Investigation of the interaction mechanism of the recombinant human antibody MDJ8 and its fragments with chromatographic apatite phases

The chromatographic behaviour of a recombinant human antibody (IgG₁-subtype, κ-light chain, MW: 149.5 kD, pI: 9.3) was investigated as a function of the buffer pH and buffer type (HEPES, phosphate, borate) on fluoroapatite and hydroxyapatite stationary phases. HEPES buffer was used at pH 7.0, phosphate buffer at pH 8.2 and borate buffer between pH 8.5 and 11. Elution was by a double gradient method of first a salt gradient from 0 to 1 M NaCl in the corresponding buffer, followed by a step gradient to 0.4 M sodium phosphate. Regardless of the pH and buffer type, the antibody eluted in the NaCl gradient; capacity factors decreased with increasing pH. At pH 11 the antibody eluted in the flow-through. Retention was thus dominated by electrostatic interaction throughout the investigated pH-range. Investigation of antibody fragments obtained by papain digestion (fc- and fab-fragments) and deglycosylated fc-fragments showed that the sugar structures had no influence on the chromatographic behaviour. Instead the chromatographic behaviour was dominated by that of the fab-fragment. ζ-Potential measurements verified that the apatite surface bore a negative surface charge in the investigated pH range, while the antibody net surface charge switched from positive to negative as the pH increased. The corresponding isoionic point was a function of both the buffer concentration and the buffer species. However, above a pH of 8.3 the ζ-potential of the antibody generally was negative. Simulations of the molecular electrostatic potential of the antibody and the two fragments revealed the presence of a positively charged patch within the fab-fragment, which only disappeared above a pH of 10. Most likely this patch was responsible for the observed behaviour.