Holistic bioprocess engineering of antibody fragment secreting Bacillus megaterium

Antibodies and antibody fragments are most important tools for therapeutic and diagnostic

applications. An increasing demand of these high potential drugs makes less cost

intensive production systems most desirable. Compared to mammalian cell culture

microbial production platforms are beneficial with regards to reaching high production

titers, scale up approaches, regulatory aspects and reduced costs of goods. Amongst

these production systems those that are having the ability of an additional product

secretion are most advantageous as the downstream processing costs are decisively

reduced.

In this work Bacillus megaterium, as a Gram positive model organism, was used to

extensively study the production and secretion of the antibody fragment D.13 scFv. For

the bioprocess optimization a holistic approach was followed. First the aim was to

establish a high productive defined cultivation medium. Different media components like

carbon sources, metal ions and ammonium concentrations were screened throughout

various cultivation platforms ranging from micro titer plates to shaking flasks. Statistical

design of experiments and a genetic algorithm approach were used to establish an

appropriate defined high production medium. As a second step the process was transferred

to the bioreactor scale of several liter cultivation volume. An optimal bioprocess

strategy based on alternating growth and starvation phases was established to gain high

product titers of antibody fragment D1.3 scFv. As a final step an up-scale to a 100 L

bioreactor was done accounting for an advanced process control and considering ¿good

manufacturing practice¿ guidelines. The advanced bioprocess monitoring tool of flow

cytometry was used to gain deeper insights on microbial physiology at single cell level

regarding cell viability, cell integrity and production intensity. This knowledge was used for

a sophisticated cell physiology based bioprocess development and optimization. Furthermore

culture heterogeneities were measured and characterized for B. megaterium

producing antibody fragment D1.3 scFv under controlled bioreactor conditions. To obtain

additional information about the regulatory processes occurring inside the cell on gene

expression level a transcriptome analysis was performed comparing cells with an

increased production and secretion status to less producing and non-producing cells.

These cutting-edge technologies of flow cytometry and transcriptome analysis revealed

possible bottlenecks of the overall bioprocess¿ performance and product secretion of

antibody fragments with B. megaterium as production platform and form a robust basis for

rational strain optimization and advanced process designs.