Dynamic process control of twin-column periodic countercurrent chromatography processes

Twin-column periodic countercurrent chromatography has become a promising solution for continuous downstream processes as chromatography equipment for both process development and GMP manufacturing has become available. Twin-column periodic countercurrent processes have been utilized successfully in many applications including purification of biologics, such as monoclonal antibodies (mAbs), bispecific antibodies and fusion proteins, the purification of peptides and also small molecules such as antibiotics and fatty acid ethyl esters. This presentation deals with the online UV-based control of two twin-column periodic countercurrent processes, 2C-PCC and MCSGP, covering applications in chromatographic capture and polishing. 2C-PCC is a capture process significantly improving the process performance (productivity, resin utilization, buffer consumption, product concentration) of affinity capture, e.g. the capture of mAbs, in comparison to traditional single column chromatography. MCSGP is a polishing process to solve difficult ternary separation challenges, allowing purification with high product yield and purity in situations where traditional single column chromatography faces a yield-purity trade-off. For robust operation in view of commercial manufacturing using these two cyclic processes, UV-based dynamic control strategies have been developed and tested. In this presentation a UV-based control strategy for 2C-PCC based on online-determination of breakthrough curve signals is introduced and case studies for its application in protein A chromatography are shown. The control strategy accounts for changes in resin capacity and, in case of continuous upstream, for changes in titer occurring over time, and adjusts the operating parameters such that capacity utilization and yield are kept constant. A second control strategy for MCSGP based on the online evaluation of the elution peak signal is presented based on a case study. The method accounts for shifts of the product peak e.g. due to changes in temperature and buffer preparation (e.g. during buffer refill). An application of the control strategy in protein purification is presented. The presented methods represent important tools for robust manufacturing using twin column processe

Aggregation Stability of a Monoclonal Antibody During Downstream Processing

ABSTRACT: Purpose: To study the effect of several operative parameters, particularly pH and salt concentration, on the stability and aggregation kinetics of IgG solutions under the conditions typically encountered in downstream processing. Methods: The time evolution of the aggregates is analyzed by a combination of dynamic light scattering, size exclusion chromatography (SEC) and field flow fractionation (FFF). Secondary structure changes are monitored by circular dichroism. Results: For the given antibody, it is found that at pH lower than 4.0 addition of salt induces a reversible aggregation to oligomers accompanied by an increase in the content of the β-sheet structure. The aggregation rate increases monotonically with the salt concentration. Both the SEC and FFF techniques are successfully applied to obtain the oligomer distributions, and their results are consistent. The modified Lumry-Eyring kinetic model can well describe the time evolutions of the oligomers. Conclusions: For the given antibody, low pH and presence of salt induce conformational changes that are responsible for the reversible aggregation, but in the investigated conditions only small soluble oligomers are formed and oligomer sizes larger than trimer are negligible. For this reason, no accompanied macroscopic changes can be observe

Dynamic process control of continuous twin-column chromatography

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Design of a twin-column countercurrent purification (MCSGP) unit for the polishing of an oligonucleotide sequence

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Development of scalable semi-continuous downstream processes

The goal of this work is to establish an intensified downstream scheme for stable, high-titer monoclonal antibody (mAb) processes to achieve increased manufacturing productivity with short cadence, reduced cost, and small facility footprint. Several continuous manufacturing technologies including multi-column chromatography for capture, automated low-pH viral inactivation (low-pH VI), and integrated pool-less polishing steps were evaluated following consistent development methodologies for several mAbs. This presentation aims to provide an overview of the approaches to developing and integrating these discrete technologies in one cohesive process flow that fits manufacturing requirements in a flexible manner. Development efforts are illustrated in three major areas. First, twin-column continuous capture chromatography (CaptureSMB) was evaluated systematically for equivalency assessment comparing to traditional batch operation for different molecules. Development data showed overall comparable chromatography performance, while certain trends were found to be molecule/process specific. For executing viral clearance studies, scalable models were developed using CaptureSMB and a surrogate system employing standard batch chromatography with flow path modifications to mimic the loading strategy of CaptureSMB. We also introduce a model-assisted process characterization approach toward validation of continuous twin-column capture chromatography owing to increased process understanding. Second, experimental studies and computational fluid dynamics (CFD) modeling were used to reduce the risk of product aggregation in low-pH VI manufacturing operation. For various mixing systems, localized low-pH zones were characterized quantitatively to avoid the undesirable conditions that could cause severe aggregate formation during acid adjustment. The modeling tool integrated with mAb aggregation measurements facilitates the optimization of operating parameters (e.g., titrant addition rate, impeller agitation) and automation strategy to ensure robust VI scale-up performance. Third, various scenarios of integrated pool-less polishing steps operated in flowthrough-flowthrough (FT-FT) or flowthrough-bind/elute (FT-B/E) mode were evaluated with or without inline adjustment between the two steps. Performance and quality attributes are compared for integrated and decoupled polishing steps, with an example describing the development and optimization workflow for a specific mAb process. Finally, implication to process development timelines, scale-up performance and practical challenges to process implementation in the new 2000-L manufacturing facility will be discussed

