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…
Downstream Processing
Development of a High-Capacity MAb Capture Step Based on Cation-Exchange Chromatography
Protein A affinity chromatography is traditionally used as the capture step for monoclonal antibodies (MAbs) (1,2,3). It yields high purity because only the fragment-crystallizable (Fc) region of an antibody (IgG1 or IgG2) or Fc-containing fusion protein can bind to the protein A ligand. The resulting specificity provides substantial reduction in impurities such as host cell proteins (HCPs) and DNA (4,5,6,7,8). The dynamic binding capacity of protein A chromatography resins is generally ≤40 g/L and depends highly on residence time because…
Increasing MAb Capture Productivity
Continually increasing bioreactor titers is placing pressure on downstream processing, especially chromatography steps, to process the greater mass of protein produced. Whereas an order of magnitude increase has been seen in titers over the last few years, no similar increase has yet been achieved in the capacity of chromatography resins. Meanwhile, the industry is coming under rising pressure to reduce manufacturing costs and the resulting cost per gram of monoclonal antibodies (MAbs) produced. Because of the specificity it offers, protein…
Use of Membrane Technology in Bioprocessing Therapeutic Proteins from Inclusion Bodies of
The ultimate goal of recombinant fermentation research is cost-effective production of desired proteins by maximizing volumetric productivity (to obtain the highest amount of protein in a given volume in the least amount of time). Bioprocessing for recombinant proteins using genetically modified organisms requires a stable, high-yielding recombinant culture, a highly productive fermentation process, and cost-effective recovery and purification procedures. Escherichia coli has been a widely used host for expression of recombinant proteins (1). Its advantages lie in the enormous data…
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…
Purifying a Recalcitrant Therapeutic Recombinant Protein with a Mixed-Mode Chromatography Sorbent
Mixed-mode chromatography sorbents can save time and money by reducing the number of steps required to purify recombinant proteins. They also have the potential to purify proteins that single-mode sorbents cannot. As the term mixed mode suggests, these sorbents contain ligands that offer multiple modes of interaction. Although mixed-mode sorbents are used extensively in solid-phase extraction for high-pressure liquid chromatography (HPLC) sample preparation — and to a more limited extent in analytical HPLC — these resins are generally unsuitable for…
MAb Contaminant Removal with a Multimodal Anion Exchanger
Monoclonal antibodies (MAbs) constitute ∼30% of the biopharmaceutical products currently under development (1). An increasing demand for MAbs during the past decade has led to intense development of high-expression cell cultures (2). Today, it is possible to see titers of 4–5 g/L, and expression levels as high as 15 g/L and greater have been reported. As a consequence, demand has increased for more efficient downstream processes. That demand, combined with its potential for reducing time-to-market, has increased interest in the…
The Reoccurrence of Mycoplasma Contamination: Prevention Strategies
The contamination of microbiological media by mycoplasmas such as Acholeplasma laidlawii is not a recent phenomenon. It has been a major problem with animal-derived sera since the 1980s and has been a concern in the management of cell cultures for decades. The main culprit of serum contamination was the inadequate blood collection methodology and was eliminated with the introduction of hollow collection needles. In addition to the introduction of an improved collection method, serum was filtered with 0.1…
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,…
Quantifying Trends Toward Alternatives to Protein A
Problems associated with affinity purification in antibody production continue to increase as upstream cell culture expression levels improve. As a result, many vendors and users in the biopharmaceutical industry are working to identify alternative technologies that can replace tried-and-true column chromatography. In the fifth annual report and survey by BioPlan Associates, 434 global respondents pointed to bottlenecks created by downstream processes as one of their most serious manufacturing problems today (1). Amost two-thirds (63.8%) said their facility is experiencing some…