Monitoring the upstream process of cell culture by time-gated Raman

With the corona pandemic, the world realized that continuous manufacturing is inevitable in the biopharmaceutical industry. This is to produce higher quantities while maintaining consistent product quality. The greatest challenge faced in bioprocesses is maintaining the cell culture for the entire process. To attain manufacturing efficiencies with rising demand, modern state-of-art technologies should be adopted by the pharmaceutical industry. The FDA has started advocating PAT since 2004 so that technologies can be employed to continuously monitor, evaluate, and validate the process by measurements, tests, and the process-end point control.

Maintaining the cell culture during the entire bioprocess has been a challenge with conventional CW Raman. Time-gated Raman offers a solution to monitor the process.

Quality control by monitoring critical process parameters

In light of this, there is a need for actively monitoring the bioprocess. Raman spectroscopy is a powerful analytical technique that can be applied to monitor critical process parameters in the upstream process of cell culture. Raman spectroscopy is a non-destructive, vibrational spectroscopy that provides molecular fingerprint information about the structure and composition of a material. This technique has gained popularity in recent years due to its ability to provide real-time, in-situ measurements without the need for complex sample preparation.

In the context of cell culture, Raman spectroscopy can be used to monitor a range of crucial process parameters including cell viability, growth rate, nutrient consumption, and metabolite production. For example, the cellular metabolic state can be monitored by measuring the levels of specific metabolites such as glucose, lactate, and amino acids.

Timegate´s VP of Service Business, Amuthachelvi Daniel in a laboratory monitoring the upstream process of cell culture with time-gated Raman.

Suppressing the fluorescence to attain the molecular fingerprint information

Fluorescence emission is the ‘Achilles’ heel’ of conventional CW (continuous wave) Raman technology. Fluorescence emission is a competing physical phenomenon with Raman scattering, and it can overlap the whole Raman signal in the measured spectra making the monitoring of the state of cellular physiology impossible. The most common way to try to avoid fluorescence during Raman measurement is to use longer wavelength excitation. However, a significant amount of Raman scattering is lost when longer excitation wavelengths are used, leading to a poor signal-to-noise ratio in spectra, lower sensitivity, and considerably longer measurement times.

The cellular matrix includes several components and structures with intrinsic fluorescence properties, such as some basic structural units of proteins (tryptophan, tyrosine, and phenylalanine), some vitamins (e.g., pyridoxine and riboflavin), and cellular cofactors (flavin adenine dinucleotide (FAD), reduced nicotinamide adenine dinucleotide (NADH) and its phosphate derivative (NADPH). Undoubtedly, fluorescence emission has been a limiting factor preventing the practical, larger-scale adoption of this technology for bioprocess monitoring. The fluorescence issue is solved in time-gated Raman technology by recording the Raman signal before the advent of fluorescence.

PicoRaman M3 spectrometer and ProbePro connected to a bioreactor.

Optimal cell growth by monitoring nutrient levels, cell morphology, and structure

This disruptive technology can also be used to monitor the nutrient levels in the culture media, which are essential for optimal cell growth and viability. Changes in the nutrient concentrations such as glucose, glutamine, and amino acids can be detected using time-gated Raman spectroscopy, providing a valuable tool for process optimization.

Another potential application in cell culture is the monitoring of cell morphology and structure. Changes in cell morphology, such as cell size and shape, can be monitored using this technology, providing insights into the cell culture conditions and the impact of process parameters.

Close-up of the Time-gated Raman green laser in a bioreactor.

Chemometric Model for the CHO fed-batch

We have tested our latest PicoRaman M3 instrument in a CHO cell culture environment. Below are the regression models for the critical process parameters and the table of the regression coefficient.

Regression model graphs for the critical process parameters.

Table of the regression coefficient.

Conclusion

In conclusion, Timegated® Raman spectroscopy is a valuable analytical technique that can be used to monitor critical process parameters in the upstream process of cell culture. The ability to provide real-time, in-situ measurements without complex sample preparation makes this technology a promising tool for cell culture process optimization and control. Read more about time-gated Raman technology in biopharmaceutical applications.

Author

This blog was written by Timegate Instruments’ VP of Service Business Dr. Amuthachelvi Daniel. Amutha has a Ph.D.  in Biophotonics with 10 years of experience in the application of Raman spectroscopy for cancer/precancer detection and Raman imaging of cells and tissues. She was also a recipient of the Marie Sklodowska Curie IF fellowship and has done extensive work in cancer diagnosis using continuous wave Raman spectroscopy and SERS. Read more about her and the whole Timegate team.