Integrated multidimensional chromatography on preparative scale for oligonucleotides purification.

Therapeutic oligonucleotides represent a recent breakthrough in the pharmaceutical industry due to their ability to regulate gene expression with great specificity. This aspect allows treatment of a wide range of diseases. However, since oligonucleotides are used for therapeutic purposes, the Active Pharmaceutical Ingredient (API) must fulfill strict purity levels which require intensive purification steps.

Continuous countercurrent chromatographic twin-column purification of oligonucleotides: The role of the displacement effect.

Oligonucleotides (ONs) are breaking through in the biopharmaceutical industry as a promising class of biotherapeutics. The main success of these molecules is due to their peculiar way of acting in the cellular process, regulating the gene expression and hence influencing the protein synthesis at a pretranslational level. Although the Food and Drug Administration (FDA) already approved a few ON-based therapeutics, their production cost strongly limits large-scale manufacturing: a situation that can be alleviated through process intensification.

Analysis and optimal design of batch and two-column continuous chromatographic frontal processes for monoclonal antibody purification.

The increasing demand for efficient and robust processes in the purification of monoclonal antibodies (mAbs) has recently brought frontal chromatography to the forefront. Applied during the polishing step, it enables the removal of high molecular weight aggregates from the target product, achieving high purities. Typically, this process is operated in batch using a single column, which makes it intrinsically subjected to a purity-yield tradeoff.

From batch to continuous chromatographic purification of a therapeutic peptide through multicolumn countercurrent solvent gradient purification.

A twin-column Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) process has been developed for the purification of a therapeutic peptide, glucagon, from a crude synthetic mixture. This semi-continuous process uses two identical columns operating either in interconnected or in batch mode, thus enabling the internal recycle of the portions of the eluting stream which do not comply with purity specifications. Because of this feature, which actually results in the simulated countercurrent movement of the stationary phase with respect to the mobile one, the yield-purity trade-off typical of traditional batch preparative chromatography can be alleviated.

Model based strategies towards protein A resin lifetime optimization and supervision.

The high cost of protein A resins drives the biopharmaceutical industry to maximize its lifetime, which is limited by several processes, usually referred to as resin aging. In this work, two model based strategies are presented, aiming to control and improve the resin lifetime. The first approach, purely statistical, enables qualitative monitoring of the column state and prediction of column performance indicators (e.

Design space and robustness analysis of batch and counter-current frontal chromatography processes for the removal of antibody aggregates.

Increasing molecular diversity and market competition requires biopharmaceutical manufacturers to intensify their processes. In this respect, frontal chromatography on cation exchange resins has shown its potential to effectively remove aggregates. However, yield losses during the wash step need to be accepted in order to ensure robust product quality.

Process-wide control and automation of an integrated continuous manufacturing platform for antibodies.

Integrated continuous manufacturing is entering the biopharmaceutical industry. The main drivers range from improved economics, manufacturing flexibility, and more consistent product quality. However, studies on fully integrated production platforms have been limited due to the higher degree of system complexity, limited process information, disturbance, and drift sensitivity, as well as difficulties in digital process integration.

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

Aggregates are amongst the most important product-related impurities to be removed during the downstream processing of antibodies due to their potential immunogenicity. Traditional operations use cation-exchange resins in bind-elute mode for their separation. However, frontal analysis is emerging as an alternative.

Understanding mAb aggregation during low pH viral inactivation and subsequent neutralization.

Monoclonal antibodies (mAbs) and related recombinant proteins continue to gain importance in the treatment of a great variety of diseases. Despite significant advances, their manufacturing can still present challenges owing to their molecular complexity and stringent regulations with respect to product purity, stability, safety, and so forth. In this context, protein aggregates are of particular concern due to their immunogenic potential.

A new flow cell and chemometric protocol for implementing in-line Raman spectroscopy in chromatography.

On-line monitoring tools for downstream chromatographic processing (DSP) of biotherapeutics can enable fast actions to correct for disturbances in the upstream, gain process understanding, and eventually lead to process optimization. While UV/Vis spectroscopy is mostly assessing the protein's amino acid composition and the application of Fourier transform infrared spectroscopy is limited due to strong water interactions, Raman spectroscopy is able to assess the secondary and tertiary protein structure without significant water interactions. The aim of this work is to implement the Raman technology in DSP, by designing an in-line flow cell with a reduced dead volume of 80 μL and a reflector to increase the signal intensity as well as developing a chemometric modeling path.

Experimental Evaluation of the Impact of Intrinsic Process Parameters on the Performance of a Continuous Chromatographic Polishing Unit (MCSGP).

The semicontinuous twin-column multicolumn countercurrent solvent gradient purification (MCSGP) process improves the trade-off between purity and yield encountered in traditional batch chromatography, while its complexity, in terms of hardware requirements and process design, is reduced in comparison to process variants using more columns. In this study, the MCSGP process is experimentally characterized, specifically with respect to its unique degrees of freedom, i.e.

Purification of Human Monoclonal Antibodies and Their Fragments.

This chapter summarizes the most common chromatographic mAb and mAb fragment purification methods, starting by elucidating the relevant properties of the compounds and introducing the various chromatography modes that are available and useful for this application. A focus is put on the capture step affinity and ion-exchange chromatography. Aspects of scalability play an important role in judging the suitability of the methods.

Continuous and Integrated Expression and Purification of Recombinant Antibodies.

This chapter introduces the necessary concepts to design continuous expression and purification processes for monoclonal antibodies. The operation of a perfusion bioreactor is discussed containing the preparation procedures, the seeding train and the preparation and control of a long-term production run. The downstream processes exploit the benefits of countercurrent chromatography.

Proteomic analysis of micro-scale bioreactors as scale-down model for a mAb producing CHO industrial fed-batch platform.

The pharmaceutical production of recombinant proteins, such as monoclonal antibodies, is rather complex and requires proper development work. Accordingly, it is essential to develop appropriate scale-down models, which can mimic the corresponding production scale. In this work, we investigated the impact of the bioreactor scale on intracellular micro-heterogeneities of a CHO cell line producing monoclonal antibodies in fed-batch mode, using a 10 mL micro-bioreactor (ambr™) scale-down model and the corresponding 300 L pilot-scale bioreactor.

Application of metabolic controls for the maximization of lipid production in semicontinuous fermentation.

Acetic acid can be generated through syngas fermentation, lignocellulosic biomass degradation, and organic waste anaerobic digestion. Microbial conversion of acetate into triacylglycerols for biofuel production has many advantages, including low-cost or even negative-cost feedstock and environmental benefits. The main issue stems from the dilute nature of acetate produced in such systems, which is costly to be processed on an industrial scale.