Fig 1.
Preliminary determination of optimal caprylic acid concentration for precipitation step of the purification protocol.
Results (total protein concentration, IgG concentration and purity) are given as mean +/- 95% CI (n = 3).
Fig 2.
The assessment of purification steps by size-exclusion chromatography.
Analysis was performed on TSK-Gel G3000SWXL column (7.8 × 300 mm) with 0.1 M phosphate-sulphate running buffer, pH 6.6, at a flow rate of 0.5 mL min-1. Heat-treated plasma (A). IgG fraction—supernatant from 2% caprylic acid precipitation before (crude IgG; B) and after diafiltration using a 100 kDa membrane (pure IgG; C). F(ab')2 fraction produced by pepsin digestion of IgG preparation before (crude F(ab')2; D) and after diafiltration using a 50 kDa membrane (pure F(ab')2; E). Ultrapure F(ab')2 preparation—flow-through fraction from anion-exchange chromatography performed at pH 5.0 (F). Detection: UV at 280 nm.
Fig 3.
SDS-PAGE analysis of representative samples from purification process (A) and pepsin preparation (B). Lane 1 and 2, hyperimmune plasma pools; lane 3, molecular weight standards; lane 4, IgG fraction obtained by caprylic acid precipitation (crude IgG); lane 5, IgG fraction after diafiltration (pure IgG); lane 6, F(ab')2 fraction produced by pepsin digestion of IgG preparation (crude F(ab')2); lane 7, F(ab')2 preparation after diafiltration (pure F(ab')2); lanes 8 and 9, F(ab')2 preparation polished using CIM QA chromatography (ultrapure F(ab')2). The analysis was done on 4–12% gel under non-reducing conditions. Staining was performed with CBB R250.
Table 1.
Purities and yields of the intermediates and the final product obtained by developed downstream processing protocol.
Fig 4.
Verification of pepsin removal by the final polishing procedure.
SEC analysis of pepsin sample on TSK-Gel G3000SWXL column (7.8 × 300 mm) with 0.1 M phosphate-sulphate running buffer, pH 6.6, at a flow rate of 0.5 mL min-1 prior (A) and post-diafiltration on a 50 kDa membrane (B). Anion-exchange chromatography of pepsin sample on CIM QA disk (V = 0.34 mL) with MES + 0.15 M NaCl buffer, pH 5.0, at a flow rate of 2 mL min-1 (C). Enzymatically active material was retained on the column and it was subsequently eluted with 100% yield. Manufacturing by-products that affect pepsin purity lacked capability of binding. Size-exclusion chromatography of flow-through (D) and elution fraction (E) from anion-exchange chromatography in (C). Detection: UV at 280 nm.
Fig 5.
Preliminary screening of pepsin digestion conditions.
(A) SDS-PAGE analysis of representative samples obtained by pepsin digestion of highly pure IgG sample (eIgG) on 4–12% gel under non-reducing conditions. Lane 1, typical digestion pattern after incubation at 20 °C; lane 2, typical digestion pattern after incubation at 37 °C when pH was set to 3.5; lane 3, molecular weight standards; lane 4, typical digestion pattern after incubation at 37 °C when pH was set to 3.2; lane 5, typical digestion pattern after incubation at 56 °C. In all lanes samples obtained by pepsin to IgG ratio of 10:300 (w/w) during 1.5 h-long incubation were presented. Staining was performed with CBB R250. (B) Optimisation of pepsin digestion with respect to duration and pepsin to IgG ratio studied according to full factorial experimental design. Mean yield (ο) of active drug obtained at higher and lower level of each experimental factor (X1—duration of enzymatic reaction; X2—pepsin to IgG ratio) in comparison to mean value (full line) and 95% CI (dashed lines) from the overall set of experiments. (C) Impact of pepsin to IgG ratio on F(ab')2 yield measured by ELISA assay.
Fig 6.
The assessment of IgG/F(ab')2 aggregation by size-exclusion chromatography.
Analysis was performed on TSK-Gel G3000SWXL column (7.8 × 300 mm) with 0.1 M phosphate-sulphate running buffer, pH 6.6, at a flow rate of 1 mL min-1. IgG substrate—supernatant from 2% caprylic acid precipitation before diafiltration (A). IgG fraction acidified to pH 3.2 in the presence of 2% caprylic acid (V/V) and incubated at +4 °C (B) or 37 °C (C). F(ab')2 fraction produced by pepsin digestion in the presence of caprylic acid (D). Detection: UV at 280 nm.
Fig 7.
SDS-PAGE analysis of F(ab')2 preparation after flow-through chromatography under different pH conditions and detection of pepsin traces.
(A) Detection of pepsin in Unosphere Q fractions by "negative" silver staining method. Lanes 1, 3 and 5, crude F(ab’)2 as starting material; lanes 2, 4 and 6, unbound fractions at pH 4.0, 5.0 and 6.0, respectively. (B) SDS-PAGE analysis of F(ab’)2 preparation before (left lane) and after CIM QA chromatography (right lane) at pH 5.0. Molecular mass markers are at right side. Staining was performed with AgNO3. "Negatively" silver stained bands corresponding to pepsin are marked.
Table 2.
Characterisation of the unbound fractions following incubation of F(ab)2 preparation containing pepsin with UNOsphere Q stationary phase under variuos pH conditions.
Fig 8.
2D gel electrophoresis of the final product.
In the first dimension F(ab')2 (350 μg) was focused using IPG strip under denaturing conditions (linear pH 3–10). Prior second dimension IPG strip was reduced, alkylated and loaded to a 4–12% gel. Proteins were detected with CBB R250 and identified by MS/MS analysis, as denoted (LC = light chain, VR = variable region; HC = heavy chain, CR = constant region). Molecular mass markers are at left side.
Table 3.
Neutralisation potencies of HHP and corresponding IgG/F(ab')2 determined in vivo together with specific activities.
Purification factors obtained through manufacturing procedure are indicated.
Fig 9.
Flow sheet of downstream processing steps with corresponding samples and performance rationales.