Improving Downstream Process Related Manufacturability Based on Protein Engineering-A Feasibility Study.

While bioactivity and a favorable safety profile for biotherapeutics is of utmost importance, manufacturability is also worth of consideration to ease the manufacturing process. Manufacturability in the scientific literature is mostly related to stability of formulated drug substances, with limited focus on downstream process-related manufacturability, that is, how easily can a protein be purified. Process-related impurities or biological impurities like viruses and host cell proteins (HCP) are present in the harvest which have mostly acid isoelectric points and need to be removed to ensure patient safety.

Mimicking continuous capture chromatography for virus clearance using a single chromatography column model.

Continuous chromatography is increasingly being used across the biotechnology industry due to its economic advantages. For adoption in commercial manufacturing, also models for virus clearance studies must be available. It is demonstrated how for a virus clearance study for a multispecific antibody, the continuous protein A capture chromatography process, being run on multiple interconnected columns, can be mimicked with only a single column.

Moving to CoPACaPAnA: Implementation of a continuous protein A capture process for antibody applications within an end-to-end single-use GMP manufacturing downstream process.

For the first time to our knowledge the implementation of a continuous protein A capture process for antibody applications (CoPACaPAnA) embedded in an end-to-end single-use 500 L GMP manufacturing downstream process of a multispecific monoclonal antibody (mAb) using a single-use SMB system was conducted. Throughout the last years, a change concerning the pipelines in pharmaceutical industry could be observed, moving to a more heterogeneous portfolio of antibodies, fusion proteins and nanobodies. Trying to adjust purification processes to these new modalities, a higher degree of flexibility and lower operational and capital expenditure is desired.

Scale-up of continuous multicolumn chromatography for the protein a capture step: From bench to clinical manufacturing.

The awareness about implementing continuous processing for biopharmaceutical products has significantly increased throughout the recent years not only at developmental scale but also for phase I supply in clinical trial manufacturing. In this study, we focused on upscaling continuous protein A chromatography from lab to pilot scale using the Cadence™ BioSMB PD and the Cadence™ BioSMB Process 80 system, respectively. Additionally, we evaluated hardware and software capability whilst running the system for 10 days non-stop using feed from a perfusion bioreactor.

Feasibility of using continuous chromatography in downstream processing: Comparison of costs and product quality for a hybrid process vs. a conventional batch process.

The protein A capture step is the main cost-driver in downstream processing, with high attrition costs especially when using protein A resin not until end of resin lifetime. Here we describe a feasibility study, transferring a batch downstream process to a hybrid process, aimed at replacing batch protein A capture chromatography with a continuous capture step, while leaving the polishing steps unchanged to minimize required process adaptations compared to a batch process. 35g of antibody were purified using the hybrid approach, resulting in comparable product quality and step yield compared to the batch process.

Mid-infrared spectroscopy-based analysis of mammalian cell culture parameters.

Within the framework of process analytical technology, infrared spectroscopy (IR) has been used for characterization of biopharmaceutical production processes. Although noninvasive attenuated total reflection (ATR) spectroscopy can be regarded as gold standard within IR-based process analytics, simpler and more cost-effective mid-infrared (MIR) instruments might improve acceptability of this technique for high-level monitoring of small scale experiments as well as for academia where financial restraints impede the use of costly equipment. A simple and straightforward at-line mid-IR instrument was used to monitor cell viability parameters, activity of lactate dehydrogenase (LDH), amount of secreted antibody, and concentration of glutamate and lactate in a Chinese hamster ovary cell culture process, applying multivariate prediction models, including only 25-28 calibration samples per model.

Development of a univariate membrane-based mid-infrared method for protein quantitation and total lipid content analysis of biological samples.

Biological samples present a range of complexities from homogeneous purified protein to multicomponent mixtures. Accurate qualification of such samples is paramount to downstream applications. We describe the development of an MIR spectroscopy-based analytical method offering simultaneous protein quantitation (0.

Feasibility of polyelectrolyte-driven Fab fragment separation.

The use of antigen-binding fragments (Fabs) as biotherapeutic agents is gaining interest and thus requires development of adequate purification strategies aimed at separating Fabs from other proteins. Thus, the feasibility of using a copolymer for separation of Fabs from monoclonal antibodies (mAbs) and fragment constant regions (Fcs) was evaluated, employing a blend of purified solutions of these proteins. The use of a copolymer exerting both hydrophobic as well as anionic properties resulted in high precipitation yields for both the mAb and Fc fragment, even at ionic strength of 150 mM NaCl.

Customization of copolymers to optimize selectivity and yield in polymer-driven antibody purification processes.

This manuscript describes customization of copolymers to be used for polymer-driven protein purification in bioprocessing. To understand how copolymer customization can be used for fine-tuning, precipitation behavior was analyzed for five target antibodies (mAbs) and BSA as model impurity protein, at ionic strength similar to undiluted cell culture fluid. In contrast to the use of standardized homopolymers, customized copolymers, composed of 2-acrylamido-2-methylpropane sulfonic acid (AMPS) and 4-(acryloylamino)benzoic acid (ABZ), exhibited antibody precipitation yields exceeding 90%.

Mid-infrared spectroscopy-based antibody aggregate quantification in cell culture fluids.

Therapeutic antibody purification involves several steps which potentially induce antibody aggregation. Currently, aggregate monitoring mainly employs chromatographic, SDS-PAGE and light scattering techniques. In this study, the feasibility of mid-infrared spectroscopy (MIR) for the quantification of soluble antibody aggregates was investigated.

Feasibility study of semi-selective protein precipitation with salt-tolerant copolymers for industrial purification of therapeutic antibodies.

We present a feasibility study for an antibody capturing process from clarified cell culture fluid using semi-selective protein precipitation with salt-tolerant copolymers. Protein precipitation is mediated by hydrophobic and electrostatic interactions with the copolymer that can be customized for the respective target. Precipitation yield with different copolymers at ionic strength of 2-22.

Matrix effects during monitoring of antibody and host cell proteins using attenuated total reflection spectroscopy.

Production of recombinant proteins, e.g. antibodies, requires constant real-time monitoring to optimize yield and quality attributes and to respond to changing production conditions, such as host cell protein (HCP) titers.

Host cell protein quantification by Fourier transform mid infrared spectroscopy (FT-MIR).

Process development in up- and downstream processing requires enhanced, non-time-consuming, and non-expensive monitoring techniques to track product purity, for example, the level of endotoxins, viral particles, and host cell proteins (HCPs). Currently, HCP amounts are measured by laborious and expensive HCP-enzyme-linked immunosorbent assay (ELISA) assays best suited for measuring HCP amounts in the low concentration regime. The measurement of higher HCP amounts using this method requires dilution steps, adding dilution errors to the measurement.