Development and scale-up of the recovery and purification of a domain antibody Fc fusion protein-comparison of a two and three-step approach.

A robust, economical process should leverage proven technology, yet be flexible enough to adopt emerging technologies which show significant benefit. Antibody and Fc-fusion processes may capitalize on the high selectivity of an affinity capture step by reducing the total number of chromatographic steps to 2. Risk associated with this approach stems from the potentially increased time frame needed for process development as well as unforeseen changes in impurity profile during first scale-up of drug substance (DS) for animal toxicology and clinical phase I trials (FIH) production, which could challenge a two-step process to the point of failure. Two different purification strategies were pursued during process development for an FIH process of a dAB-Fc fusion protein. A two-step process was compared to a three-step process. The two-step process leveraged additives to maximize impurity reduction during affinity capture. While wash additives in combination with a mixed mode chromatography met all impurity reduction requirements, HCP levels were still higher as compared to the three-step process. The three-step process was implemented for manufacture of clinical material to mitigate risk.

Downstream Processing Technologies/Capturing and Final Purification : Opportunities for Innovation, Change, and Improvement. A Review of Downstream Processing Developments in Protein Purification.

Increased pressure on upstream processes to maximize productivity has been crowned with great success, although at the cost of shifting the bottleneck to purification. As drivers were economical, focus is on now on debottlenecking downstream processes as the main drivers of high manufacturing cost. Devising a holistically efficient and economical process remains a key challenge. Traditional and emerging protein purification strategies with particular emphasis on methodologies implemented for the production of recombinant proteins of biopharmaceutical importance are reviewed. The breadth of innovation is addressed, as well as the challenges the industry faces today, with an eye to remaining impartial, fair, and balanced. In addition, the scope encompasses both chromatographic and non-chromatographic separations directed at the purification of proteins, with a strong emphasis on antibodies. Complete solutions such as integrated USP/DSP strategies (i.e., continuous processing) are discussed as well as gains in data quantity and quality arising from automation and high-throughput screening (HTS). Best practices and advantages through design of experiments (DOE) to access a complex design space such as multi-modal chromatography are reviewed with an outlook on potential future trends. A discussion of single-use technology, its impact and opportunities for further growth, and the exciting developments in modeling and simulation of DSP rounds out the overview. Lastly, emerging trends such as 3D printing and nanotechnology are covered. Graphical Abstract Workflow of high-throughput screening, design of experiments, and high-throughput analytics to understand design space and design space boundaries quickly. (Reproduced with permission from Gregory Barker, Process Development, Bristol-Myers Squibb).

Buffer exchange path influences the stability and viscosity upon storage of a high concentration protein.

High concentration protein solutions are generally produced by spin column concentration (SCC) during early development and by tangential flow filtration (TFF) during later stages, when greater quantities of protein become available. This is based on the assumption that the protein generated by the SCC process would be fairly similar to the TFF process material. In this study, we report the case of high concentration solutions of an Fc fusion protein produced by the two processes using the same upstream drug substance (DS) with very different storage stability. The TFF and SCC batches were characterized for aggregation, viscosity, and hydrodynamic radius before and after storage at different temperatures (5°C, 25 °C, and 40 °C). Aggregation and viscosity of the solutions processed by TFF were higher than those processed by SCC upon storage at 25 °C and 40 °C for three months. Differential scanning fluorimetry (DSF) revealed differences in initial protein conformation. Upon exposure to shear stress, protein solutions showed conformational instability and increased aggregation upon storage at 35 °C. In addition, protein solution showed higher aggregation upon shearing under mixed (downstream purification process and final formulation) buffer conditions - which are more likely to be encountered during the TFF, but not SCC, process. These results were further confirmed in an independent experiment by Fourier transform-infrared (FT-IR) spectroscopy and aggregation analysis. Taken together, these data indicate that shearing the protein in intermediate, unstable buffer conditions can lead to conformational perturbation during TFF processing, which led to higher rate of aggregation and viscosity upon storage. This study highlights the importance of testing shear stress sensitivity in the transitional buffer states of the TFF process early in development to de-risk process related product instability.

Adaptation to high throughput batch chromatography enhances multivariate screening.

High throughput process development offers unique approaches to explore complex process design spaces with relatively low material consumption. Batch chromatography is one technique that can be used to screen chromatographic conditions in a 96-well plate. Typical batch chromatography workflows examine variations in buffer conditions or comparison of multiple resins in a given process, as opposed to the assessment of protein loading conditions in combination with other factors. A modification to the batch chromatography paradigm is described here where experimental planning, programming, and a staggered loading approach increase the multivariate space that can be explored with a liquid handling system. The iterative batch chromatography (IBC) approach is described, which treats every well in a 96-well plate as an individual experiment, wherein protein loading conditions can be varied alongside other factors such as wash and elution buffer conditions. As all of these factors are explored in the same experiment, the interactions between them are characterized and the number of follow-up confirmatory experiments is reduced. This in turn improves statistical power and throughput. Two examples of the IBC method are shown and the impact of the load conditions are assessed in combination with the other factors explored.

Purification of native dehydrin from Glycine Max cv., Pisum sativum, and Rosmarinum officinalis by affinity chromatography.

An improved method for the purification of dehydrin from soy (glycine max) is described. Acidic extraction of soy whey was followed by a three step chromatographic process: capture on copper charged Chelating Sepharose Big Beads, intermediate hydrophobic interaction chromatography on Source 15 PHE, and a polishing step on blue Sepharose. The 32-kDa native soy dehydrin was purified to a purity of greater than 98.5% with an overall recovery of 63%. When compared to a previously published purification procedure, recovery, time requirements, and sample preparation steps were improved. The developed method is readily scaleable. Preliminary results show that the process can be used for dehydrins from rosemary (Rosmarinum officinalis) and pea (Pisum sativum).