Rapid process monitoring & control in mammalian cell culture using off-gas mass spectrometry analysis

In a concerted effort to investigate the implementation of a process analytical technology (PAT), we have evaluated the applicability of off-gas mass spectrometry (MS) analysis in mammalian cell culture based on the inlet and outlet gases from bioreactors. The limitations of the system & potential applications are also discussed. Compared to other invasive methods that require physical probes inserted directly into the culture (e.g. bio-capacitance probe, Raman spectrometry probe, etc.), the MS only requires gas going in and out of the cell culture which means it has minimal impact on the culture itself. With the Prima BT magnetic sector MS (Thermo Fisher Scientific), multiple gas streams can be analyzed (i.e. multiple bioreactors can be connected to the same Prima BT MS) which could reduce analytical equipment footprint (c.f. other equipment that may require 1 equipment per bioreactor). Distance between bioreactors and the MS is also not an issue given a high enough gas flow and a long enough gas tubing connection to the MS, i.e. the MS can be placed a distance away or even a different site from where all the bioreactors are situated. Our data showed that small changes in the gas traces (O2, CO2, N2 & Ar) on the MS, down to 0.001% mol, can be captured during the following events: 1) feed media addition (containing surfactant) 2) antifoam addition 3) any gas perturbation caused by external factors (non-culture related; gas supply leak or loss) 4) metabolic changes in the cell culture The MS data from two replicate 5L benchtop glass bioreactor runs are very comparable, as is the offline data between them; this indicates that the MS data can be used reliably to identify process deviations during bioreactor runs and be used as a convenient way to evaluate batch to batch variation, within predefined specifications, for robust manufacturing. We have also demonstrated that the same MS setup in the benchtop experiments, with minor modifications, can be applied in a 50L single-use bioreactor (SUB). We would also report that there are technical considerations with respect to the feasibility of integrating this system to the various cell culture platforms. The data so far suggests that the off-gas MS analysis can be: 1) incorporated as a feedback control to determine feed addition rates; 2) used to correlate the growth and perhaps viability of the culture with the MS gas traces; 3) identify cell culture contamination; 4) used to correlate cellular metabolic activity in the culture with the MS gas traces from the O2 & CO2; 5) used to elucidate more detailed metabolic states by analyzing volatile organic carbons (VOCs). In conclusion, we believe that our work presented here will be of significant relevance to the cell culture community who are keen adopters of PAT and its practical implementation

Discovering Novel Small Molecule Compound for Prevention of Monoclonal Antibody Self-Association

Designing an antibody with the desired affinity to the antigen is challenging, often achieved by lengthening the hydrophobic CDRs, which can lead to aggregation and cause major hindrance to the development of successful biopharmaceutical products. Aggregation can cause immunogenicity, viscosity and stability issues affecting both the safety and quality of the product. As the hydrophobic residues on the CDR are required for direct binding to antigens, it is not always possible to substitute these residues for aggregation-reduction purposes. Therefore, discovery of specific excipients to prevent aggregation is highly desirable for formulation development. Here, we used a combination of in silico screening methods to identify aggregation-prone regions on an aggregation-prone therapeutic antibody. The most aggregation-prone region on the antibody was selected to conduct virtual screening of compounds that can bind to such regions and act as an aggregation breaker. The most promising excipient candidate was further studied alongside plain buffer formulations and formulations with trehalose using coarse-grained molecular dynamics (CGMD) simulations with MARTINI force field. Mean interaction value between two antibody molecules in each formulation was calculated based on 1024 replicates of 512 ns of such CGMD simulations. Corresponding formulations with an excipient:antibody ratio of 1:5 were compared experimentally by measuring the diffusion interaction parameter kD and accelerated stability studies. Although the compound with the highest affinity score did not show any additional protective effects compared with trehalose, this study proved using a combination of in silico tools can aid excipient design and formulation development

Stability of two competing populations in chemostat where one of the population changes its average mass of division in response to changes of its population.

This paper considers a novel dynamical behaviour of two microbial populations, competing in a chemostat over a single substrate, that is only possible through the use of population balance equations (PBEs). PBEs are partial integrodifferential equations that represent a distribution of cells according to some internal state, mass in our case. Using these equations, realistic parameter values and the assumption that one population can deploy an emergency mechanism, where it can change the mean mass of division and hence divide faster, we arrive at two different steady states, one oscillatory and one non-oscillatory both of which seem to be stable. A steady state of either form is normally either unstable or only attainable through external control (cycling the dilution rate). In our case no external control is used. Finally, in the oscillatory case we attempt to explain how oscillations appear in the biomass without any explicit dependence on the division rate (the function that oscillates) through the approximation of fractional moments as a combination of integer moments. That allows an implicit dependence of the biomass on the number of cells which in turn is directly dependent on the division rate function

Computational and Experimental Evaluation of the Stability of a GLP-1-like Peptide in Ethanol–Water Mixtures

Aggregation resulting from the self-association of peptide molecules remains a major challenge during preformulation. Whereas certain organic solvents are known to promote aggregation, ethanol (EtOH) is capable of disrupting interactions between peptide molecules. It is unclear whether it is beneficial or counterproductive to include EtOH in formulations of short peptides. Here, we employed molecular dynamics simulations using the DAFT protocol and MARTINI force field to predict the formation of self-associated dimers and to estimate the stability of a GLP-1-like peptide (G48) in 0–80% aqueous EtOH solutions. Both simulation and experimental data reveal that EtOH leads to a remarkable increase in the conformational stability of the peptide when stored over 15 days at 27 °C. In the absence of EtOH, dimerisation and subsequent loss in conformational stability (α-helix → random coil) were observed. EtOH improved conformational stability by reducing peptide–peptide interactions. The data suggest that a more nuanced approach may be applied in formulation decision making and, if the native state of the peptide is an α-helix organic solvent, such as EtOH, may enhance stability and improve prospects of long-term storage