SUPPORTING LATER MANUFACTURING CHANGES
The ability for later changes in manufacturing equipment and processes is a prerequisite for most customers. To meet that
need, early product characterization efforts for investigational medicinal proteins were significantly increased at the pilot
plant. A dedicated process science team was established. For complex, sensitive products, a wide pattern of analysis including
in-process glycosylation analyses and bioactivity tests was agreed upon with the customers. Product-specific databases were
produced to characterize final, purified product and to control process consistency in each type of system and culture mode.
Unique flexibility, speed of material supply, and significant savings in QC and QA efforts at the pilot plant far outweigh
early increased product characterization activities and later reference comparability costs, if manufacturing changes in later
phases of product development cycle are necessary.
HIGH-EXPRESSION CELL LINES BOOST PLANT CAPACITIES
One of the major obstacles for protein manufacturing in the early stages of the product life cycle is the low expression rate
of early research cell lines of usually less than 5 pg/c-d in standard cell culture. Experienced teams for cell line and process
development were integrated into the company's Berlin pilot plant to develop and realize strategies for fast expression enhancement.
Table 2. Features of the Disposable-Based Bioreactor Systems
At least three major issues contribute to cell specific and volumetric productivity in final mammalian manufacturing processes.
Figure 5 schematically summarizes the contribution of each issue to the cell-specific productivity in the final manufacturing
The issues are:
- Molecular engineering of the manufacturing cell line (bars 1, 2, and 3)
- Initial media selection
- Process optimization in bioreactors including media optimization.
Over the last 15 years, validated fed-batch processes became the gold standard for antibody production. Volumetric productivities
of more than 100 mg/L-d were described for individual proprietary fed-batch processes in the late 90s.9,10 More than 80 mg/L-d could be reached in generic fed-batch processes by 2000.11 Corresponding cell-specific productivities referred to in different sources over the years constantly grew up to ranges of
20 to 60 pg/c-d. Key industrial players such as Amgen, Genentech, Lonza, Biogen IDEC, PDL, and Sigma-Aldrich Biotechnology
share the latest developments in this area at meetings such as the European Society of Animal Cell Culture Technology (ESCAT).12-17 Significantly lower cell-specific productivities were observed for other human proteins such as erythropoietin.18 Efforts in therapeutic protein production in cultivated mammalian cells were reviewed in 2004.19
Figure 5. Issues of Cell Specific Productivity in Mammalian Cell Culture
The cell-line development team at our pilot plant concentrated on engineering production-quality cell lines based on optimization
and adaptation of the starting cell line; vector design including a sequential selection strategy, complex promoters and translation
enhancement, as well as elements stabilizing expression. Care was taken to transfer the complete process to a serum-free environment.
Moreover, we developed technologies for targeted gene insertion of Chinese hamster ovary (CHO) cells as well as for a human
and mouse heterohybridoma. Based on a proprietary strategy, the IgH locus of heterohybridoma known to account for stable,
high-level expression was used to express a protein of choice.