Staphylococcal protein A affinity chromatography is a well-established platform for purification of clinical-grade antibodies. The wild type ligand has been mutated to improve caustic stability, elution behavior, and/or to increase binding capacity. Several modified protein A ligands are nowadays commercially available, one of them being the thermosensitive chromatography medium Byzen Pro from Nomadic Bioscience Co., Ltd. According to the manufacturer, Byzen Pro has the ability to release IgG upon a change in temperature. It is based on a thermosensitive mutant of protein A which should allow elution at neutral pH by changing the temperature from binding at 5 °C to elution conditions at 40 °C. We determined equilibrium binding capacities of the thermosensitive protein A medium (Byzen Pro), MabSelect SuRe (GE Healthcare), and TOYOPEARL AF-rProtein A HC-650F (Tosoh Bioscience LLC) for antibodies of the subclass IgG1 and IgG2 at five different temperatures from 4 °C to 40 °C to elucidate the temperature effect. We also observed a temperature dependence of the dynamic binding capacities which were determined for the subclass IgG2 at three temperatures from 4 °C to 40 °C. However, for Byzen Pro, the temperature dependence was only present at a low flow rate and vanished at high flow rates indicating that pore diffusion is the rate-limiting step. Binding of the antibody to MabSelect SuRe and TOYOPEARL AF-rProtein A HC-650F stabilized the conformations as shown by an increase in melting temperature in differential scanning calorimetry measurements. The antibody conformation was slightly destabilized upon binding to the thermosensitive ligand. The conformation change upon binding was fully reversible as shown by circular dichroism, differential scanning calorimetry and size exclusion chromatography. Isothermal titration calorimetry was used to measure the raw heat of adsorption for the IgG2 molecule. The thermosensitive ligand can also be used for antibodies with low stability, because elution can also be effected by salt.

BACKGROUNDIntegrated continuous production technology is of great interest in biopharmaceutical industry. Efficient, flexible and cost effective methods for continuous cell removal have to be developed, before a fully continuous and integrated product train can be realized. The paper describes the development and testing of such an integrated continuous and disposable set‐up for cell separation by flocculation combined with depth filtration.RESULTSScreening of multiple flocculation agents, depth filters, and conditions demonstrated that the best performance was obtained with 0.0375% polydiallyldimethylammonium chloride (pDADMAC; a polycationic flocculation agent) in combination with Clarisolve® depth filters. Using this set‐up, a 4‐fold decrease of filtration area was achieved relative to standard filtration without flocculation, with yields of ≥97% and DNA depletion of up to 99%. Continuous operation was accomplished using a simple tubular reactor design with parallelization of the filtration. The reactor length was selected to allow a 13.2‐min residence time, which was sufficient to complete flocculation in batch experiments. Continuous flocculation performance was monitored on‐line using focused beam reflectance measurement. Filter switch cycles based on upstream pressure were controlled by in‐line pressure sensors, and were stable from one filter to the next.CONCLUSIONIt was demonstrated that stable and efficient continuous flocculation associated with depth filtration can be easily accomplished using tubular reactors and parallelization. Continuous cell separation is essential for the development of fully continuous integrated process trains. This cost‐efficient disposable design run in continuous mode significantly reduces facility foot print, process costs and enables great flexbility. © 2017 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Protein and in particular antibody precipitation by PEG is a cost‐effective alternative for the first capture step. The 3D structure of precipitates has a large impact on the process parameters for the recovery and dissolution, but current technologies for determination of precipitate structures are either very time consuming (cryo‐TEM) or only generate an average fractal dimension (light scattering). We developed a light microscopy based reconstruction of 3D structures of individual particles with a resolution of 0.1–0.2 µm and used this method to characterize particle populations generated by batch as well as continuous precipitation in different shear stress environments. The resulting precipitate structures show a broad distribution in terms of fractal dimension. While the average fractal dimension is significantly different for batch and continuous precipitation, the distribution is broad and samples overlap significantly. The precipitate flocs were monofractal from micro‐ to nanoscale showing a random but consistent nature of precipitate formation. We showed that the fractal dimension and 3D reconstruction is a valuable tool for characterization of protein precipitate processes. The current switch from batch to continuous manufacturing has to take the 3D structure and population of different protein precipitates into account in their design, engineering, and scale up.

Antibodies of the IgG2 subclass were captured from the clarified cell culture fluid either by protein A chromatography or by polyethylene glycol precipitation. The captured intermediates were stored as neutralized eluates (protein A chromatography) or in solid form as polyethylene glycol precipitates over a period of 13 months at three temperatures, −20°C, 5°C, and room temperature to compare the capture technologies in regard of the resulting product storability. Monomer content, high molecular mass impurities product loss and changes in the composition of the charge variants were determined at six time points during the storage. At the beginning and end of the study, samples were additionally tested by differential scanning calorimetry, differential scanning fluorimetry, and circular dichroism to determine structural alterations occurring during storage. Protein A purified material was highly stable at all tested temperatures in regard of monomer content and product losses. A transient, acidic isoform was formed during the chromatography step which re‐converted to the main charged variant upon storage within a matter of days. Precipitated antibodies could be stored at −20 or 5°C for 3 months without product losses but afterwards recovery yields dropped to 65%. At room temperature, the precipitated antibody was not stable and degraded within 3 months.