| In recent years,protein drugs have the advantages of high specificity,clear mechanism of action,remarkable effect and great economic benefits,and become an important growth point of the biomedical industry.However,unlike small molecule drugs,protein drugs not only have large molecular weight and complex structure,but also can be glycosylated or modified after translation,resulting in a high degree of heterogeneity of their structure.Especially,compared to traditional small molecule drugs,pIs of protein drugs have obvious influence on their activities.So,it is very important to establish characterization analysis and quality control of protein drugs.Among them,capillary isoelectric focusing with immobilized pH gradient has been proved to be applicable to the separation of proteins and the determination of isoelectric points,with the characteristics of fast speed,stable baseline,without the addition of carrier ampholytes(CAs)in the sample solution and easy to couple with other instruments.Nevertheless,there are few studies on IPG columns,and their preparation methods,resolution and sensitivity are still to be improved.On account of this reason,this paper aims to develop a new type of IPG column in order to provide higher separation degree and higher resolution capillary columns for capillary electrophoresis apparatus.So,the production process and quality control of protein biological drugs could be better monitored in this new cIEF system.Meanwhile,a platform was built on the basis of these capillary columns with immobilized pH gradient,and an efficient electric micro-separation device was used to realize isoelectric point determination evaluation of protein biological drugs,so as to provide a reliable and stable detection technology for the production process` stability and quality control of protein biological drugs.This dissertation in divided into the following sections:The first chapter is the introduction.This chapter introduces the principle,classification,application and existing problems of isoelectric focusing and capillary isoelectric focusing.In this chapter,the development status and problems in preparation technology of capillary isoelectric focusing column with immobilized pH gradient were analyzed,and synthesis strategies of this column were summarized.The second chapter is the preparation of a porous layer open-tubular capillary column with immobilized pH gradient(PLOT-IPG).The monolithic column prepared in the previous chapter has a large backpressure,which is not conducive to rapid injection of samples.At the same time,during the migration process of sample zones,they will be interacted with the matrix of the monolithic column,which of this result will widen the focused protein peaks.In this chapter,a new strategy for the preparation of IPG-cIEF columns is proposed.Through optimizing the ratio of monomers to each other,monomers to porogens,and porogens to each other,in situ polymerization is adopted to realize the prepartion of PLOT column.Scanning electron microscopy images show that this PLOT column produced by our method has a thicker polymer layer than that of the common PLOT column.Therefore,when this PLOT column was further modified,more CAs could be immobilized on it,as well as maintaining a lower backpressure.In the preparation of this PLOT-IPG column,we attempted to immobilize CAs with different pH ranges on this PLOT column.Meanwhile,in the process of electromigration,we also attempted to prepare the PLOT-IPG column with normal and reversed pH order.Finally,based on the CE platform,the macromolecular proteins and smallmolecular amino acids were successfully separated using this PLOT-IPG column.The third chapter is about the preparation of capillary isoelectric focusing column with monolithic immobilized pH gradient column(M-IPG).According to the characteristic that proteins are easy to precipitate in reversed-phase chromatography,poly(ethylene glycol)diacrylate was used as the crosslinking agent in the preparation of monoliths.Meanwhile,in order to further modify the monolithic column,the functional monomer of glycidyl methacrylate containing the epoxy group was introduced.In the preparation of the monolith,the capillary was partially filled with the polymers.IPG-cIEF columns with a pH range of 3-10 were prepared by accurately controlling the volume of carrier ampholytes(CAs)injected into the monolithic column.In addition,due to the high backpressure of this IPG column,an analytical separation platform that is suitable for this IPG column was established to realize the “on column” detection.After optimizing the experimental conditions,the newly developed IPG columns were used to separate the mixture of 7 model proteins,human serum proteins,and a mixture of human hemoglobin and its glycosylated protein.In chapter 4,the new online cIEF platform was applied to determine the pI value of two kinds of protein drugs.In this chapter,a capillary coated with hydroxypropyl cellulose(HPC capillary)was prepared.And its system suitability,linearity and repeatability were verified.On this basis,the HPC capillary was used to determine the pI values of trastuzumab and etanercept.In the meantime,the M-IPG column was prepared according to the method in chapter 2,and the pI values of trastuzumab and etanercept were measured under the self-built cIEF platform.The results of the comparison show that the newly developed IPG column(M-IPG)can be successfully determine the pI of protein drugs in our online capillary isoelectric focusing platform.In chapter 5,the preparation of a capillary isoelectric focusing column based on silica particles and its IPG column is presented.Silica particles are widely used as filling materials for capillaries due to their high mechanical strength,stable chemical properties and uniform particle size.In this chapter,485 nm silica particles were used to prepare an IPG open tubular capillary with pH range of 4.0-6.5.The effective separation of three model proteins(trypsin inhibitor,bovine serum albumin,and carbonic anhydrase)proved the successful preparation of this IPG column.Next,3 ?m silica particles were selected as matrix for the packed capillary.In the preparation of this packed column,on the one hand,a confined-surface atomic free radical polymerization method was introduced to increase the active reaction sites on the surface of silica particles.On the other hand,free radical reaction was introduced to make CAs bond faster and firmer.Based on this strategy,we prepared a free solution packed cIEF column and an IPG packed cIEF column.Besides,we also improved the online cIEF platform in chapter 2.By introducing the eight-way injection valve and the three-way valve,the whole process of the sample injecting,focusing and field or free-field mobilization can be completed online.In this platform,four model proteins(trypsin inhibitor,pH=4.5;carbonic anhydrase,pH=5.9;myoglobin,pH=6.8,7.3;cytochrome c,pH=10.2)could be successfully separated with the two mode cIEF columns.Compared to open tubular capillary and M-IPG column,it can be found that higher electric field could be applied to the packed capillaries.The sixth chapter is the conclusion and prospect.Firstly,this paper developed four new types of capillary isoelectric focusing columns based on polymer and silica microspheres: 1)polymer-based M-IPG,2)polymer-based PLOT-IPG,3)silicon-based OT-IPG,4)silicon-based Free-cIEF and P-IPG.Meanwhile,we used scanning electron microscope,infrared spectrum and other means to characterize their morphology and structure.Also,we used model protein to characterize them,and used these IPG columns to separate the real samples.Secondly,by introducing the eight-way injection valve,three-way switching valve,four-way connection unit,HPLC pump and other modules,we can successfully complete the continuous online operation of quantitative sample injecting,focusing and mobilization in the focusing process.Finally,on our online cIEF platform,the results of parallel experiments show that our newly developed IPG columns can be used for separation and isoelectric point determination of protein drugs. |
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