Hydrophobic interaction chromatography (HIC) is commonly used as a polishing step in monoclonal antibody purification processes HIC offers an orthogonal selectivity to ion exchange chromatography and can be an effective step for aggregate clearance and host cell protein reduction HIC, however, suffers from the limitation of use of high concentrations of kosmotropic salts to achieve the desired separation These salts often pose a disposal concern in manufacturing facilities and at times can cause precipitation of the product Here, we report an unconventional way of operating HIC in the flowthrough (FT) mode with no kosmotropic salt in the mobile phase A very hydrophobic resin is selected as the stationary phase and the pH of the mobile phase is modulated to achieve the required selectivity Under the pH conditions tested (pH 60 and below), antibodies typically become positively charged, which has an effect on its polarity and overall surface hydrophobicity Optimum pH conditions were chosen under which the antibody product of interest flowed through while impurities such as aggregates and host cell proteins bound to the column This strategy was tested with a panel of antibodies with varying pI and surface hydrophobicity Performance was comparable to that observed using conventional HIC conditions with high salt

Purification of monoclonal antibodies by hydrophobic interaction chromatography under no-salt conditions

The biopharmaceutical industry is at a turning point moving toward a more customized and patient-oriented medicine (precision medicine). Straightforward routines such as the antibody platform process are extended to production processes for a new portfolio of molecules. As a consequence, individual and tailored productions require generic approaches for a fast and dedicated purification process development. In this article, different effective strategies in biopharmaceutical purification process development are reviewed that can analogously be used for the new generation of antibodies. Conventional approaches based on heuristics and high-throughput process development are discussed and compared to modern technologies such as multivariate calibration and mechanistic modeling tools. Such approaches constitute a good foundation for fast and effective process development for new products and processes, but their full potential becomes obvious in a correlated combination. Thus, different combinatorial approaches are presented, which might become future directions in the biopharmaceutical industry.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/elsc.201600033

Downstream process development strategies for effective bioprocesses: Trends, progress, and combinatorial approaches.

The present invention relates to methods for modulating the glycosylation profile of recombinantly-expressed proteins. In particular, the present invention relates to methods of controlling the galactosylation profile of recombinantly-expressed proteins by supplementing production medium, e.g., a hydrolysate-based or a chemically defined medium, with manganese and/or D-galactose.

Methods for controlling the galactosylation profile of recombinantly-expressed proteins

Screening of effective column rinse solvent for Protein-A chromatography.

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.

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

Hydrophobic interaction chromatography (HIC) uses weakly hydrophobic resins and requires a salting-out salt to promote protein-resin interaction. The salting-out effects increase with protein and salt concentration. Dynamic binding capacity (DBC) is dependent on the binding constant, as well as on the flow characteristics during sample loading. DBC increases with the salt concentration but decreases with increasing flow rate. Dynamic and operational binding capacity have a major raw material cost/processing time impact on commercial scale production of monoclonal antibodies. In order to maximize DBC the highest salt concentration without causing precipitation is used. We report here a novel method to maintain protein solubility while increasing the DBC by using a combination of two salting-out salts (referred to as dual salt). In a series of experiments, we explored the dynamic capacity of a HIC resin (TosoBioscience Butyl 650M) with combinations of salts. Using a model antibody, we developed a system allowing us to increase the dynamic capacity up to twofold using the dual salt system over traditional, single salt system. We also investigated the application of this novel approach to several other proteins and salt combinations, and noted a similar protein solubility and DBC increase. The observed increase in DBC in the dual salt system was maintained at different linear flow rates and did not impact selectivity.

Hydrophobic interaction chromatography in dual salt system increases protein binding capacity

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Hydrophobic interaction chromatography in dual salt system increases protein binding capacity