Measurement of yield at different elution conditions was performed in PreDictor 96-well filter plates. Were VX% = load volume (mL) at x% breakthrough, V0 = void volume (mL), C0 = MAb concentration in the sample (mg/mL) and Vc = volumetric bed volume (mL). Before frontal analysis, the MAb solution was injected by-passing the column to obtain a maximum absorbance value. The UV-absorbance at 280 nm was used for determination of breakthrough. Where Cint = MAb concentration in sample, Cout = MAb concentration in FT fraction, and Vmedium = medium volume in each well (i.e., 6 μL).ĭetermination of dynamic binding capacityĭynamic binding capacity (DBC) was determined by frontal analysis using. SBC = MAbbound /Vmedium = MAbbound /0.006 Unbound material (FT fraction) was removed by centrifugation for 3 min, and MAb concentration was determined by measurement of absorbance at 280 nm. MAb solution (200 μL volume, 4 mg/mL sample load, corresponding to 133 mg MAb/mL chromatography medium) was added to each well followed by agitation for 90 min. The equilibration step was performed three times. Equilibration of wells in the filter plates was performed by addition of 200 μL of loading buffer per well followed by agitation at 1100 rpm for 1 min, after which the buffer was removed by vacuum extraction.

Static binding capacity (SBC) was determined in 6 μL PreDictor™ 96-well filter plates. Some characteristics of the MAbs are shown in Table 1. The two MAbs used in this study were initially purified from CHO cell supernatant by protein A affinity chromatography. The studies include measurement of static- and dynamic binding capacities at various binding conditions, as well as screening and optimization of gradient- and step-elution conditions.ġ Capto adhere ImpRes is also be used for purification of recombinant proteins and other biomolecules. This application note describes development of polishing steps for two different MAbs in B/E mode using Capto adhere ImpRes. Contaminants such as DNA, HCP, leached protein A, aggregates, and viruses are efficiently separated from monomeric MAbs1 in B/E or FT modes. The high resolution possible with Capto adhere ImpRes enables reduced buffer consumption and improved product yield compared with Capto adhere, a related product with the same ligand but with a larger bead size. The small bead size of Capto adhere ImpRes enables high-resolution purification of target protein. The medium enables operation in either B/E or nonbinding (flowthrough, FT) modes and results in either two- or three-step purification schemes. One of these options, Capto adhere ImpRes, is a cost-effective and flexible chromatography medium (resin) designed for high-resolution polishing of MAbs.Ĭapto adhere ImpRes is a multimodal anion exchange medium with a ligand (Fig 1) that displays high selectivity compared with traditional ion exchange polishing media. After the initial protein A capture step, there is a wide range of options for intermediate and polishing purification steps. GE Healthcare Life Sciences’ MAb production toolbox employs protein A chromatography media such as MabSelect SuRe™ or MabSelect SuRe LX for capture of the target. A platform approach is desirable as it saves both time and money in process development. The relative homogeneity of MAbs makes them well-suited for platform processes, which are sets of unit operations, conditions, and methods applied to molecules of a given class.

As a result, more cost-effective, efficient, and flexible process purification schemes are one of the highest priorities for MAb manufacturers.

MAbs and MAb conjugates are today in great demand for use as biopharamaceuticals. The results showed high yields of monomeric MAb, as well as good clearance of aggregates, host cell proteins (HCP), and leached protein A. The effects of buffer, pH, conductivity, and sample load were investigated. The study presents results from optimization of the loading conditions using the Design of Experiments (DoE) approach. In this study, the binding capacity for MAbs and the efficiency in the clearance of impurities using Capto adhere ImpRes in bind/elute (B/E) mode was evaluated.

Capto adhere ImpRes is a strong ion exchanger with multimodal functionality designed for polishing of monoclonal antibodies (MAbs).

Capto impres