Concluding remarks

5 Summary

estimate the state of compound dissolution the FBRM technology is limited by its lower limit of detection range. Additionally, it is sensitive on incident flow angle, mounting position in the tank and the kind of powder addition. In order to characterize a newly developed CDM recipe a preferably complete set of probes is recommended as each measurement principle allows conclusions on different chemical parameters. For a routine batch to batch comparability monitoring tool, for example after process transfer to manufacturing scale, conductivity is the most suitable of the sensors evaluated in this thesis. The reasons are it shows fast reaction to most of the CDM compound additions and is simple and robust to handle.

The value of characterizing many chemical parameters during medium preparation has been emphasized by the discovery of a tremendously oxygen consuming chemical reaction in CDM upon iron addition. Each unknown chemical reaction is not under control because the impact of educts and products on cell culture robustness cannot be assessed and the risk for process robustness is difficult to estimate. Even though to date it is not realistic to characterize each single chemical reaction taking place in a complex multi component mixture like CDM, at least the major reactions with such outstanding traits should be investigated in more detail. The approach with simplified basal powder mixtures enabled to limit the compounds responsible for the oxygen consumption upon iron addition. This example underlines that simplified basal powders or grouping of chemical compounds can be promising to either conclude on chemical reactions occurring in CDM or to decrease reactivity during preparation and storage. Due to this strategy the oxygen consuming reaction upon iron addition to basal powder could be restricted to an oxidation of ascorbic acid that is very likely accelerated by phosphate. The presumably emerging radicals could be scavenged by compounds such as pyruvate. Furthermore, the investigation of metal free basal powders showed that further oxygen consuming reaction mechanisms upon pH adjustment were present in CDM.

The study of simplified basal powders during preparation further revealed that the chemical composition of CDM impacts key chemical parameters as pH, redox potential, dissolved oxygen and conductivity. The application of the dMRM method developed showed that compounds like L-cystine reacted with a concentration increase upon iron addition. Others like thiamine, vitamin B6 and L-cysteine were either matrix dependent in MS measurement or had an impacted stability profile. Another interesting observation was the effect of glucose addition on L-2-aminobutyric acid concentration. The concentration decrease upon glucose addition suggests that the expected glycation reactions are happening in CDM.

A further major concern in present day CDM preparation, especially in large scale, is the control of preparation temperature. Since currently used manufacturing plants are equipped with medium preparation tanks made of stainless-steel without temperature control option the controllability of preparation temperature will remain limited in the future 10 to 20 years.

Therefore, understanding the impact of CDM hydration temperature on chemical composition is of high interest. The results of a chemical composition investigation of media prepared at different temperatures clearly shows that this critical parameter does not alter CDM composition.

Thus, an impact of preparation temperature in a range from 25 to 40°C on cell culture robustness is highly unlikely. In improbable events of process deviations this finding helps to specify trouble shooting activities.

The versatile results of CDM behavior during preparation emphasize the chemical complexity of CDM. This underlines the need to investigate chemical balances in complex mixtures to better understand the key raw material of bioprocesses.

Chemical characterization of CDM during and after storage

As modern cell culture facilities are filled to capacity they are run with high efficiencies and minimized warehousing of hydrated CDM. However, in order to increase flexibility it is still inevitable to store the liquid formulations for some time. Even though the standard storage temperature for liquid medium is 2-8°C a prolonged exposure to room temperature for example during cell cultivation in fed-batch processes is typical. Therefore, cell culture medium characterization experiments were conducted in liquid feed medium at room temperature.

The regular sampling of feed medium during storage showed that the concentrations of measured CDM compounds were not impacted of the storage vessel materials glass and plastic. The independence of vessel material is of great importance during process development where different scale cultivations require different format storage vessels. Another important observation was that in particular L-cysteine, L-proline, vitamin B6, thiamine and cyanocobalamin were unstable during medium preparation and storage. These findings bring a better understanding of the maturation of CDM and show that these compounds should be in focus during future media development. For example, oxidation products like L-hydroxyproline could be completely removed from basal powders. Vitamin B6 could be added in the stable pyridoxine form only and cyanocobalamin and thiamine should be tested if they are really essential for CHO cells. Furthermore, L-cysteine should be investigated for more stable derivatives or if a balance could be found with antioxidant compounds. The investigation of reaction products identified in literature proved the 6 oxidation products lumichrome, N-formylkynurenine, 5-hydroxy-L-tryptophan, DL-O-tyrosine, L-citrulline and DL-methionine sulfoxide being formed during medium storage. Knowledge about reaction products allows investigations for their impact on cell culture. The example of lumichrome as an oxidation product of tryptophan that harms cell culture performance clearly shows the importance to understand reaction mechanisms in CDM.³⁰⁹ Understanding the impact of reaction products is important to understand effects that are negative for cell culture. But in the same time there could be degradation products that are beneficial and positively impact medium development. If such cases exist, they would be as interesting for cell culture engineers.

Another phenomenon happening during cell culture media storage is the formation of solid precipitates. Understanding the identity of these precipitates and the underlying reaction mechanisms will further benefit the chemical understanding of CDM. The industry will especially benefit from this if the mechanisms that form precipitates in CDM are so deeply understood that they and the subsequent blockage of filters and loss of nutrients can be avoided. A first step in that endeavor is the establishment of suitable analytical tools. Over the course of this thesis FTIR, Raman microscopy, SEM-EDX, ICP-MS and ICP-OES have been found to be suitable tools to gain information. However, special care has to be taken on sample preparation and handling. By application of these tools proteinaceous material could be identified in a multitude of solid precipitates. Raman spectra of some of these precipitates could be clearly matched to insulin.

Another distinct identification of precipitate forming material was iron oxide. Furthermore, a silver precipitate could be identified as elemental sulfur with three orthogonal methods. This undoubtable precipitate identification with ICP-OES, SEM-EDX and Raman spectroscopy allowed to postulate a potential reaction mechanism responsible for silver precipitate formation.