Downstream Development

Protein A Intermediate Wash Strategies

Protein A affinity chromatography offers efficient monoclonal antibody (MAb) purification and is used extensively in large-scale MAb production. As is the case with most chromatography media, protein A resins often have some degree of nonspecific binding, which causes host-cell proteins (HCPs) to coelute with a MAb. To reduce nonspecific binding interactions, an intermediate wash step can be performed before product elution. Doing so can improve product purity, extend column lifetime, and potentially eliminate a subsequent polishing step. For large- scale…

Advances in Chromatography Automation

Not long ago, chromatography automation meant strip recorders and peristaltic pumps. Today, few people would consider that to be true automation, and even fewer would settle for binders full of strip-recorder paper reels. Automation is becoming intelligent and in the process is making our workflows smarter. But how close is automation to being as smart as an experienced scientist? Bio-Rad Laboratories spoke with academics, biotechnology R&D scientists, and industrial process engineers about the evolution of chromatography automation — where it…

Evaluating Freeze–Thaw Processes in Biopharmaceutical Development – Small-Scale Study Designs

Regulations mandate that biopharmaceutical product quality be controlled throughout manufacturing, storage, transportation, and delivery to patients (1). Operations often include freezing and thawing of a bulk drug substance, dilution of that purified substance to a target concentration, filtration, filling into a selected container–closure system, additional processing (e.g., lyophilization), inspection, packaging, storage, transport, and delivery (2). Freezing is a common processing step used to maintain stability and quality of a drug substance during development and production of biopharmaceutical products. It is…

Accelerated Product Development: Leveraging Industry and Regulator Knowledge to Bring Products to Patients Quickly

A Chemistry, Manufacturing and Controls (CMC) Strategy Forum titled “Accelerated Product Development: Leveraging Combined Industry and Regulator Knowledge to Bring Products to Patients More Quickly” was held in Washington, DC, on 27 January 2014. Biological therapeutics in development are demonstrating remarkable results in the clinic for many indications. So companies are seeking ways to accelerate the approval of these therapies and rapidly bring them to market. Many such products take the form of well-characterized proteins (e.g., IgG1 or IgG2 monoclonal…

Upstream Efficiencies, Economic Forces, and Changing Technologies Complicate Separation and Purification

When it comes to biotherapeutics manufacturing, downstream processing groups tend to get “dumped on.” Advances in cell lines, bioreactors, and culture media formulations have increased production output, providing both higher expression titers and greater volumes, but the filters and chromatography columns on the downstream side haven’t kept pace. These century-old technologies haven’t evolved as much and are reaching their limits. Regulatory agencies have contributed to innovation stagnation because they are cautious about manufacturing process changes for fear of undermining quality…

Evolving Clarification Strategies to Meet New Challenges

Increasingly efficient bioreactors allow biopharmaceutical manufacturers to achieve higher cell densities. That improved upstream efficiency has led to new purification challenges resulting from high product and contaminant concentrations as well as complex components. Therefore, harvest and clarification techniques are evolving to incorporate feed pretreatment, flocculation, and different filtration technologies such as normal-flow, tangential-flow, and depth filtration. The objective is to increase process capacities and filtrate quality, ultimately reducing biomanufacturing costs. New strategies for clarification of recombinant proteins (in particular, monoclonal…

Cost Estimation for Protein A Chromatography: An In Silico Approach to MAb Purification Strategy

Monoclonal antibody (MAb) production has adopted an accepted technology platform for downstream processing (1). The need for more economic processes has been addressed by increasing MAb titers in fermentation and aiming toward greater bioreactor volumes to increase productivity. Consequently, cost pressures are now passed on to downstream process groups. Membrane and chromatography resin savings are more important for MAb processes than ever before, with highly productive cell cultures generating large volumes of process fluid to purify (2). Traditionally, protein A…

Site-Specific Characterization of Glycosylation on Protein Drugs

A large proportion of biotherapeutic products are glycoproteins. These include erythropoietin and other cytokines, antibodies, glycosyltransferases, and glycosidases, which together generate billions of dollars in sales worldwide. Such drugs are inherently complex. As new treatments emerge and biosimilars are evaluated, the need to better understand their molecular structures is more acute than ever. Therapeutic glycoproteins are typically produced as recombinant products in cell culture systems. Glycosylation is of major importance during development of these drugs because their glycan chains markedly…

Enabling Greater Process Control and Higher Protein Titers: Advances in Downstream Single-Use Technologies

Downstream protein purification (the stage in which a protein is isolated and purified) is one of the last steps in biotherapeutic manufacturing. Single-use technologies are an increasingly popular choice for both upstream and downstream bioprocessing because they offer significant benefits over traditional multiuse manufacturing systems. Single-use technologies also provide an array of logistical benefits, including reduced costs, minimized risk of cross-contamination, and improved operational efficiency (1). Challenges remain, however, in designing a complete, streamlined, single-use process for downstream protein purification.…

Development of a Single-Use Filling Needle

Single-use components such as tubing, connectors, and filters have been widely used for many decades in bioprocess unit operations. Users have been able to identify and quantify the specific benefits of single-use over cleanable systems. In more recent years, many other process components have been designed for disposability such as bioreactors, mixers, and chromatography and ultrafiltration systems. Those and other advances have made it possible to incorporate multicomponent, presterilized manifolds into both existing and new processes, realizing benefits such as…