Separation/Purification

Modeling Perfusion Processes in Biopharmaceutical Production

    Perfusion processes are considered more difficult to model than batch-based fermentation processes because up to a third of a perfusion-based campaign is spent outside “steady-state” production mode. Variabilities in cell density, titer, and harvest rate (HR) during ramp-up necessitate planning and explicit modeling of variabilities in these processes and their subsequent downstream operations. Longer continuous fermentation times require more rigorous attention to risk than do batch-based systems. A flexible purification platform must respond to changing fermentation conditions. Here…

Improving Process Economy with Expanded-Bed Adsorption Technology

    Most biopharmaceutical processes involve purifying proteins and peptides from various sources. Typically, purification schemes contain multiple unit operations, including several chromatographic steps to ensure safe removal of critical impurities and contaminants. Each step affects the overall process economy by increasing operational cost and process time and by causing product losses. Carefully designing a purification procedure to reduce the number of steps is an efficient way to reach high process economy. Expanded-bed adsorption (EBA) technology is a powerful alternative…

Rapid and Scalable Microplate Development of a Two-Step Purification Process

    High-throughput screening and process development methods are becoming more widely used in the biopharmaceutical industry. Recent development of high-expression (high–target-titer) recombinant culture methods has enhanced the need to also develop more effective separation products, methods, and processes (1). Part of the solution would be chromatographic resins offering higher capacities and flow rates.       However, developing an optimized purification process that involves several chromatographic steps can consume significant time and samples. In addition, a purification process developed…

Primary Clarification of Very High-Density Cell Culture Harvests By Enhanced Cell Settling

    In recent years biopharmaceutical manufacturing has demonstrated major improvements in MAb production, exhibiting product titers as high as 25 g/L often associated with very high cell densities (1). High-density cell cultures with >150 million cells/mL pose a great challenge in clarification and further downstream processing because of a need to remove a large amount of biomass and increased levels of contaminants from cell debris generated during cell culture and harvesting. Production of biological substances (MAbs, in particular) usually…

PAT-Based In-Line Buffer Dilution

    Technological advancement has taken protein expression titers from concentrations measured in mg/L to those measured in g/L over just a few years (1). Annual demand for antibodies has reached several metric tons, which has spurred production of >100 kg batches of protein at a time (2). As upstream yields continue to increase, downstream purification involving process solution preparation and delivery must increase in proportion to keep pace with demand. That has placed facility and instrumentation capacity constraints front…

Questioning the Downstream Bottleneck

In preparing for our October supplement on bioprocess design, BPI’s contributing editor Lorna D. McLeod spoke with Bayer Healthcare’s Harald Dinter (vice president of global biological development) and Jens Vogel (CMC development team leader and head of isolation and purification in global biological development) about the downstream bottleneck. Is it or isn’t it a real problem? Does the answer depend on your point of view? BPI: “Does a company’s downstream capacity place practical constraints on increasing production titers? Is that…

Development of a Turn-Key Harvest Solution for Small-Volume Bioreactors

Over the past 10 years, disposable bioreactors have grown from a niche tool servicing small-scale projects to a common and essential component in the CGMP production of human therapeutics (1). Recent advances in filter integration, aseptic connectors, and disposable sensing allow entire cell culture processes to be performed using only single-use components. However, harvest and clarification operations remain largely dependent on centrifugation, cross-flow filtration, and depth filtration (2), which are all techniques that have not been widely adapted to single-use…

Rapid Purification of Lys-C from Cultures

Endoproteases specific for cleavage of peptidyl bonds on the C-terminal side of lysine residues (e.g., Lys-C) are produced from a number of bacterial species, including Achromobacter lyticus (1), Pseudomonas aeruginosa (2), and Lysobacter enzymogenes (3). The Achromobacter protease 1 (API) protein has been substantially characterized (4,5,6) and shown to be a resilient enzyme that can specifically cleave after lysine residues under a wide range of buffer conditions, including high concentrations of denaturing agents such as urea and sodium dodecyl sulphate…

Implementing Cost Reduction Strategies for HuMab Manufacturing Processes

    The combination of innovative and traditional process technologies has resulted in major advancements in the antibody industry, such as accelerated process development and time-to-market. In addition, this paper examines the avenues that have opened as a result of exploring established process technologies for new applications, as in the case of perfusion cell cultures to amplify dhfr-based expression cell lines by incrementally increasing selection markers in the perfusion medium for the faster generation of stable and high-productivity clones. Furthermore,…