Fractional Factorial Analysis Of Immunoglobulin G Anti-Diphtheria Serum Fragmentation By Pepsin

Abstract

. The number of diphtheria outbreaks was observed to be quite high in the last 5 years. One way to treat diphtheria at the time of an outbreak is to use an anti-diphtheria serum. The availability of anti-diphtheria serum products worldwide has been reduced for years because manufacturers in several countries have stopped producing. The cessation of production is partly due to a decrease in demand, production failures and regulatory requirements in the manufacture of increasingly stringent biological products from the blood. In addition, the use of certain types of pepsin as a component of production becomes problematic with the issuance of Law No. 34 of 2014 concerning Guarantees of Halal Products. This study aims to analyze the use of pepsin A as a substitute for pepsin B, get parameters that have a significant effect on IgG fragmentation using fractional factorial design, get the optimum parameter value of IgG fragmentation (highest titer value, shortest Kf time and absence of IgG bands in SDS PAGE ) and increase anti-diphtheria serum product antibody titers. Risk analysis was carried out to find out several factors that influence the process of pepsin A fragmentation against IgG anti-diphtheria serum products using fishbone and FMEA methods. Factors belonging to the unacceptable and intolerable classification are purification methods (RPN 60 value), pH termination of the fragmentation process (RPN 100 value), fragmentation temperature (RPN 60 value), number of pepsin (RPN 125 value), and fragmentation time (RPN 125 value ) Changes in the method carried out by replacing the ammonium sulfate fractionation process to diafiltration using a 100kDa filter membrane on the initial plasma showed insignificant results. Fractional factorial analysis was carried out on 4 factors, namely: pH; temperature; amount of pepsin and time. Based on pareto analysis and main effect, the four factors showed a very significant effect on the IgG fragmentation process by pepsin A (p-value <0.05) by using antibody titers as the main response. Characterization of the results of fragmentation is done using the BCA test and SDS PAGE. The conclusion of this study is that pepsin A can replace pepsin B, a factor that has a significant effect is the amount of pepsin, time, temperature, and pH of the incubation termination; optimum value (highest titer, shortest Kf time and no IgG band) IgG fragmentation process by pepsin A is the amount of pepsin 1163mg / L, temperature 37ºC, time 135 minutes, and pH 5.95; and increasing antibody titer at IgG fragmentation stage to 2.67 times than the previous method using pepsin B. This study aims to analyze the use of pepsin A as a substitute for pepsin B, obtain parameters that have a significant effect on fragmentation of IgG by using fractional factorial design, obtain optimum parameters for IgG fragmentation (highest titer value, shortest Kf time and absence of IgG bands in SDS PAGE ) and increase the antibody titer of anti diphtheria serum products. Optimized parameters in the digestion process are the amount of pepsin A, incubation temperature, pH of digestion process termination, and digestion time. The response used in assessing a significant factor (P-value <0.05) is a quantitative parameter of the IgG fragmentation process, which is the antibody titer value. Characterization of IgG fragmentation results used bichinconic acid (BCA) test to determine the total protein content produced and SDS-PAGE profile to see the character of IgG fragmentation into antibody fragment F (ab) 2. acceptable (score 13-36); unacceptable (score 37-64); and intolerable (score 65-125). RPN scores that are in the unacceptable and intolerable matrices are analyzed using factorial fractional designs to determine the influence of these factors and the interactions between them.