Integration of a complete downstream process for the automated lab-scale production of a recombinant protein.

In this work, an automated downstream process for the purification and formulation of a recombinant protein was integrated at lab scale in a single chromatography unit. The purification chain consists of three bind-and-elute chromatography columns, a flow-through membrane chromatography step, and a final ultrafiltration-diafiltration (UFDF) step to concentrate and formulate the sample. An integrated downstream process increases productivity and decreases process time and hold-up volume. In addition, the automation of the process allows reducing the manual work and increases reproducibility. To integrate the downstream steps, all the intermediate tanks are removed, and the eluate of a column is loaded directly onto the next one. This makes it necessary to design the process in order to minimize the column volumes and the process time. A research software called Orbit was used to automate the purification process and implement a UFDF step in the chromatography unit. The whole downstream sequence was successfully implemented at lab scale, getting a pure concentrated and formulated product with a productivity of 1.09 mg mL-1 h-1, achieving a time reduction from almost two to one working day, while getting a similar yield and purity. Regarding the UFDF operation, the sample was concentrated 10 times, and 97% of the old buffer was exchanged by the formulation buffer with a sequential diafiltration.

Designing an Autonomous Integrated Downstream Sequence From a Batch Separation Process - An Industrial Case Study.

This work is a proof of concept of how a sequence of industrial batch separation steps together are used to form an integrated autonomous downstream process. The sequence in this case study consisted of an anion chromatography step, virus inactivation and finally a hydrophobic chromatography step. Moving from batch to integrated separation minimizes hold-up times, storage tanks, and required equipment. The conversion from batch to integrated mode is achieved by extracting operating points and separation data from batch chromatograms. The integrated separation process is realized on an ÄKTA Pure controlled by an open research software called Orbit, making it possible to operate complex process configurations including multiple steps. The results from this case study is the principle and method of the steps taken to automation, achieving a more continuous and efficient downstream process.

Mitigation of chromatography adsorbent lot performance variability through control of buffer solution design space.

The separation of undesired product-related impurities often poses a challenge in the purification of protein therapeutic species. Product-related impurity species, which may consist of undesirable isoforms, aggregated, or misfolded variants of the desired monomeric form of the product, can be challenging to remove using preparatory scale chromatographic techniques. When using anion exchange chromatography to remove undesirable product-related impurities, the separation can be highly sensitive to relatively small changes in the chromatography operating conditions, including changes to buffer solution pH, buffer solution conductivity protein loading, and operating temperature. When performing difficult separations, slight changes to the chemical and physical properties of the anion exchange adsorbent lot may also impact the separation profile. Such lot-to-lot variability may not be readily measurable by the adsorbent manufacturer, since variability can be highly dependent on a specific protein separation. Consequently, manufacturers of chromatographic adsorbents may not be able to control adsorbent lot to lot variability tightly enough to prevent differences from occurring when performing difficult product-related separations at the preparatory scale. In such cases, it is desirable to design a chromatography step with a control strategy which accounts for adsorbent lot to lot variability in the separation performance. In order to avoid the undesired changes to process consistency and product quality, a proper adjustment of the column operating conditions can be implemented, based on the performance of each adsorbent lot or lot mixture. In this work, we describe how the adjustment of the column buffer solution composition can be used as a design space based-control strategy used to ensure consistent process performance and product quality are achieved for an anion exchange chromatography step susceptible to adsorbent lot to lot performance variability. In addition, a "use test" is described that can be employed to determine the optimal buffer solution compositions for different anion exchange adsorbent lots based on the retention volume of the therapeutic protein during a gradient elution.