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

Abstract

An efficient and consistent method of monoclonal antibody (mAb) purification can improve process productivity and product consistency. Although protein A chromatography removes most host-cell proteins (HCPs), mAb aggregates and the remaining HCPs are challenging to remove in a typical bind-and-elute cation-exchange chromatography (CEX) polishing step. A variant of the bind-and-elute mode is the displacement mode, which allows strongly binding impurities to be preferentially retained and significantly improves resin utilization. Improved resin utilization renders displacement chromatography particularly suitable in continuous chromatography operations. In this study we demonstrate and exploit sample displacement between a mAb and impurities present at low prevalence (0.002%-1.4%) using different multicolumn designs and recycling. Aggregate displacement depends on the residence time, sample concentration, and solution environment, the latter by enhancing the differences between the binding affinities of the product and the impurities. Displacement among the mAb and low-prevalence HCPs resulted in an effectively bimodal-like distribution of HCPs along the length of a multi-column system, with the mAb separating the relatively more basic group of HCPs from those that are more acidic. Our findings demonstrate that displacement of lowprevalence impurities along multiple CEX columns allows for selective separation of mAb aggregates and HCPs that persist through protein A chromatography. K E Y W O R D S aggregates, continuous chromatography, displacement, frontal chromatography, host-cell proteins, multicolumn chromatography 1 | INTRODUCTION Displacement was classified as one of three forms of chromatography, together with elution and frontal, in 1943 (Tiselius, 1943). Separation via elution chromatography is achieved after multiple components of the sample first bind to the column, then are eluted sequentially in the order of increasing affinity by a buffer of gradually increasing eluent strength. Separation by displacement chromatography, in contrast, uses a high-affinity displacer to displace and separate the transiently bound product. A variant of displacement chromatography, sample-or self-displacement chromatography, refers to the displacement of a lower-affinity product component by a higher-affinity counterpart in a multicomponent system. Similar to self-displacement chromatography, frontal chromatography refers to the separation of components based on their relative affinities. In a variant of frontal chromatography, termed flow-through chromatography, the operational parameters are chosen to let the impurities bind, allowing a purer product to exit the column unbound (Hill, Mace, & Moore, 1990). While elution and flow-through chromatography employ different approaches to achieve purification, displacement simply indicates that a competitor with a stronger affinity replaces the more weakly bound rival. Given these general

Combinatorial peptides: A library that continuously probes low‐abundance proteins

“…Examples have been published on hydrophobic resins [28] as well as on anion- [29] and cation-exchangers [30]. It has been demonstrated as being very effective for the enrichment of certain low-abundance proteins in lectin affinity chromatography, [31] and in the detection of protein impurity traces [32] as well as in the removal of host cell proteins from purified antibodies [33].…”

Improved adeno‐associated virus empty and full capsid separation using weak partitioning multi‐column AEX chromatography

“…[ 12 ] In addition, the same author also reported the displacement of low‐prevalence impurities along multiple CEX columns allows for selective separation of mAb aggregates and HCPs that persist through Protein A chromatography. [ 13 ] Although WPC has not been fully applied in the rAAV purification process yet, it is reasonable to believe that this technology could also enable AEX chromatography to achieve improved separation of empty and full rAAV during column overloading.…”

Process development exploiting competitive adsorption‐based displacement effects in monoclonal antibody aggregate removal—A new high‐throughput screening procedure for membrane chromatography

“…In addition, the high mass transfer rates observed in MAs furthermore promote competitive binding‐based displacement effects and maintain the typically high productivity. During the last decades, several efforts in research and process development were carried out investigating competitive‐based displacement effects and their use 14–16,23,24,27–31 . However, currently no screening strategy exists to investigate potential displacement effects applying membrane chromatography on a robotic screening platform.…”

“…During the last decades, several efforts in research and process development were carried out investigating competitive-based displacement effects and their use. [14][15][16]23,24,[27][28][29][30][31] However, currently no screening strategy exists to investigate potential displacement effects applying membrane chromatography on a robotic screening platform.…”