Pepsin digestion optimisation

Preliminary optimisation of pepsin digestion was done using a model IgG substrate—highly pure IgG sample (eIgG) isolated from HHP by protein A based affinity chromatography. Generally, substrate aliquots (2 mg mL^-1) were pH adjusted using 0.4 M HCl and tempered according to planned experiments. Pepsin solution (5 mg mL^-1) in 0.15 M NaCl (saline) was added at different enzyme to IgG ratios (as specified in "Results" section) while gently mixing (350 rpm). The final volume of reaction mixture, prepared in saline, was 1 mL. Digestion was terminated at timed intervals with a 0.4 M NaOH solution until slightly acidic to neutral pH was achieved.

Initially, preliminary screening of four factors—pepsin to IgG ratio (1:300 or 10:300, w/w), duration of incubation (45, 60 or 90 min), pH of the reaction mixture (3.2 or 3.5), and temperature (20, 37 or 56 °C), was performed according to a general factorial experimental plan that consisted of a total number of 40 runs, four of which were assessed in duplicates. Since it was not possible to execute all runs simultaneously, their order was randomised to avoid systemic errors. We used a regression function model covering linear contribution of each factor, but also non-linear for selected experimental area.

In the second experiment duration of enzymatic reaction (X1) and pepsin to IgG ratio (X2), each at two levels (marked with minus (-) for the low and plus (+) for the high level), were further selected to study their impact on the digestion outcome. Their values were 1 or 3 h for X1 and 1:300 (w/w) or 10:300 (w/w) for X2. The full factorial design was employed resulting in 4 experimental runs, each performed in triplicate (2² × 3). The main effect of each factor was calculated according to Eq (1),

where index X represents factors 1 or 2, n is the total number of experimental runs (4) and Y¯j are F(ab')₂ yields obtained at - and + level of each factor. The significance of the given factors was determined by means of ANOVA using Statistica 13.4 software.

Fine-tuning of enzyme quantity with respect to IgG was tested by preparing reaction mixtures with a wide range of discrete pepsin concentrations (from 1:300 to 10:300, w/w) that were tested under conditions giving highest yield, according to results from previous experimental sets.

All subsequent experiments were performed using real process IgG substrate—IgG fraction from the optimised caprylic acid fractionation step, and examining one variable at a time. The common approach involved acidification to pH 3.2 and addition of pepsin solution in saline until desired enzyme to IgG ratio in a 1.5-fold diluted reaction mixture (V = 1 mL) was reached. Incubation was performed at 37 °C for 1.5 h. When optimal conditions were set, the procedure was scaled up 20-fold.

Samples from each experimental set were analysed by SDS-PAGE. The quantity of F(ab')₂ fragments from reaction mixtures in which complete IgG cleavage occurred was measured by ELISA (detailed description is given in "ELISA assays for IgG and F(ab')₂ content determination" section) and used for yield estimation [%]. For each run mean value and 95% confidence interval (CI) were calculated.