Ageing-associated changes in transcriptional elongation influence longevity

Physiological homeostasis becomes compromised during ageing, as a result of impairment of cellular processes, including transcription and RNA splicing1-4. However, the molecular mechanisms leading to the loss of transcriptional fidelity are so far elusive, as are ways of preventing it. Here we profiled and analysed genome-wide, ageing-related changes in transcriptional processes across different organisms: nematodes, fruitflies, mice, rats and humans. The average transcriptional elongation speed (RNA polymerase II speed) increased with age in all five species. Along with these changes in elongation speed, we observed changes in splicing, including a reduction of unspliced transcripts and the formation of more circular RNAs. Two lifespan-extending interventions, dietary restriction and lowered insulin-IGF signalling, both reversed most of these ageing-related changes. Genetic variants in RNA polymerase II that reduced its speed in worms5 and flies6 increased their lifespan. Similarly, reducing the speed of RNA polymerase II by overexpressing histone components, to counter age-associated changes in nucleosome positioning, also extended lifespan in flies and the division potential of human cells. Our findings uncover fundamental molecular mechanisms underlying animal ageing and lifespan-extending interventions, and point to possible preventive measures

In Vitro Aggregation Behavior of a Non-Amyloidogenic λ Light Chain Dimer Deriving from U266 Multiple Myeloma Cells

Excessive production of monoclonal light chains due to multiple myeloma can induce aggregation-related disorders, such as light chain amyloidosis (AL) and light chain deposition diseases (LCDD). In this work, we produce a non-amyloidogenic IgE λ light chain dimer from human mammalian cells U266, which originated from a patient suffering from multiple myeloma, and we investigate the effect of several physicochemical parameters on the in vitro stability of this protein. The dimer is stable in physiological conditions and aggregation is observed only when strong denaturating conditions are applied (acidic pH with salt at large concentration or heating at melting temperature Tm at pH 7.4). The produced aggregates are spherical, amorphous oligomers. Despite the larger β-sheet content of such oligomers with respect to the native state, they do not bind Congo Red or ThT. The impossibility to obtain fibrils from the light chain dimer suggests that the occurrence of amyloidosis in patients requires the presence of the light chain fragment in the monomer form, while dimer can form only amorphous oligomers or amorphous deposits. No aggregation is observed after denaturant addition at pH 7.4 or at pH 2.0 with low salt concentration, indicating that not a generic unfolding but specific conformational changes are necessary to trigger aggregation. A specific anion effect in increasing the aggregation rate at pH 2.0 is observed according to the following order: SO4−≫Cl−>H2PO4−, confirming the peculiar role of sulfate in promoting protein aggregation. It is found that, at least for the investigated case, the mechanism of the sulfate effect is related to protein secondary structure changes induced by anion binding

Continuous counter-current chromatography for capture and polishing steps in biopharmaceutical production

The economic advantages of continuous processing of biopharmaceuticals, which include smaller equipment and faster, efficient processes, have increased interest in this technology over the past decade. Continuous processes can also improve quality assurance and enable greater controllability, consistent with the quality initiatives of the FDA. Here, we discuss different continuous multi-column chromatography processes. Differences in the capture and polishing steps result in two different types of continuous processes that employ counter-current column movement. Continuous-capture processes are associated with increased productivity per cycle and decreased buffer consumption, whereas the typical purity-yield trade-off of classical batch chromatography can be surmounted by continuous processes for polishing applications. In the context of continuous manufacturing, different but complementary chromatographic columns or devices are typically combined to improve overall process performance and avoid unnecessary product storage. In the following, these various processes, their performances compared with batch processing and resulting product quality are discussed based on a review of the literature. Based on various examples of applications, primarily monoclonal antibody production processes, conclusions are drawn about the future of these continuous-manufacturing technologies

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. In this study, we address this problem by developing an efficient and continuous chromatographic purification process for ONs. In particular, we considered the chromatographic purification of an ON crude prepared by chemical synthesis using anion exchange resins. We demonstrate that in this system the competitive adsorption of the various species on the same sites of the resin leads to the displacement of the more weakly adsorbing species by the more strongly adsorbing ones. This phenomenon affects the behavior of the chromatographic units and it has been investigated in detail. Then, we developed a continuous countercurrent solvent gradient purification (MCSGP) process, which can significantly improve the productivity and buffer consumption compared to a classical single-column, batch chromatographic process