One of the challenges in downstream processing is the design of robust process steps to efficiently isolate instable products, such as recombinant proteins, from complex fermentation broths to the required pharmaceutical degree of purity. Downstream operations usually represent 50-80% of the total processing cost, therefore, optimization of downstream processing technologies is considered the central element in appropriate process design.
The purification process includes chromatographic and filtration technologies. The combination and design of the single steps is of paramount importance for economic process development. In the overall process design the engineer has to consider different aspects such as elimination of contaminants, process scalability, automation, capacity of the production line, and regulatory compliance. Every single step of the process has to be validated and optimized in terms of yield, purity, and stability.
Protein purification is still the bottleneck of process development, and it is the improvement in chromatographic matrices that accounts for modern downstream processing.
At Rentschler Biotechnologie(www.rentschler.de), process design additionally comprises the implementation of new technologies such as expanded bed adsorption (EBA) and membrane adsorbers. These technologies increase yield, cut overall processing time and costs, and shorten time-to-market.
State-of-the-art chromatographic matrices. The requirements for novel matrices are demanding. Novel high throughput and high-capacity chromatographic matrices must meet GMP-quality requirements and be suitable for upscaling. The supplier must be able to provide the batches with tight specifications in order to assure a supply with consistent quality over a long period of time.
Furthermore, the matrices have to be stable over a broad pH range, permit column regeneration and cleaning, and should support increasing cycle numbers for economic considerations. In the case of nonadherence, the contract manufacturer cannot guarantee the manufacturing of a product over several years.
Expanded Bed Adsorption. With purification technologies, the challenge is to recover the target protein in its active form and in high yields from the hundreds or thousands of other proteins found in the host organism, as well as from the many components of the medium in which the cells were grown. Manufacturers have traditionally tackled this problem with a multi-step combination of techniques, commonly involving centrifugation and filtration.
Once clarified, the target protein can be concentrated via ultrafiltration/diafiltration and then purified further, usually by fixed-bed chromatography. More recently, however, expanded bed adsorption has emerged as a single-step alternative that achieves cell separation, clarification, concentration, and initial purification in one operation. EBA increases yield, cuts overall processing time, decreases labor and running costs, and reduces capital expenditure compared with conventional steps.
Membrane adsorbers. Membrane adsorbers are chromatographic membranes carrying functional groups for the binding of biomolecules. They are not filters, although the structure looks similar. Separation is achieved by reversibly binding the protein to the ligand.
Methods and buffers known from conventional gel chromatography can be directly applied. Compared to a conventional chromatographic resin, the major kinetic effect of the macroporous membrane adsorbers is the convective flowmolecules are transported to the binding sites by pump pressureand rapid film diffusion. The diffusion limitation in chromatographic beads is generated mainly by poor diffusion due to a small pore size in the nanometer range.
Adsorbers allow large flow rate ranges and show high binding capacity for large proteins. However, the use of innovative products, such as membrane adsorbers, can also be a risk rather than a blessing for process development. Therefore, Rentschler Biotechnologie is co-developing membrane adsorbers with Sartorius, among others.