Miroslav Soos

Process performance and product quality in an integrated continuous antibody production process.

Continuous manufacturing is currently being seriously considered in the biopharmaceutical industry as the possible new paradigm for producing therapeutic proteins, due to production cost and product quality related benefits. In this study, a monoclonal antibody producing CHO cell line was cultured in perfusion mode and connected to a continuous affinity capture step. The reliable and stable integration of the two systems was enabled by suitable control loops, regulating the continuous volumetric flow and adapting the operating conditions of the capture process. For the latter, an at‐line HPLC measurement of the harvest concentration subsequent to the bioreactor was combined with a mechanistic model of the capture chromatographic unit. Thereby, optimal buffer consumption and productivity throughout the process was realized while always maintaining a yield above the target value of 99%. Stable operation was achieved at three consecutive viable cell density set points (20, 60, and 40 × 106 cells/mL), together with consistent product quality in terms of aggregates, fragments, charge isoforms, and N‐linked glycosylation. In addition, different values for these product quality attributes such as N‐linked glycosylation, charge variants, and aggregate content were measured at the different steady states. As expected, the amount of released DNA and HCP was significantly reduced by the capture step for all considered upstream operating conditions. This study is exemplary for the potential of enhancing product quality control and modulation by integrated continuous manufacturing. Biotechnol. Bioeng. 2017;114: 298–307.

Analysis of site-specific N-glycan remodeling in the endoplasmic reticulum and the Golgi

The hallmark of N-linked protein glycosylation is the generation of diverse glycan structures in the secretory pathway. Dynamic, non-template-driven processes of N-glycan remodeling in the endoplasmic reticulum and the Golgi provide the cellular setting for structural diversity. We applied newly developed mass spectrometry-based analytics to quantify site-specific N-glycan remodeling of the model protein Pdi1p expressed in insect cells. Molecular dynamics simulation, mutational analysis, kinetic studies of in vitro processing events and glycan flux analysis supported the defining role of the protein in N-glycan processing.

Controlling the time evolution of mAb N‐linked glycosylation ‐ Part II: Model‐based predictions

N‐linked glycosylation is known to be a crucial factor for the therapeutic efficacy and safety of monoclonal antibodies (mAbs) and many other glycoproteins. The nontemplate process of glycosylation is influenced by external factors which have to be tightly controlled during the manufacturing process. In order to describe and predict mAb N‐linked glycosylation patterns in a CHO‐S cell fed‐batch process, an existing dynamic mathematical model has been refined and coupled to an unstructured metabolic model. High‐throughput cell culture experiments carried out in miniaturized bioreactors in combination with intracellular measurements of nucleotide sugars were used to tune the parameter configuration of the coupled models as a function of extracellular pH, manganese and galactose addition. The proposed modeling framework is able to predict the time evolution of N‐linked glycosylation patterns during a fed‐batch process as a function of time as well as the manipulated variables. A constant and varying mAb N‐linked glycosylation pattern throughout the culture were chosen to demonstrate the predictive capability of the modeling framework, which is able to quantify the interconnected influence of media components and cell culture conditions. Such a model‐based evaluation of feeding regimes using high‐throughput tools and mathematical models gives rise to a more rational way to control and design cell culture processes with defined glycosylation patterns.

Microarray-based MALDI-TOF mass spectrometry enables monitoring of monoclonal antibody production in batch and perfusion cell cultures.

Cell culture process monitoring in monoclonal antibody (mAb) production is essential for efficient process development and process optimization. Currently employed online, at line and offline methods for monitoring productivity as well as process reproducibility have their individual strengths and limitations. Here, we describe a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)-based on a microarray for mass spectrometry (MAMS) technology to rapidly monitor a broad panel of analytes, including metabolites and proteins directly from the unpurified cell supernatant or from host cell culture lysates. The antibody titer is determined from the intact antibody mass spectra signal intensity relative to an internal protein standard spiked into the supernatant. The method allows a semi-quantitative determination of light and heavy chains. Intracellular mass profiles for metabolites and proteins can be used to track cellular growth and cell productivity.

