides and monoclonal antibodies under the influence of IAM or NEM.In the second part of the work,non-reducing sodium dodecyl sulfate capillary gel electrophoresis(CE-SDS)and liquid chromatography-mass spectrometry(LC-MS)were used to analyze the degradation of site-specific glycation samples.Results:The results of the first part showed that iodoacetamide(IAM)could react with Lys and Ser residues in the recombinant protein X molecule through nucleophilic substitution reaction,and N-ethyl maleimide(NEM)could react with Lys through nucleophilic addition reaction,thus increasing the molecular weight and decreasing the electrophoretic mobility.The content of shoulder peak impurity was significantly dependent on sample preparation conditions,such as incubation temperature,incubation time,p H and the present or absent of SDS.In addition,the analysis of polypeptides and monoclonal antibodies of different molecular weight showed that they would also be affected by IAM and NEM,which significantly change their hydrophilicity and hydrophobicity.The results of the second part showed that when the recombinant protein X was incubated in a system containing reducing sugars such as glucose and galactose for a certain period of time,the shoulder peak was formed in the non-reducing CE-SDS.The degradation was confirmed by liquid/mass spectrometry and enzyme-digested peptide mapping techniques.The specific reaction between the lysine(Lys)residues in the recombinant protein X molecule and the reducing sugar was the root cause.Conclusions:The results of the first part of the study indicated that although IAM is widely used as an alkylation agent in conventional non-reducing CE-SDS analysis of monoclonal antibodies and other proteins,alkylation of lysine(Lys)and serine(Ser)residues in protein molecules with IAM may lead to additional impurity peaks.Similarly,when NEM is used as an alkylation agent,lysine(Lys)residues could also undergo potential degradation reactions to form impurities.This kind of degradation was also found in a variety of protein samples,including peptides,monoclonal antibodies,fusion proteins,and antibody-drug conjugates.Therefore,the influence of sample preparation process on the analysis results must be evaluated before non-reducing CE-SDS analysis,and the use of alkylation agents like IAM and NEM should be paid careful attention.The results of the second part suggested that in the cell culture and production process of protein-based products,the use of reducing sugars will inevitably cause some degradation of the products,so it is necessary to fully purify the products and use relevant technologies to fully analyze and test the quality of the products.Among them,non-reducing CE-SDS combines with a variety of other technologies,such as reverse phase chromatography and liquid mass spectrometry,can play a certain role.