Maduramycin, a novel glycosylation modulator for mammalian fed-batch and steady-state perfusion processes.

Controlling high-mannose (HM) content of therapeutic proteins during process intensification, reformulation for subcutaneous delivery, antibody-drug conjugate or biosimilar manufacturing represents an ongoing challenge. Even though a range of glycosylation levers to increase HM content exist, modulators specially increasing M5 glycans are still scarce. Several compounds of the polyether ionophore family were screened for their ability to selectively increase M5 glycans of mAb products and compared to the well-known α-mannosidase I inhibitor kifunensine known to increase mainly M8-M9 glycans. Maduramycin, amongst other promising polyether ionophores, showed the desired effect on different cell lines. For fed-batch processes, a double bolus addition modulator feed strategy was developed maximizing the effect on glycosylation by minimizing impact on culture performance. Further, a continuous feeding strategy for steady-state perfusion processes was successfully developed, enabling consistent product quality at elevated HM glycan levels. With kifunensine and maduramycin showing inverse effects on the relative HM distribution, a combined usage of these modulators was further evaluated to fine-tune a desired HM glycan pattern. The discovered HM modulators expand the current HM modulating toolbox for biotherapeutics. Their application not only for fed-batch processes, but also steady-state perfusion processes, make them a universal tool with regards to fully continuous manufacturing processes.

Raman-controlled pyruvate feeding to control metabolic activity and product quality in continuous biomanufacturing.

BACKGROUND: Despite technological advances ensuring stable cell culture perfusion operation over prolonged time, reaching a cellular steady-state metabolism remains a challenge for certain manufacturing cell lines. This study investigated the stabilization of a steady-state perfusion process producing a bispecific antibody with drifting product quality attributes, caused by shifting metabolic activity in the cell culture. MAIN METHODS: A novel on-demand pyruvate feeding strategy was developed, leveraging lactate as an indicator for tricarboxylic acid (TCA) cycle saturation. Real-time lactate monitoring was achieved through in-line Raman spectroscopy, enabling accurate control at predefined target setpoints. MAJOR RESULTS: The implemented feedback control strategy resulted in a three-fold reduction of ammonium accumulation and stabilized product quality profiles. Stable and flat glycosylation profiles were achieved with standard deviations below 0.2% for high mannose and fucosylation. Whereas galactosylation and sialylation were stabilized in a similar manner, varying lactate setpoints might allow for fine-tuning of these glycan forms. IMPLICATION: The Raman-controlled pyruvate feeding strategy represents a valuable tool for continuous manufacturing, stabilizing metabolic activity, and preventing product quality drifting in perfusion cell cultures. Additionally, this approach effectively reduced high mannose, helping to mitigate increases associated with process intensification, such as extended culture durations or elevated culture densities.

Co-current filtrate flow in TFF perfusion processes: Decoupling transmembrane pressure from crossflow to improve product sieving.

Hollow fiber-based membrane filtration has emerged as the dominant technology for cell retention in perfusion processes yet significant challenges in alleviating filter fouling remain unsolved. In this work, the benefits of co-current filtrate flow applied to a tangential flow filtration (TFF) module to reduce or even completely remove Starling recirculation caused by the axial pressure drop within the module was studied by pressure characterization experiments and perfusion cell culture runs. Additionally, a novel concept to achieve alternating Starling flow within unidirectional TFF was investigated. Pressure profiles demonstrated that precise flow control can be achieved with both lab-scale and manufacturing-scale filters. TFF systems with co-current flow showed up to 40% higher product sieving compared to standard TFF. The decoupling of transmembrane pressure from crossflow velocity and filter characteristics in co-current TFF alleviates common challenges for hollow fiber-based systems such as limited crossflow rates and relatively short filter module lengths, both of which are currently used to avoid extensive pressure drop along the filtration module. Therefore, co-current filtrate flow in unidirectional TFF systems represents an interesting and scalable alternative to standard TFF or alternating TFF operation with additional possibilities to control Starling recirculation flow.

Controlling the time evolution of mAb N-linked glycosylation, Part I: Microbioreactor experiments.

N-linked glycosylation is of key importance for the efficacy of many biotherapeutic proteins such as monoclonal antibodies (mAbs). Media components and cell culture conditions have been shown to significantly affect N-linked glycosylation during the production of glycoproteins using mammalian cell fed-batch cultures. These parameters inevitably change in modern industrial processes with concentrated feed additions and cell densities beyond 2 × 107 cells/mL. In order to control the time-dependent changes of protein glycosylation, an automated microbioreactor system was used to investigate the effects of culture pH, ammonia, galactose, and manganese chloride supplementation on nucleotide sugars as well as mAb N-linked glycosylation in a time-dependent way. Two different strategies comprising of a single shift of culture conditions as well as multiple media supplementations along the culture duration were applied to obtain changing and constant glycosylation profiles. The different feeding approaches enabled constant glycosylation patterns throughout the entire culture duration at different levels. By modulating the time evolution of the mAb glycan pattern, not only the endpoint but also the ratios between different glycosylation structures could be modified. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1123-1134, 2016.

