A novel method for continuous chromatographic separation of monoclonal antibody charge variants by combining displacement mode chromatography and step elution.

Charge heterogeneity of monoclonal antibodies is considered a critical quality attribute and hence needs to be monitored and controlled by the manufacturer. Typically, this is accomplished via separation of charge variants on cation exchange chromatography (CEX) using a pH or conductivity based linear gradient elution. Although an effective approach, this is challenging particularly during continuous processing as creation of linear gradient during continuous processing adds to process complexity and can lead to deviations in product quality upon slightest changes in gradient formation. Moreover, the long length of elution gradient along with the required peak fractionation makes process integration difficult. In this study, we propose a novel approach for separation of charge variants during continuous CEX chromatography by utilizing a combination of displacement mode chromatography and salt-based step elution. It has been demonstrated that while the displacement mode of chromatography enables control of acidic variants ≤26% in the CEX eluate, salt-based step gradient elution manages basic charge variant ≤25% in the CEX eluate. The proposed approach has been successfully demonstrated using feed materials with varying compositions. On comparing the designed strategy with 2-column concurrent (CC) chromatography, the resin specific productivity increased by 95% and resin utilization increased by 183% with recovery of main species >99%. Further, in order to showcase the amenability of the designed CEX method in continuous operation, the method was examined in our in-house continuous mAb platform.

Development of continuous processing platform utilizing aqueous two-phase extraction for purification of monoclonal antibodies.

Monoclonal antibody downstream processing typically entails chromatography-based purification processes beginning with Protein A chromatography, accounting for 50 % of the total manufacturing expense. Alternatives to protein A chromatography have been explored by several researchers. In this paper, aqueous two-phase extraction (ATPE) has been proposed for continuous processing of monoclonal antibodies (mAbs) as an alternative to the traditional protein A chromatography. The PEG-sulfate system has been employed for phase formation in ATPE, and the mAb is separated in the salt phase, while impurities like high molecular weight (HMW) and host cell proteins (HCPs) are separated in the PEG phase. Following ATPE of clarified cell culture harvest, yield of ≥ 80 % and purity of ≥ 97 % were achieved in the salt phase. Considerable (28 %) reduction in consumable cost has been estimated when comparing the proposed platform to the traditional protein A based platform. The outcomes demonstrate that ATPE can be a potentially effective substitute for the traditional Protein A chromatography for purification of mAbs. The proposed platform offers easy implementation, delivers comparative results, and offers significantly better economics for manufacturing mAb-based biotherapeutics.

Refolding of Proteins Expressed as Inclusion Bodies in E. coli.

Microbial-based biotherapeutics that are produced in Escherichia coli (E. coli) can be generated intracellularly in the form of inclusion bodies (IBs) or in soluble active form in periplasmic space or extracellularly. Overexpression of these biotherapeutics in E. coli leads to formation of insoluble aggregates called inclusion bodies. These IBs contain misfolded and inactive form of proteins which need to be refolded to obtain a functionally active form of proteins. Here, we discuss refolding of E. coli-based recombinant human granulocyte colony-stimulating factor (GCSF), expressed as IBs, and highlight some of the key features associated with the refolding kinetic reaction.

Enablers of continuous processing of biotherapeutic products.

The benefits of continuous processing over batch manufacturing are widely acknowledged across the biopharmaceutical industry, primary of which are higher productivity and greater consistency in product quality. Furthermore, the reduced equipment and facility footprint lead to significantly lower capital costs. Technology enablers have a major role in this migration from batch to continuous processing. In this review, we highlight the various enablers that are facilitating adoption of continuous upstream and downstream bioprocessing. This includes new bioreactors and cell retention devices for upstream operations, and on-column and continuous flow refolding, novel continuous chromatography, and single-pass filtration systems for downstream processes. We also elucidate the significant roles of process integration and control as well as of data analytics in these processes.