Liquid scintillation counting (LSC)

Radioactive tracers are commonly used to follow the behavior of elements or chemical species in chemical processes.⁵⁴⁹ Methods which use radioactivity to trace elements offer advantages as very high sensitivity, robustness to changing conditions and the possibility to quantify materials that stem from discrete classes of chemicals.⁵⁵⁰ The radioactivity can be measured with different detectors as the liquid scintillation counter. The LSC is a universal method that can measure several types of nuclear radiation (e.g. α-, β⁺-, β^--, γ- and ε-nuclides) simultaneously. The basic principle of LSC is the transformation of radioactive energy to light by a liquid scintillation cocktail (Figure 9 A). For example in the case of an α- or β-decay a solvent molecule absorbs a portion of the energy of the emitted particle and passes it over to other solvent molecules until it reaches e.g. a phosphor that absorbs the energy and re-emits it as light and relaxes to its ground state. For the sake of completeness, it shall be mentioned that most scintillation cocktails contain not only primary scintillation molecules that emit light but also secondary scintillators that function as a wave length shifter to improve signal. The light is then captured by an optical system and the signal gets amplified in a photomultiplier that converts light to an electric signal by a photosensitive surface. The light is emitted in pulses as for example β-decay events happen in pulses with a correspondingly short time and path within the scintillation cocktail, too. Therefore, the burst of photons resulting from one emission event is derived from a small local area and arrives at the photomultiplier with sufficient simultaneity to be considered as one pulse of light.

The energy of the light pulses corresponds with the energy of the emission events and the number of pulses per time corresponds to the number of radioactive emissions. This is of course only true if quenching is not considered. In reality, concentration-, self-absorption-, optic-, chemical- and color-quench can have a strong impact on counting yields. A LSC classifies each pulse of photons according to the energy of the β-emission event (number of photons) and collates them into channels (Figure 9 B). A common approach is to divide the energy spectrum into three energy ranges, where the lowest corresponds to ³H emission and the highest corresponds to ³²P emission.

In case the element of interest is the Sulphur of L-cysteine a radioactively labelled derivative of the amino acid can be used to do radioactive tracing experiments. Since the energy of S-35 and C-14 is nearly the same it would be tracked in the C-14 range (Figure 9 C).⁵⁵¹

Figure 9: Liquid Scintillation Counting. A) Radioactivity detection principle. B) Pulse channels divide the spectrum into isotope specific windows that collate the pulses of specific energy. C) The similar energy of ¹⁴C and ³⁵S decays (Graph was adapted from Moljk et al.⁵⁵¹).

2 Objectives

For a long time, the two only CDM quality parameters assessed were osmolality and final pH after preparation. However, the importance of the medium as the nutrition source for cells and as source of chemical components for recombinant protein expression underlines the outstanding importance for robust bioprocess engineering. In the same time the CDM is an aqueous solution of high chemical complexity and the versatile factors it is exposed to during preparation and storage emphasize the need to shed light on the chemical behavior. For years, medium at Boehringer Ingelheim (BI) has been developed with a focus on chemically defined formulation, cell growth and productivity. This has led to the situation that the development of specific analytics for the quantification of media compounds has not kept pace and that the media formulations showed difficult to explain phenomena like drastic oxygen consumption during preparation and precipitate formation over the course of storage time.

As instable medium is a major concern for prolonged CHO cell fed-batch processes a major focus of this thesis was to evaluate and develop analytical technologies for the characterization of CDM.

An ideal analytical technology for the highly specific and sensitive quantification of compounds added to the CDM solution is triple quadrupole MS. If evaluated for matrix effects, it is ideal to measure several compound classes in parallel and can be adapted to cover different compound concentration levels. Therefore, a part of this thesis aims at the development of a LC-QqQ-MS method especially adapted to the Boehringer Ingelheim media formulations and the specific laboratory situation.

Furthermore, this thesis aims at the application of the LC-QqQ-MS method to develop a chemical understanding of CDM liquid formulations. Additionally, multiple probe technologies have been evaluated on their applicability as online monitoring tools with the goal to estimate their usefulness as live media quality estimator during powder hydration. In parallel, the goal to assess the meaningfulness of the measured on-line profiles to compare the reproducibility of media batches was followed. Moreover, the application of online probes during raw material dissolution has the potential to describe the chemical behavior of liquid media. An example is the major oxygen consuming reaction happening in Boehringer Ingelheim feed medium after iron salt addition.

Another major concern that occurred over the time working with Boehringer Ingelheim proprietary media formulations was the formation of precipitate during storage. The criticality of liquid media forming precipitate is not only caused by the concern that cells may lack nutrients but also by process related problems like filter clogging and batch to batch variability. A first major step in understanding the impact of precipitation on cell culture robustness and more importantly to avoid the solid formation is to collect information about chemical identity.

Since the feed media have a tremendous impact on process performance in fed-batch cultivation and because of the high concentration they are expected to be especially prone for chemical instability. This, and their comparability to other CDM types, makes them an ideal subject for the characterization of media. Thus, in this thesis exclusively feed media were investigated.

3 Results and Discussion

This work has been structured into three main parts. The first and most important step of characterization studies is the development and implementation of suitable analytics. Therefore, the first chapter describes the LC-QqQ-MS method development and validation for CDM matrix.

In the second part, the focus lies on the application of on-line and off-line analytics for the generation of insight to the chemical behavior of CDM during medium preparation. Consequently, the chemical characterization of CDM over the storage period with versatile analytical technologies is described in the third part.

3.1 Development of dynamic multiple reaction monitoring (dMRM) method on an