Modulation of mAb quality attributes using microliter scale fed-batch cultures.

A high-throughput DoE approach performed in a 96-deepwell plate system was used to explore the impact of media and feed components on main quality attributes of a monoclonal antibody. Six CHO-S derived clonal cell lines expressing the same monoclonal antibody were tested in two different cell culture media with six components added at three different levels. The resulting 384 culture conditions including controls were simultaneously tested in fed-batch conditions, and process performance such as viable cell density, viability, and product titer were monitored. At the end of the culture, supernatants from each condition were purified and the product was analyzed for N-glycan profiles, charge variant distribution, aggregates, and low molecular weight forms. The screening described here provided highly valuable insights into the factors and combination of factors that can be used to modulate the quality attributes of a molecule. The approach also revealed specific intrinsic differences of the selected clonal cell lines - some cell lines were very responsive in terms of changes in performance or quality attributes, whereas others were less affected by the factors tested in this study. Moreover, it indicated to what extent the attributes can be impacted within the selected experimental design space. The outcome correlated well with confirmations performed in larger cell culture volumes such as small-scale bioreactors. Being fast and resource effective, this integrated high-throughput approach can provide information which is particularly useful during early stage cell culture development.

A high-throughput media design approach for high performance mammalian fed-batch cultures.

An innovative high-throughput medium development method based on media blending was successfully used to improve the performance of a Chinese hamster ovary fed-batch medium in shaking 96-deepwell plates. Starting from a proprietary chemically-defined medium, 16 formulations testing 43 of 47 components at 3 different levels were designed. Media blending was performed following a custom-made mixture design of experiments considering binary blends, resulting in 376 different blends that were tested during both cell expansion and fed-batch production phases in one single experiment. Three approaches were chosen to provide the best output of the large amount of data obtained. A simple ranking of conditions was first used as a quick approach to select new formulations with promising features. Then, prediction of the best mixes was done to maximize both growth and titer using the Design Expert software. Finally, a multivariate analysis enabled identification of individual potential critical components for further optimization. Applying this high-throughput method on a fed-batch, rather than on a simple batch, process opens new perspectives for medium and feed development that enables identification of an optimized process in a short time frame.

Effect of hydrocortisone on the production and glycosylation of an Fc-fusion protein in CHO cell cultures.

Glucocorticoids are known to modulate various cellular functions such as cell proliferation, metabolism, glycosylation, and secretion of many proteins. We tested the effect of hydrocortisone (HC) on cell growth, viability, metabolism, protein production, and glycosylation of an Fc-protein expressing Chinese hamster ovary (CHO) cell culture. HC extended cell viability but impaired cell growth. The inhibitory effect on cell growth was dose-dependent and decreased when the glucocorticoid addition was delayed. When HC was added after 2 or 3 days of culture, an increase in glutamate consumption was observed, which was reversed by the glucocorticoid receptor antagonist mifepristone (Mif). Titer and specific productivity increased in the presence of HC. The increase in titer was only slightly reversed by Mif. On the other hand, Mif by itself induced an increase in titer to a level comparable to or higher than HC. Protein glycosylation was altered by the glucocorticoid in a dose- and time-dependent manner, with a shift to more acidic bands, which correlated with an increase in sialic acid moieties. This increase, which was not linked to a decrease in extracellular sialidase activity in HC-treated cultures, was reversed by Mif. Predictive models based on design of experiments enabled the definition of optimal conditions for process performance in terms of viability and titer and for the quality of the Fc-fusion protein in terms of glycosylation. The data obtained suggest a use of glucocorticoids for commercial production of Fc-fusion proteins expressed in CHO cells.

Minimizing hydrodynamic stress in mammalian cell culture through the lobed Taylor-Couette bioreactor.

The objective of the present study was to investigate the effect of hydrodynamic stress heterogeneity on metabolism and productivity of an industrial mammalian cell line. For this purpose, a novel Lobed Taylor-Couette (LTC) mixing unit combining a narrow distribution of hydrodynamic stresses and a membrane aeration system to prevent cell damage by bubble bursting was developed. A hydrodynamic analysis of the LTC was developed to reproduce, in a uniform hydrodynamic environment, the same hydrodynamic stress encountered locally by cells in a stirred tank, particularly at the large scale, e.g., close and far from the impeller. The developed LTC was used to simulate the stress values near the impeller of a laboratory stirred tank bioreactor, equal to about 0.4 Pa, which is however below the threshold value leading to cell death. It was found that the cells actively change their metabolism by increasing lactate production and decreasing titer while the consumption of the main nutrients remains substantially unchanged. When considering average stress values ranging from 1 to 10 Pa found by other researchers to cause physiological response of cells to the hydrodynamic stress in heterogeneous stirred vessels, our results are close to the lower boundary of this interval.