Reducing Cell Batch Losses by Improving Process Monitoring
Graham Lewis, Technical Sales Consultant: Process Mass Spectrometers Environmental and Process Monitoring
Thermo Fisher Scientific

Laboratories that develop biosimilars can go through thousands of fermentations before they successfully reproduce their target molecule. The goal, however, is to shorten the development lifecycle, eliminating errors and inefficiencies that lead to lost time and material. This article examines how improved fermentation monitoring can play a role in reducing batch loss and accelerating biosimilars development.

The advent of biologic medicines has exponentially increased the complexity of drug development processes. Unlike traditional small molecule drugs produced by chemical processes, biologics are made using genetically engineered microorganisms that generate protein-based medicines through fermentation and other biological processes. The resulting medicines are much more complex than small molecule drugs, often containing more than one thousand times the total number of atoms.

Due to the inherent complexity of large molecule drugs, the production of generic biologics is difficult. Small molecule drugs are relatively simple to characterise, and with some effort scientists can almost always engineer a process that results in an identical molecule. Large biologic medicines, on the other hand, are made primarily of proteins that are much more difficult to characterise and nearly impossible to reproduce identically. The difficulty is compounded by the fact that labs attempting to reverse engineer biologics rarely (if ever) have access to the original cell line that produced the drug. For the above reasons, generic versions of biologic drugs are never exactly the same as their branded counterparts. To make that difference clear, the generic versions are referred to as "biosimilars" and are evaluated on how reliably they achieve the same effect as the original product. Structural similarity of the two products is also considered, but does not have to be identical.

Developing Biosimilars
Successfully designing organisms that produce a biosimilar and creating an environment in which they can operate at maximum efficiency requires rapid prototyping over many iterations. Research and development scientists typically begin with a bacteria, yeast or other living cell that produces something close to the target product. The cells are then separated into several groups, and new DNA designed to produce a particular protein is inserted into each. Scientists then evaluate the output of each of the different cell lines to see which produces a product most similar to the target biologic. This line is then expanded and refined again, and the process repeats until an acceptable biosimilar is produced. The cell line that produced the biosimilar is then divided into batches and developed using various growth media. Once the most efficient growth medium is found, the process can begin to scale up to production.

The speed of this development process largely depends on the lab's ability to manage the healthy development of multiple groups of cells simultaneously - more lines and batches means faster iteration, which in turn means arriving at a finished product more rapidly. To achieve this speed, labs must have a reliable system in place to monitor the development of multiple different groups simultaneously and ensure that they are healthy, errors are detected quickly and remedies are administered as soon as possible. In biosimilar research and development, the failure of a batch of cells can be slow down the process and increase costs significantly, so recognising errors before they do significant damage should be a top priority.

The Importance of Monitoring Instruments
Instruments that monitor drug production in real time are becoming increasingly important as pharmaceutical companies incorporate biological systems into their drug development processes. The development of comparatively simple small molecule drugs, produced using chemical reactions alone, does not require monitoring techniques as comprehensive as those necessary for large molecule biologic development. The living cells that produce biologics and biosimilars are much more sensitive to environmental factors and process changes. For that reason, they are also more prone to failure and their condition must be monitored very closely.

In addition to pressure from the processes themselves, labs that develop biosimilars are also under pressure from regulators to ensure the quality of their processes using analytical technology. Over the past decade, regulatory agencies (including the US FDA) have introduced quality by design (QbD), process analytical technologies (PAT) and current good manufacturing practices (cGMP) regulations and initiatives that require drug development labs to monitor their processes more closely. This means that quality process monitoring is more than just a good business practice - in many cases, it is also necessary for regulatory compliance.

Mass Spectrometry for Cell Monitoring
One of the most reliable technologies for monitoring the health of multiple cell groups developing simultaneously is gas analysis mass spectrometry. Familiar to almost all laboratory professionals, mass spectrometry is a very common analytical technique that determines the composition of a sample by analysing the mass-to-charge ratio and relative abundance of gaseous ions .

Bench top mass spectrometers designed specifically to monitor fermentation processes work by analysing the effluent produced as cell batches develop. Mass spectrometers designed specifically for analysis of developing cell batches feature multi-point inlets that can monitor up to 15 bioreactor effluent streams simultaneously, allowing biosimilar development laboratories to drastically reduce cell batch losses, increase development speed and minimise costly rework. Very small changes in the concentration of CO2, O2 and other gases in the effluent of a developing batch provide valuable diagnostic information on the batch's overall health. Because cells used in biosimilar development are subject to damage by a wide range of variables, these diagnostic data are critical for preventing the loss of valuable batches.

In addition to batch health information, precise effluent monitoring allows scientists to review respiration metrics and determine how efficiently each batch is converting its growth medium into its expected output. This information can be used to optimise growth media and improve the overall efficiency of the process. More detailed the respiration data allows development teams to realise smaller and smaller efficiencies - and while some of these efficiencies may seem small in the development lab, they become huge when they’re applied production scale.

Benefits of Magnetic Sector Technology
To ensure reliable real-time detection of effluent composition changes, biosimilar research laboratories must select monitoring technologies that produce as few false positives as possible. The analysers within some gas analysis mass spectrometers are prone to drift over time - without calibration, this drift will generate false positive readings in effluent composition. For this reason, lab professionals should select instruments that incorporate analysers with minimal tendency to drift.

One of the least drift-prone mass spectrometry technologies is magnetic sector analysis. Unlike quadrupole gas analysis mass spectrometers, which control the trajectory of effluent ions using a combination of DC and AC electric fields, magnetic sector mass spectrometers use a variable magnetic field. This field influences the path of each ion as it passes through the magnetic sector analyser, influencing their trajectory and separating them by their mass-to-charge ratios. The separated ions then land on a single detector that analyses the whole effluent sample.

The analyser's output signal is a series of flat-topped peaks (figure 1) where each amplitude is proportional to the concentration of ions at each mass. Each peak represents a large target with consistent amplitude, meaning that the detector is not thrown off even by relatively large variations in the incoming ions' mass position. This capability makes magnetic sector mass spectrometers intrinsically fault-tolerant and perfectly suited for continuous monitoring of bioreactor effluent. The quadrupole MS, on the other hand, produces a round-topped Gaussian peak which is much more susceptible to drift.

The monitoring capabilities provided by multi-inlet magnetic sector mass spectrometers, such as the Thermo Scientific Prima BT, can significantly reduce the loss rate of biosimilar development cell batches. In addition to saving time and preventing unnecessary rework, this monitoring data can also help developers of biosimilars simplify their regulatory compliance obligations by providing clear documentation of quality processes. Finally, the highly detailed respiration metrics collected by the spectrometer allow development scientists to optimise the cell batches' growth media, reducing time to scale up and increasing the overall efficiency of the final process.