Small, genetically engineered immunological constructs are being developed industry-wide for a growing range of in vivo applications. Examples include Fab, F(ab’)2, single-chain (sc) Fv, bis-scFV, diabodies, minibodies, and single-domain antibodies (1). Their small size potentially gives them access to tissues that are poorly accessible by intact antibodies; rapid clearance from blood and nontargeted tissues; lower immunogenic response; and eye-drop, inhalant, or oral administration. We report here on purification of an affinity-matured, humanized, antiprostate stem-cell antigen (PSCA) minibody for…
Chromatography
Novel Affinity Ligands Provide for Highly Selective Primary Capture
Downstream processing of biopharmaceuticals is costly and time-consuming, often involving multiple steps with significant time and energy expended on maximizing product quality and yield. Affinity chromatography is one of the simplest and most effective methods for purifying protein and peptide therapeutics, offering reduced process steps and therefore higher yields than nonaffinity methods can provide. Protein A is widely used for affinity purification of monoclonal antibodies (MAbs), Fc fragments, and Fc fusion proteins. But it is a challenge to…
50 Years of Sephadex Media
It has been 50 years since the first Sephadex paper was published (1). Readers of BioProcess International work in a field that was fundamentally affected by what happened after that paper appeared in 1959. So this anniversary is certainly worthy of a party and a few speeches. But there are lessons to be learned, too. Here we take a look at threads connecting events before and after the discovery of gel filtration chromatography and introduction of the Sephadex product. Interdisciplinary…
The Road to a Fully Disposable Protein Purification Process
What’s keeping senior biopharmaceutical executives awake late at night? According to BioPlan Associates, Inc., which publishes an annual comprehensive survey of the state of worldwide biopharmaceutical manufacturing, capacity constraints are among the key issues at hand (1). And one of the most important constraints is the lack of physical capacity in purification equipment. Bioreactors are producing a lot more protein than current downstream purification steps are designed for. Overcoming the resulting bottlenecks may require increasing the productivity of…
Single-Use, Continuous-Countercurrent, Multicolumn Chromatography
Manufacturing processes for biopharmaceuticals have undergone significant changes over the past decade. One of the most striking results of improved process sciences is the dramatic rise in expression levels from animal cell cultures. Figure 1 shows how some monoclonal antibody titers have increased about 30-fold over the past 15 years. These increasing titers have allowed current biomanufacturing facilities to produce larger product quantities than anticipated at the time they were designed and built. Figure 1: As a…
Hydrophobic-Interaction Membrane Chromatography for Large-Scale Purification of Biopharmaceuticals
Biopharmaceutical manufacturing is divided into two areas: upstream fermentation or cell culture and downstream purification processes. Each area contains multiple unit operations. A unit operation is defined as a step in processing using a particular type of equipment. Here, we focus on downstream process development, which must reliably produce a highly purified drug substance (often >99%). Downstream processing includes recovery, capturing, and polishing steps. The primary downstream unit operation is chromatography because of its simplicity and high resolving…
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…
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…