Analysis Of Protein Aggregates In Nano Size At Single-Particle Level By High-Sensitivity Flow Cytometry

Protein drugs are an important part of bio-pharmaceutical,including enzyme,interferon,recombinant cytokine,monoclonal antibody,recombinant antibody,recombinant vaccine,etc.Compared with traditional small-molecule chemical drugs,protein drugs have the advantages of high activity,high specificity and low toxicity.Currently,it is widely used in the treatment of malignant tumors,cardiovascular diseases,viral infections and other major diseases.The global market of protein drugs has grown to more than $140 billion and continues to grow steadily.Therefore,the development of protein drugs is of great significance to medicine,society and economy.However,protein drugs are flawed,especially if they are far less stable than small-molecule chemical drugs.Protein drugs are susceptible to changes in surface residue oxidation,disulfide bond rupture,peptide chain solution folding and conformation change due to environmental stimuli or their own stability factors in the process of production,purification,transportation and storage.These changes may eventually lead to the formation of protein aggregates.Protein aggregation can reduce the therapeutic effect of protein drugs.More seriously,protein aggregates can induce an immune response in patients with intravenous injection into the patient,and even cause anaphylactic shock,which seriously endangers the patients’ lives.Therefore,it is very important to detect and study the protein aggregation in protein drugs.In recent 40 years,with the continuous development of protein aggregates detecting techniques and methods,greatly promoted to improve the stability of the protein modification and structure improvement,exploration of inhibitor of protein aggregation,study of protein aggregation mechanism,and improvement of the quality control,etc.According to the testing results of electron microscopy in the literature,the size of protein aggregates are mainly distributed in 20-300 nm,therefore,it is very important to establish the detection method of nano-scale protein aggregates for protein drug quality control and aggragation mechanism.This work will use the high-sensitivity flow cytometer(HSFCM)developed by our laboratory,in which the side scattering detection has high sensitivity as beautiful as the resolution of electron microscopy to establish the single-particle level detection method of nanoscale for protein aggregates,and to provide advanced and fast detecting platform for the quality control to the research of therapeutic protein.We firstly selected bovine serum albumin(BSA)as the protein model.By heating stimulation to form protein aggragates and testing,we found that HSFCM could effectively detect the protein aggregates of nanoscale.It had been proved that protein aggregates is mainly distributed in the nanometer scale,and the size of aggregates is just in the range of side scattering detection of HSFCM.After examining the dilution effect on detecting,we use nanoparticles with known concentration and particle size as the taste standards to establish a single-particle level concentration and size characterization methods for protein aggregates of nanoscale.Then,we optimized the preparation conditions of BSA aggregation.With BSA as the object,the effects of the type and concentration of metal salts,sugars,polyols,amino acids and cyclodextrins on protein aggregation were investigated.A new method of detection,optimization and screening was provided in order to study the effect of excipients on protein aggregation.Finally,we applied this method to optimize and screen the inhibition effect of excipients for protein aggregation under oxidation and reduction conditions.