Optimising biopharmaceutical processes with respiratory gas analysis

Fermentation and mammalian cell culture are critical processes for the production of vaccines and therapeutic monoclonal antibodies, and underpin many exciting advances in cell and gene therapies.

These complicated processes require careful monitoring of the environment, as well as the culture, to ensure high yields and avoid unwanted by-products. Even small changes in oxygen and carbon dioxide concentrations can have a significant impact on the quality, safety and consistency of the final biotherapeutic, meaning that processes must be closely monitored at every stage.

Analysing the gases being fed into and removed from the fermenter or bioreactor is an ideal and non-invasive way to achieve this, helping to characterise the behaviour and productivity of the cells, as well as providing information on the optimum point to cease the fermentation for maximum yield.

Information in real time

Online process analytical technologies are increasingly used for monitoring the sparge gases going into fermenters and the respiratory gases emitted in the biomanufacturing process. These methods can measure a variety of gases — including oxygen, carbon dioxide, nitrogen and argon — which must all be analysed to calculate the respiratory quotient (RQ), as well as the rate of oxygen consumption and carbon dioxide evolution. Knowing the RQ is essential to understand the health of the culture, indicating the metabolism efficiency and type of nutrients being consumed.

Precise evaluation of the concentrations of a bioreactor’s inlet and outlet gases — including volatile gases — provides an ideal approach to accurately track a culture’s growth kinetics and substrate consumption in a non-invasive manner, without compromising the sterility of the environment. This data provides invaluable insights to help optimise the process, feed times and the start of induction, as well as to determine the ideal time to stop fermentation for maximum viable cell mass.

Real-time gas analysis also provides opportunities to identify contamination prior to inoculation, as well as to detect unwanted by-products and the onset of poisoning. These factors improve overall manufacturing efficiency, reducing over-processing and waste, contributing to higher biopharmaceutical yields and profits.

Magnetic sector mass spectrometry

Mass spectrometry (MS) is ideal for the real-time monitoring of fermentation and cell cultures, largely owing to its speed, accuracy and versatility. MS platforms also offer more flexibility than alternative gas analysers, because their analytical methods are primarily defined in the software, allowing the analysis of a wide range of sample streams with different compositions. Furthermore, they require very low maintenance and are self-calibrating, which reduces downtime and allows for continuous use.

Magnetic sector MS — where charged particles are separated in a variable magnetic field — has emerged as a preferred method for fermentation monitoring, and many of the world’s leading biotechnology and pharmaceutical companies are successfully using this technology. This technique offers numerous advantages over quadrupole MS — including higher linearity, accuracy and precision — depending on the gases being analysed and the complexity of the mixture. It also uses a high ion acceleration voltage to produce high energy ions, reducing its susceptibility to scattering by contamination from residual molecules in the vacuum system. This enables the analyser to continuously operate for long periods between calibrations and achieve excellent stability.

Another benefit of magnetic sector MS is that instruments are less influenced by surface charging effects due to imperfect electrode surfaces, which can result in misalignment or drift in the mass axis. The signal intensity at any specific mass position appears as a flat-top peak, removing the need to measure the middle of the peak, making the system intrinsically fault tolerant. This allows magnetic sector MS systems to have long intervals between calibrations, making them extremely beneficial for prolonged fermentation processes, which can last days or even weeks.

Boost productivity by analysing multiple streams

Modern magnetic sector MS instruments come in many forms and are amenable to both laboratory use and large-scale production. For example, the Thermo Scientific Prima BT benchtop mass spectrometer is designed for continuous use in the laboratory environment. It is also equipped with a rapid multi-stream sampler (RMS), enabling it to switch between up to 15 different sample streams without compromising the quality of the sample presented to the analyser. In comparison, the Thermo Scientific Prima PRO process mass spectrometer is made for full-scale production and is capable of monitoring up to 64 fermenter and bioreactor sample streams. These RMS systems have been tested to switch streams up to six million times a year — for multiple years — with little or no maintenance.¹

Summary

The ever-increasing need for reliable, real-time gas analysis in the biopharmaceutical industry has led to the development of innovative and intuitive MS devices. These systems offer a simple and non-invasive way to analyse the gases involved in the fermentation process and don’t require sample collection or the use of sensors placed inside the sterile fermentation area, preventing any risk of contamination. Crucially, magnetic sector MS provides essential data — such as the RQ — allowing operators to make informed decisions about feed times and when to halt fermentation. This helps to significantly boost yields and profits, while contributing to a safer and more consistent biotherapeutic product.

^{1. Thermo Fisher Scientific. Process Mass Spectrometry in Biotechnology. (2010). https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fbrochures%2FD19632.pdf. Accessed May 5, 2023.}