Dynamic Response Studies on Aggregation and Breakage Dynamics of Colloidal Dispersions in Stirred Tanks

Aggregation and breakage of aggregates of fully destabilized polystyrene latex particles in turbulent flow was studied experimentally in both batch and continuous stirred tanks using small‐angle static light scattering. It was found that the steady‐state values of the root‐mean‐square radius of gyration are fully reversible upon changes of stirring speed as well as solid volume fraction. Steady‐state values of the root‐mean‐square radius of gyration were decreasing with decreasing solid volume fraction as well as with increasing stirring speed. Moreover, it was found that the steady‐state structure and shape of the aggregates is not influenced by the applied stirring speed.

Isotope labeling to determine the dynamics of metabolic response in CHO cell perfusion bioreactors using MALDI-TOF-MS

The steady‐state operation of Chinese hamster ovary (CHO) cells in perfusion bioreactors requires the equilibration of reactor dynamics and cell metabolism. Accordingly, in this work we investigate the transient cellular response to changes in its environment and their interactions with the bioreactor hydrodynamics. This is done in a benchtop perfusion bioreactor using MALDI‐TOF MS through isotope labeling of complex intracellular nucleotides (ATP, UTP) and nucleotide sugars (UDP‐Hex, UDP‐HexNAc). By switching to a 13C6 glucose containing feed media during constant operation at 20 × 106 cells and a perfusion rate of 1 reactor volume per day, isotopic steady state was studied. A step change to the 13C6 glucose medium in spin tubes allowed the determination of characteristic times for the intracellular turnover of unlabeled metabolites pools, τST (≤0.56 days), which were confirmed in the bioreactor. On the other hand, it is shown that the reactor residence time τR (1 day) and characteristic time for glucose uptake τGlc (0.33 days), representative of the bioreactor dynamics, delayed the consumption of 13C6 glucose in the bioreactor and thus the intracellular 13C enrichment. The proposed experimental approach allowed the decoupling of bioreactor hydrodynamics and intrinsic dynamics of cell metabolism in response to a change in the cell culture environment.

Breakup of Individual Colloidal Aggregates in Turbulent Flow Investigated by 3D Particle Tracking Velocimetry

Aggregates grown in mild shear flow are released, one at a time, into homogeneous isotropic turbulence where their breakup is recorded by three-dimensional particle tracking velocimetry (3D-PTV). T ...

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. For each scale, we measured the time evolution of the proteome, which enabled us to compare the impact of the bioreactor scale on the intracellular processes. Nearly absolute accordance between the scales was verified by data mining methods, such as hierarchical clustering and in-detail analysis on a single protein base. The time response of principal enzymes related to N-glycosylation was discussed, emphasizing major dissimilarities between the glycan fractions adorning the heavy chain and the corresponding protein abundance. The enzyme expression displayed mainly a constant profile, whereas the resulting glycan pattern changed over time. It is concluded that the enzymatic activity is influenced by the changing environmental conditions present in the fed-batch processes leading to the observed time-dependent variation.

Probing Coagulation and Fouling in Colloidal Dispersions with Viscosity Measurements: In Silico Proof of Concept

Colloidal dispersions in a flow can undergo the unwanted processes of coagulation and fouling. Prevention of these processes requires their proper understanding and the ability to monitor their extent. Currently, neither of these requirements is sufficiently fulfilled and this motivates the development of detailed models that capture the nature of the dispersion processes operating at the scale of primary colloidal particles. We model coagulation and fouling in colloidal dispersions using the dynamic discrete element method (DEM), with an interaction model accounting for particles that are elastic, adhesive, and stabilized by electrostatic charge. At the same time, the particles can adhere to the wall. Flow-field computation captures the mutual influence between particles and flow. The model also includes a pair-wise implementation of lubrication forces. The modeling results indicate that viscosity is highly sensitive to the formation of clusters, reflecting not only the larger size of clusters with increasing surface energy, but also the slower kinetics of coagulation in charge-stabilized dispersions. By contrast, viscosity is not sensitive to the attachment of particles to the wall. The mechanism of fouling determined from the simulation results comprises the initial bulk formation of clusters and subsequent dynamic wall attachment and detachment of the clusters. The presented work improves understanding of the dynamic behavior of colloidal dispersions, which is strongly relevant for industrial applications as well as for on-line monitoring and control.