Capture of human monoclonal antibodies from a clarified cell culture supernatant by phenyl boronate chromatography

Abstract

In this work, we investigated the feasibility of using phenyl boronate (PB) chromatography for the direct capture of monoclonal antibodies from a CHO cell supernatant. Preliminary results, using pure protein solutions have shown that PB media can bind to human antibodies, not only at strong alkaline conditions but also at acidic pH values. In fact, antibodies have been found to bind in the pH range 5.5-8.5. On the other hand, insulin and human serum albumin did not bind at alkaline pH but at lower pH, which reflects the importance of non-specific interactions with the matrix. Different binding and eluting buffers were evaluated for the capture of immunoglobulin G (IgG) from a CHO cell supernatant and the most promising results were obtained using 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid at pH 8.5 as binding buffer and 1.5 M Tris-HCl as eluting buffer. Using a step elution, all IgG was recovered in the elution pool with a maximum purification factor of 56. A gradient elution allowed a further increase of the final purity, yet achieving a slightly lower yield. IgG recovery was around 85% and the purification factor was 76. The highest purity was obtained when the pH of the cell supernatant feed was previously adjusted to 8.5. Starting from an initial protein purity of 1.1% and high-performance liquid chromatography (HPLC) purity of 2.2%, after PB adsorption, a final protein purity of 85% and a HPLC purity of 88% was achieved.

Key steps towards the oriented immobilization of antibodies using boronic acids

“…Considering the intrinsic properties of an Ab, a key factor for its successful immobilization is the composition of the N-glycans at its Fc chain. The affinity of Abs for boronic acids may also directly be assessed by boronate affinity chromatography using a commercially available boronate column, 32,33 although the results will also depend on the properties of the column and the binding conditions (see section 2.3). Many monosaccharides bind with boronic acids, but when they are attached to each other, relatively weak interactions between boronic acids and the formed glycans are enabled.…”

“…33 As these interactions would surely result in a partly random Ab immobilization, optimization of the binding conditions is essential. Depending on the binding conditions, (glyco)proteins can bind to boronic acids not only via the boronic acid-diol complexation, but also via secondary interactions, such as hydrophobic, ionic 20 and charge-transfer interactions.…”

“…Depending on the binding conditions, (glyco)proteins can bind to boronic acids not only via the boronic acid-diol complexation, but also via secondary interactions, such as hydrophobic, ionic 20 and charge-transfer interactions. 33 These effects are illustrated in the article by Azevedo et al, who studied the binding of pure human IgG, human recombinant insulin (non-glycosylated) and human serum albumin (HSA, poorly glycosylated) by boronate affinity chromatography under various conditions (Fig. For example, the ionic strength should be low enough to minimize hydrophobic interactions and high enough to minimize ionic interactions.…”

“…For example, the ionic strength should be low enough to minimize hydrophobic interactions and high enough to minimize ionic interactions. 33 When PBS with pH 7 was used, almost all the IgG was bound, as well as a significant amount of HSA and insulin. 33 These effects are illustrated in the article by Azevedo et al, who studied the binding of pure human IgG, human recombinant insulin (non-glycosylated) and human serum albumin (HSA, poorly glycosylated) by boronate affinity chromatography under various conditions (Fig.…”

Development of production and purification processes of recombinant fragment of pneumococcal surface protein A in Escherichia coli using different carbon sources and chromatography sequences

“…Studies of up-and downstream processing are usually performed separately (Hortsch and Weuster-Botz 2010;Goyal et al 2009;Azevedo et al 2010); however, if such studies were performed in an integrated manner, they can provide suitable results in order to develop more effective process (Balbas 2001;Gottschalk 2008;Roush and Lu 2008). Here, the influence of distinct carbon sources, glucose, and glycerol, at high cell density of E. coli BL21 (DE3) was compared for production and purification of the recombinant fragment of PspA3 in an attempt to obtain an effective process amenable to industrial scale-up.…”