Mass Spectrometry (MS) is critical to both the development and manufacture of modern biological medical products, supporting the recent rise in approved biological license applications (BLAs).1,2
The pharmaceutical industry and regulatory bodies are pushing toward Quality by Design (QbD) and Process Analytical Technologies (PAT), and the information-rich data provided by MS is ideally suited to facilitate this transition.3
However, the utilization of conventional MS for real-time process monitoring in biologics manufacturing has been restricted to centralized analytical laboratories as a result of its size, complexity, and cost, despite the potential of MS to enhance process efficiency, safety, and economics.
This article outlines the use of the Microsaic Metabolite Monitoring PAT system in the upstream manufacturing of a biotherapeutic. This system comprises a Microsaic 4500 MiD® compact Mass Spectrometer and a Hydrophobic Interaction Chromatography (HILIC) workflow.
To enable the system to work automatically in an upstream bioprocessing workflow, it also utilized the Cogent Datahub, Seg-Flow automated sampling system (Flownamics), and PharmaMV software (Perceptive Engineering).
This facilitated the monitoring of metabolites as well as the control of glucose in a large 10 L upstream cell culture in almost real-time and at a continuous high frequency.
Methodology
The Microsaic Metabolite Monitoring PAT system includes a HILIC Chromatography workflow and a miniaturized microflow electrospray ionization (ESI) source MS. The 4500 MiD® miniaturized point-of-need MS from Microsaic, supplied with a chip-based single quadrupole, was employed as the MS detector in this workflow.
This 4500 MiD® system measures 55 x 35 x 25 cm and contains all the vacuum pumps and control PC integrated into a single unit. A modified Microsaic MiDas™ was employed to perform the HILIC workflow and sample delivery.
This modified Microsaic MiDas™ comprised a 2-position 10-port switching valve, as well as a ceramic pump head for biocompatibility and an extra column oven. The modified MiDas™ allowed receipt of samples from the Seg-Flow automated sampling system and control of an isocratic HILIC separation.
The compact sizes of both the 4500 MiD® MS and the MiDas™ allowed the system to be used on the bench, at the point-of-need, next to a bioreactor autosampler, allowing close to real-time monitoring.
Figure 1. Experimental configuration of the Microsaic Metabolite Monitoring PAT system – The autosampler, consisting of a Seg-Flow autosampler coupled to a Sample-Mod 300 (right), sampled from the 10 L bioreactor at a pre-defined time interval and delivered these filtered cell culture samples to the 1 µL injection loop on the MiDasTM equipped with a ceramic pump head (left). Samples were separated using HILIC chromatography prior to analysis by the point-of-need 4500 MiD®. Image Credit: Microsaic Systems plc
Masscape® is Microsaic’s control software. Here, it was utilized for controlling the Microsaic Metabolite Monitoring PAT system.
The workflow was set up to communicate with the Open Platform Communications (OPC) server with different system states and to automatically perform tasks, such as system start-up, column equilibration, and loop cleaning, upon receipt of a trigger from the OPC server.
The Flownamics Seg-Flow 4800 autosampler, equipped with a Flownamics Sample-Mod 300, was employed to sample the cell media from the bioreactor at regular intervals. Filtration was used to clarify the samples before they were delivered to the modified Microsaic MiDasTM (see Figure 1).
Filtered samples were delivered directly to the 1 µl sample loop on the modified MiDasTM and then injected into a HILIC column to allow the separation of metabolites of interest before detection by the 4500 MiD® MS for analysis.
The processes were communicated and controlled using the OPC server in combination with Remote Operations Protocol (ROP) commands. Prior to analyzing the cell broth samples from the bioreactor, a 10-point calibration was performed using matrix-matched standard samples.
For this work, only lactate, glucose, glutamate, and glutamine were monitored and quantified regularly using the Microsaic Metabolite Monitoring PAT system.
Masscape® was employed to automatically calculate the concentrations using specifically designed algorithms for the automatic analysis. The resulting data was exported directly to the PharmaMV software.
Anti-Her2 IgG1 expressing CHO cells were cultivated in the 10 L Biostat B-DCU bioreactor (Sartorius Stedim Biotech, Germany) at the Centre for Process Innovation (CPI).
