Purification of Monoclonal Antibodies by Hydroxylapatite HPLC and Size Exclusion HPLC

Abstract

Monoclonal antibodies of both the IgG and the IgM type were purified by hydroxylapatite HPLC (HA-HPLC) under very mild conditions. The IgM type antibodies, which were isolated from ascites fluid and separated from other proteins also by means of size exclusion HPLC.It was shown that the most frequently observed disadvantage of HA-HPLC, that is the relative short life of the columns (P. Steifen (1989) GIT Fachz. Lab., Suppl. 3/89 (Chromatogr.}, 50-90), is due to microbial contamination rather than lower mechanical stability. In order to monitor column performance, a test was developed based on the use of standard proteins under isocratic separation conditions. This allows a direct comparison between the respective performances of columns made from different materials, hydroxylapatite or fluoroapatite, from different sources and with different particle sizes. A problem which often occurs with HA-HPLC in the case of IgM antibody isolation, namely precipitation of the antibodies at low salt concentrations at the beginning of a Chromatographie run, was avoided by adding sodium chloride to both separation buffers. Reinigung monoklonaler Antikörper durch HPLC an Hydroxylapatit und durch Size-Exclusions-HPLC

Monoclonal antibody purification with hydroxyapatite

“…Intermediate and polishing purification HA has been used for intermediate purification of many IgG [14,15,[19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36], IgA [37][38][39][40][41], and IgM antibodies [17,37,38,[42][43][44]. The majority of these applications employ simple phosphate gradients.…”

“…HA elution with phosphate gradients has been known to support effective aggregate removal from IgG monoclonal antibodies since the 1990s [19]. The simplicity of making all of the process buffers with dilutions of the 500 mM phosphate-cleaning buffer is attractive for antibodies that are accommodated by this approach, but that unfortunately includes only a small proportion of IgGs.…”

PEG enhances viral clearance on ceramic hydroxyapatite

“…In general, the much larger surface area provided by viruses would be expected to result in significantly tighter binding as compared with much smaller bio-molecules (such as antibodies). Such an effect has been noted for IgG antibody aggregates, which generally elute later on hydroxyapatite in a gradient than monomeric IgG, and for larger antibody types such as IgA and IgM [5][6][7]14]. The effect is similar to that observed on cation exchange resins (see, for example, [19]).…”

“…The mechanism of binding can be due to either synergistic effects of multi-site binding [5][6][7], strong interactions of clustered surface phosphates (in the case of lipid-enveloped viruses) with calcium [8,9] or both. The original eluant used almost exclusively for hydroxyapatite was phosphate.…”

Isolation and purification of recombinant proteins, antibodies and plasmid DNA with hydroxyapatite chromatography

“…Even elevated aggregate levels have been reduced to less than 0.1% in a single HA step [5]. Simple phosphate gradients can be used [51,52]; however, chloride gradients in the presence of small amounts of phosphate (< 20 mM) often show better results [5] (Table 4). Standard approaches for method development using phosphate or NaCl gradients for antibody purification are given in the Supporting information, Protocols 1 and 2, respectively.…”