Polishing approach with fully connected flow‐through purification for therapeutic monoclonal antibody

Abstract

The biopharmaceutical industry is evolving toward process intensification that can offer increased productivity and improved economics without sacrificing process robustness. A semi‐continuous downstream process linking purification/polishing unit operations in series can reduce or eliminate intermediate holding tanks and reduce overall processing time. Accordingly, we have developed a therapeutic monoclonal antibody polishing template comprised of a connected flow‐through polishing technologies that include activated carbon, cation exchange, and anion‐exchange chromatography. In this report, we evaluated fully‐connected pool‐less polishing with three flow‐through technologies, operating as a single skid to streamline and improve an mAb purification platform. Laboratory‐scale pool‐less processing was achieved without utilizing in‐line pH adjustment and conductivity dilution based on the previously optimized single process parameter. Two connected flow‐through configurations of polishing steps were evaluated: a two‐step process using anion exchange and cation exchange and a three step process using activated carbon, anion exchange and cation exchange chromatography. Laboratory‐scale proof of concept studies showed comparable performance between the batch purification process and the pool‐less process configuration. Three step polishing highly intensified the processes and provided higher process loading and achieved bulk drug specification with higher impurity clearance (>95%) and high overall mAb yield (>95%).

Process intensification by frontal chromatography: Performance comparison of resin and membrane adsorber for monovalent antibody aggregate removal

“…While this is different from the membrane's optimum, it can be attributed to the different ligand chemistry of the two stationary phases as described above. Furthermore, the found optimum is about 2 pH units lower than the antibody's pI, which is in line with the findings of Ichihara et al () and Ichihara, Ito, and Gillespie (), and confirms this pH region to be a good starting point for screening studies. Conductivities lower than 4 mS/cm might yield even better results (Stone et al, ); however, these conditions were not investigated due to considerations regarding the entire downstream processing cascade.…”

Displacement to separate host‐cell proteins and aggregates in cation‐exchange chromatography of monoclonal antibodies

“…Although CEX is routinely employed for impurity separation, the effect of displacement during sample application on the separation performance is seldom leveraged or highlighted. Whether explicitly stated or not, methods employing self‐displacement chromatography are gradually emerging (Brown, Bill, Tully, Radhamohan, & Dowd, 2010; Ichihara et al, 2018, 2019; Khanal et al, 2019; H. F. Liu et al, 2011; Vogg et al, 2020a; Vogg, Pfeifer, Ulmer, & Morbidelli, 2020b) as they offer higher productivity with similar and adaptable sequences of operations. We have demonstrated various chromatography design strategies for the enhancement of displacement among the mAb product, aggregates and HCPs on CEX resins.…”

“…For example, protein aggregates (Khanal, Kumar, Westerberg, Schlegel, & Lenhoff, 2019; Reck, Pabst, Hunter, & Carta, 2017) and various charge variants (Khanal et al, 2019; Kumar, Leweke, von Lieres, & Rathore, 2015) are retained differently on CEX resins. This difference in retention has been leveraged to separate aggregates through salt gradient elution (Suda et al, 2009; Zhou et al, 2007), hybrid pH‐salt gradient elution (Khanal et al, 2019; Zhou et al, 2007), and flow‐through operation (Ichihara, Ito, & Gillespie, 2019; Ichihara, Ito, Kurisu, Galipeau, & Gillespie, 2018; Khanal et al, 2019; Suda et al, 2009; Ulmer, Vogg, Müller‐Späth, & Morbidelli, 2019; Vogg, Müller‐Späth, & Morbidelli, 2020a). Separation of mAb aggregates using flow‐through ion‐exchange chromatography is enabled by the displacement of bound monomer by incoming aggregates (Carta & Jungbauer, 2010).…”

“…Although some studies have evaluated displacement for mAb aggregate removal (Ichihara et al, 2018(Ichihara et al, , 2019Reck et al, 2017;Stone et al, 2019;Suda et al, 2009;Vogg et al, 2020a;Wollacott et al, 2015), investigation of how displacement affects the population of HCPs has not extended beyond a secondary analysis validating the effectiveness of proposed methods (Ichihara et al, 2018(Ichihara et al, , 2019H. Liu & Tang, 2008;Ulmer et al, 2019;Wollacott et al, 2015).…”

Monitoring bioactive and total antibody concentrations for continuous process control by surface plasmon resonance spectroscopy

“…Rapid advancements in mAb production technologies, including development of mAb producing cell‐lines, new bioreactor systems, and novel purification technologies as well as improvements in the diagnostic and therapy of diseases resulted in an explosive growth of the mAb market . World‐wide sales will have an estimated volume of $125 billion by 2020 .…”