The dissolved oxygen (DO), pH, agitation, and temperature were controlled at constant values, while the Microsaic Metabolite Monitoring PAT system monitored the glucose level in the bioreactor. The PharmaMV software utilized the results to control glucose concentrations to a pre-defined target of 4000 mg/L.
Additional metabolites, including amino acids, were not quantified but were separated on the column and could be examined with the 4500 MiD® MS.
Results
Using the Microsaic Metabolite Monitoring PAT system meant that the concentrations of the four metabolites could be concurrently monitored from a single injection, reducing the required sample volume and analytical time for each analysis.
The metabolite measurements with orders of magnitude difference in concentrations, such as glucose and glutamine, were able to be taken concurrently because of the broad dynamic range (3-4 orders of magnitude) of the 4500 MiD® mass spectrometer.
This allows the direct injection of filtered cell broth samples into the system without the need for additional dilution steps, as displayed in Figure 2.
Figure 2. Typical chromatogram showing the simultaneous metabolite profiling of the filtered cell broth sample delivered from the bioreactor via the autosampler. Image Credit: Microsaic Systems plc
During the cell culture, the Microsaic Metabolite Monitoring PAT system displayed a start-up success rate of 100% following the receipt of 68 triggers from the OPC server.
The system’s error-handling ability was also tested and demonstrated during the experiment. This is an important feature for enabling a truly automated PAT system with minimal human input. The constant monitoring of the metabolites in the bioreactor is vital for optimizing the cells’ condition.
Measurements were taken at regular intervals during the experiment. The metabolite concentration profiles are displayed in Figure 3 for lactate, glucose, glutamine, and glutamate.
Figure 3. Metabolite concentration profiles during the 10 L cell culture when testing the Microsaic Metabolite Monitoring PAT system. Glucose feeding (large bolus) interventions are shown as arrows. Image Credit: Microsaic Systems plc
With this increase in sampling frequency afforded by automation, the changes in metabolite concentrations can be monitored in more detail. For glucose feeding, the higher sampling frequency also allows feeding more frequently, which prevents the glucose from deviating too far from the target value of 4000 mg/L.
This allows improved control of glucose concentrations as well as better consistency of biologic Critical Quality Attributes (CQAs).
Conclusion
This article presents the integration of an affordable and compact mass spectrometer, the 4500 MiD®, together with a HILIC workflow for the automated monitoring of metabolites in upstream bioprocessing.
The Microsaic Metabolite Monitoring PAT system allows measurements to be taken at the point of need, outside of centralized laboratories and allows near real-time monitoring.
This PAT provides the opportunity for lower running costs than the currently used photometric assays. The methodology described in this article also has the potential to be employed in combination with workflows for monitoring the biologic product CQAs alongside cell culture metabolites.
References and further reading
- A Retrospective Evaluation of the Use of Mass Spectrometry in FDA Biologics License Applications, J. Am. Soc. Mass Spectrom. (2017), 28, 786-794.
- Biosimilar, Biobetter, and Next Generation Antibody Characterization by Mass Spectrometry, Anal. Chem. (2012), 84, 4637−4646.
- PAT — A Framework for Innovative Pharmaceutical Development, Manufacturing, and Quality Assurance, Food and Drug Administration, September 2004.
About Microsaic Systems
Founded in 2001, Microsaic Systems plc (AIM: MSYS) develops real-time, point-of-need mass spectrometers. Microsaic offers fast, accurate cutting-edge solutions to multiple industries across the world.
Core products, such as the compact MiD series of mass detectors, are designed to integrate seamlessly with a wide range of third-party OEM equipment or used as a standalone system. At the forefront of our design ethos is to deliver fast, easy to use, powerful mass spectrometry (MS) performance.
Patented chip-based MS technology and intuitive software enables real-time data generation at the point-of-need, not just in a centralised laboratory. Designed for both pharmaceutical and biopharmaceutical applications, continuous data is accessible at any stage of your workflow.
Over 20 years’ experience in mass spectrometry, microfluidics, vacuum systems, analytical processes, and miniaturised instrumentation has led to the development of our outsourced services. Laboratory, engineering, and monitoring issues can now be solved with a world-class team of chemists, physicists, and engineers by your side.
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