Gas analysis mass spectrometer applications in fermentation and cell culture processes

Importance of the topic

Monitoring and controlling gas exchange in fermentation and cell culture is central to modern biotechnology process control. Off‑gas analysis provides rapid, non‑invasive insight into culture physiology (growth rate, substrate consumption, metabolic shifts and contamination), enabling optimization of yield, quality and run time. Accurate, fast gas analytics are especially critical where small concentration changes in O2 and CO2 or low‑level volatile organics (e.g., methanol, ethanol) indicate key transitions in microbial or mammalian cultures.

Objectives and overview of the application note

This application note describes the use of gas analysis mass spectrometers (Thermo Scientific Prima BT and Prima PRO) as online PAT tools for fermentation and mammalian cell culture processes. It summarizes why magnetic sector mass spectrometry (MS) is preferred for off‑gas analysis, practical sampling hardware (Rapid Multi‑Stream Sampler), software capabilities (GasWorks) and representative performance and process examples showing respiratory metrics (OUR, CER, RQ) and volatile detection.

Methodology and analytical approach

• Analytical principle: magnetic sector MS separates ions by momentum in a variable magnetic field. Compared with quadrupole MS, laminated magnetic sector analyzers combine high scanning speed with exceptional mass stability and low drift.
• Sampling strategy: the Rapid Multi‑Stream Sampler (RMS) enables heated, low‑dead‑volume selection of many sample streams (configurable settling times) with digital flow monitoring. RMS supports multi‑bioreactor monitoring without compromising response or sterility.
• Online calculations: GasWorks software computes O2 uptake rate (OUR), CO2 evolution rate (CER) and Respiratory Quotient (RQ) using inlet/outlet concentration and flow, with inert gases (N2 or Ar) used to correct for humidity and dilution effects.
• Volatile analysis: low‑ppm detection of methanol and ethanol is achieved by measuring the CH2OH+ fragment (m/z 31) while correcting for O2 tailing from m/z 32; the magnetic sector’s peak shape and mass stability minimize interference and enable reproducible ppm‑level measurements.

Used instrumentation

• Prima BT Bench Top Process Mass Spectrometer — compact solution for laboratory‑scale fermentors (e.g., 5 L to several tens of litres), multiple sample and calibration ports.
• Prima PRO Process Mass Spectrometer — production‑capable system with magnetic sector analyzer, supports monitoring of 60+ bioreactors when paired with RMS.
• Rapid Multi‑Stream Sampler (RMS) — heated multi‑stream inlet, up to 64 streams selectable, digital flow recording, designed for fast switching and low dead volume.
• GasWorks software (Data Review Plus module) — real‑time acquisition, RQ and respiratory rate calculations, long‑term data review.
• Support equipment referenced: single‑use bioreactors (HyPerforma S.U.B.) and standard sample conditioning components (desiccant towers, heated lines, filters).

Main results and discussion

• Long‑term stability: a Prima BT magnetic sector MS measured air cylinder components continuously for seven days (4–5 s cycle for four gases). Day‑to‑day variation for N2 and O2 was ≤ 0.005 %mol and CO2 varied by ~1 ppm or less, demonstrating exceptional baseline stability without frequent recalibration.
• Precision and speed: magnetic sector instruments demonstrated analytical precision multiple times better than quadrupole devices (typical oxygen precision reported on the order of 0.005 %mol; oxygen stability reported as ~0.05 % relative over 16 h in examples). Typical cycle times: 5 s for four gases; ~30 s per stream for six components including stream switching; can be reduced to ~10 s per stream when volatile organics are omitted.
• Respiratory monitoring: continuous inlet/outlet measurements of O2, CO2, N2 and Ar were used to compute OUR, CER and RQ. RQ was used to identify carbon sources and metabolic shifts (e.g., switch from ethanol to glucose in S. cerevisiae), and to trigger process interventions. Accurate RQ calculation depends on reliable flow correction using inert gases to compensate for humidity changes introduced by sparging.
• Volatile detection: measurement of methanol and ethanol at ppm levels is feasible due to the magnetic sector’s sharp, stable peaks and reduced mass‑scale drift; the instrument can reproducibly measure methanol/ethanol down to roughly 10 ppm by correcting for O2 tailing at m/z 31.
• Mammalian cell culture: mammalian fermentations (CHO, hybridomas) impose variable and broader inlet gas compositions (O2, CO2, N2 plus overlay gases). MS monitoring revealed how differing CO2 feed profiles affected viable cell count (VCC) and culture duration, informing changes to gas control strategies that extended culture lifetime and increased VCC.
• Scale‑up correlation: the application note emphasizes the need for correlation between lab and production analyzers to ensure consistent process control during scale‑up from bench to pilot and bulk production (up to 20,000 L), with MS providing continuous physiologic monitoring during each stage.

Benefits and practical applications of the method

• High precision and stability enable detection of small metabolic changes, early contamination screening and more accurate determination of optimal harvest times.
• Fast analysis and multi‑stream sampling support monitoring of many parallel bioreactors, improving throughput in process development and enabling centralized monitoring on production floors.
• Direct calculation of process‑relevant metrics (OUR, CER, RQ) facilitates tighter advanced process control (APC) and rapid tuning of feeding, pH or gas overlay strategies.
• Reliable low‑ppm volatile detection allows monitoring of solvent carryover or metabolic byproducts relevant for product quality and safety.

Future trends and potential applications

• Integration with advanced process control and digital twins: combining high‑resolution off‑gas data with machine learning models and PAT frameworks to predict culture trajectory and automate control actions.
• Expanded multi‑analyte monitoring: adding targeted volatile and trace‑gas monitoring to link metabolic signatures to specific product quality attributes or stress responses.
• Single‑use and distributed manufacturing: compact, robust MS and heated RMS configurations tailored for single‑use bioreactors and decentralized production sites.
• Higher throughput sampling and miniaturized sensors: innovations in stream multiplexing and local preconditioning to increase effective sampling density without compromising sterility or response time.

Conclusion

Magnetic sector gas analysis mass spectrometers (Prima BT and Prima PRO) paired with a Rapid Multi‑Stream Sampler provide a robust, high‑precision PAT solution for fermentation and mammalian cell culture monitoring. Their combination of speed, long‑term stability and low drift enables reliable calculation of respiratory metrics and detection of low‑level volatiles—capabilities that support improved process understanding, faster optimization and safer scale up from lab to production.

Reference

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4. P.C. van der Aar, A.H. Stouthamer, H.W. van Verseveld, Possible misconceptions about O2 consumption and CO2 production measurements in stirred microbial cultures, Journal of Microbiological Methods, Volume 9, Issue 4, 1989.