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…
Downstream Processing
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…
Non-Invasive Sensors as Enablers of “Smart†Disposables
Disposable bioprocessing has come of age. Economic and regulatory conditions are driving the widespread adoption of disposable equipment at all stages of bioprocessing. This review considers the entire bioprocess chain and assesses the status of disposables. In particular, we focus on the current availability and need for additional sensors that will enable the disposable process to be integrated — in compliance — with the latest Process Analytical Technologies. Traditional bioprocessing is highly compartmentalized into upstream and downstream operations.…
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…
Better Positioned Than Ever
In June 2007, Sartorius AG acquired the French company, Stedim S.A., and a global solution provider for the biopharmaceutical industry was created. Since then, the new company — Sartorius Stedim Biotech (SSB) — has entered into collaborations with a number of other key industry players, including Metroglas, Bayer Technology Services GmbH and the recently acquired Wave Biotech AG to bring technologies such as single-use bioreactors, UVC irradiation and the first disposable–integrated electrochemical pH sensor into its rapidly expanding…
Implementing Gamma-Stable RFID Tags in Single-Use Fluid Management Systems
This paper describes how RFID (Radio Frequency Identification) technologies can be implemented into single-use systems to generate electronic records for both bag manufacturers and bag end-users. RFID technology will enable the user to both read and write all relevant product and process information directly onto the single-use bag, providing instantaneous data recall. The tag can also provide the user with immediate access to the bag’s original part number, lot number, date of manufacture, expiration date and other critical…
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,…
Applying Good Engineering Practices to the Design of Single-Use Systems
Significant changes are being incorporated into biopharmaceutical manufacturing processes as a result of drivers such as increasingly strict regulatory demands, reduction of manufacturing costs, and outsourcing to contract manufacturing organizations (CMOs). Historically, many biopharmaceutical processes were designed and built based on cleanable, reusable stainless steel systems and unit operations. Today several industry drivers are shifting some unit operations toward single-use technologies, namely lowered cross-contamination, reduced capital investments, and desired further reduction in manufacturing costs and shortened drug development times (1,2,3).…