The production of biotherapeutics involves many unit operations, and it is common practice to store in-process material as dictated by the manufacturing setup and scheduling of unit operations.
The stability of in-process materials can vary widely, and it is important to maintain parameters such as bioactivity and stability throughout the entire process. When a target molecule is expressed as extracellular protein that is secreted into the bioreactor or fermentation broth, the unit operations of protein harvest and initial column capture can be combined to protect proteins with limited stability.
The elimination of storage requirements and susceptibility to any cooling or freeze/thaw cycles between these two unit operations can contribute to a more robust process. Already proven at bench-scale and now running at pilot-scale, scientists at GE Healthcare Life Sciences have used automated hardware and software to link two such processes together.
We found that both the initial clarification/diafiltration, using a microfilter, and the initial column capture step, using adsorptive chromatography, were completed in less than eight hours. If run traditionally as individual unit operations these steps would have required twice the time and storage of the target protein in between the two operations. In addition, we reduced the process time by 50% by using standard UNICORN™ software for both crossflow filtration on UniFlux™ to clarify and ÄKTApilot™ to purify a target protein from yeast in one day.
The process to clarify/diafilter the target protein followed by column capture consisted of:
- Preproduct: Equilibrate both the filter and chromatography column.
- Product: Clarify/diafilter the sample while simultaneously loading the permeate onto the chromatography column. Terminate column load via air detection.
- Postproduct: At completion of clarification/diafiltration, run postproduct steps on the filter including water flush, clean-in-place (CIP), and storage.
- Postproduct: At completion of purification run postproduct steps on the column including CIP and storage.
Sample: BSA 1 g/L, 10% WCW baker’s yeast (locally sourced) in PBS buffer
Crossflow Filter: CFP2E5A hollow fiber (0.2 µm, 0.12 m2, 30 cm length)
Column: BPG™ 100/500 column with Q Sepharose™ Fast Flow media, 1 L
Normal Flow Filter: ULTA™ Cap CG 2" capsule column guard
UniFlux 10 is a 10 liter per minute crossflow filtration system that can accommodate flat sheet cassettes or hollow-fiber filters for micro- or ultrafiltration applications. ÄKTApilot is a benchtop pilot-scale chromatography system capable of purifying 10 grams or more of product per cycle depending upon media capacity. Both systems are controlled by UNICORN software, a 21 CFR Part 11 compliant platform.
Figure 1 shows the configuration of the two systems for simultaneous processing. The outlet from the UniFlux 10 permeate is sent to an intermediate vessel where the inlet from the ÄKTApilot sample valve block is also placed. The intermediate vessel could be a single-use bag and tubing from ReadyToProcess™ products, or a cleanable container. Ports can also be configured to enable sampling during processing for analysis.