Date Published: June 1, 2018
Publisher: Springer Berlin Heidelberg
Author(s): Alexandra Hofer, Christoph Herwig, Oliver Spadiut.
Complex raw materials are widely used as supplements in biopharmaceutical production processes due to their positive effect on biomass growth and productivity at low cost. However, their use negatively impacts process reproducibility due to high lot-to-lot variability which contradicts current regulatory guidelines. In this study we investigated crude soy bean oil (SBO) which is a common complex raw material for filamentous fungi. We demonstrated that lecithin, which we define as phosphatidylcholines, is in fact the key material attribute in crude SBO positively affecting fungal growth and consequently productivity. The methodological toolbox we present here allows the straightforward isolation of lecithin from crude SBO, its semi-quantification by HPLC and the consequent supplementation thereof in defined amounts. Thus, over-dosage and potential resulting negative impacts on fungal growth and productivity can be omitted.
Complex raw materials are commonly used as cheap media supplements in bacterial, fungal and mammalian bioprocesses (Gao and Yuan 2011; Millis et al. 1963; Reese and Maguire 1969). These raw materials are typically of biological origin and thus underlie a high lot-to-lot variability. Furthermore, the specific substance responsible for the positive impact on the bioprocess is often unknown. However, during the International Conference of Harmonization, guidelines have been established demanding for the evaluation of critical material attributes (CMAs) and their impact on product quality (ICH 2009). Material attributes do not only affect product quality but also process performance and productivity. We define these material attributes that have an impact on productivity as key material attributes (kMAs). For both, identification of CMAs and kMAs, sound science-based knowledge of raw material quality has become a necessity.
Complex raw materials, which are multicomponent mixtures of biological origin, are often used as cheap media supplements for different organisms. However, the specific kMA in these mixtures triggering the positive effects on biomass growth and productivity are mostly unknown. Furthermore, these raw materials underlie high lot-to-lot variability (Hofer and Herwig 2017; Hofer et al. 2018) which naturally leads to variations in the processes and contradicts QbD guidelines (Saha and Racine 2010) (ICH 2009). Vegetable oils, such as soy bean oil (SBO), belong to these complex raw materials and are often used as media supplements in fungal bioprocesses. However, current QbD guidelines demand for science-based knowledge of raw material quality and the knowledge of mechanistic correlations between raw material attributes and their effects. In this study we hypothesized that lecithin is the kMA in SBO triggering the positive effects on fungal growth and productivity. To test our hypothesis we adapted a rather simple extraction method for lecithin from SBO based on lecithin degumming and developed an analytical method allowing the supplementation of defined amounts of either crude SBO or extracted lecithin. In a series of supplementation experiments with a P. chrysogenum strain we could demonstrate that lecithin is in fact the kMA in crude SBO causing beneficial effects on fungal growth. We assume an equal positive effect on productivity due to the correlation between growth an productivity in this process (Douma et al. 2010). For both crude SBO an extracted lecithin, these effects were concentration-dependent. In reproducibility studies we found that extracted lecithin caused less variation in biomass growth than crude SBO, which would be highly favorable in production processes. As the method of lecithin degumming is already used in large scale refinery processes, we believe that it can also be introduced as raw material preparation step for bioprocesses. Alternatively, we propose to quantify the amount of lecithin in crude SBO to have a means of evaluation for this complex raw material and to be able to avoid over-dosage of the supplement and thus negative effects.