Abstract The development of purification processes for protein biopharmaceuticals is challenging due to compressed timelines, long experimental times, and the need survey a large parameter space. Typical methods chromatography step evaluate several dozen chromatographic column runs optimize conditions. An efficient batch‐binding method screening conditions in 96‐well format with robotic liquid‐handling system described evaluated. dispenses slurries resins into filter plates, which are then...

Weak partitioning chromatography (WPC) is an isocratic chromatographic protein separation method performed under mobile phase conditions where a significant amount of the product binds to resin, well in excess typical flowthrough operations. The more stringent load and wash lead improved removal tightly binding impurities, although at cost reduction step yield. yield can be restored by extending column incorporating short end stage. use WPC with anion exchange resins enables two-column cGMP...

Ion-exchange (IEX) chromatography steps are widely applied in protein purification processes because of their high capacity, selectivity, robust operation, and well-understood principles. Optimization IEX typically involves resin screening selection the pH counterion concentrations load, wash, elution steps. Time material constraints associated with operating laboratory columns often preclude evaluating more than 20-50 conditions during early stages process development. To overcome this...

Abstract A high‐throughput screen (HTS) was developed to evaluate the selectivity of various hydrophobic interaction chromatography (HIC) resins for separating a mAb from aggregate species. Prior resin screen, solubility protein assessed determine allowable HIC operating region by examining 384 combinations pH, salt, and concentration. The then incorporated 480 batch‐binding elution conditions with eight in combination six salts. results were reproducible, demonstrated quantitative recovery...

Therapeutic monoclonal antibody (mAb) development and the processes for manufacturing drug substance have evolved since first approval of mAb in 1986. As past is often prologue to future, history these technologies has been classified here into three eras, leading speculation about what next era may hold with regard strategies, as well potential impacts patients. The substantial increase production culture titers bioreactor volumes availability large-scale contract facilities could translate...

Factor VIII-specific affibodies were selected from phage displayed libraries constructed by combinatorial mutagenesis of an alpha helical bacterial receptor domain derived staphylococcal protein A. Bead-immobilized recombinant human factor VIII (rVIII) (80 and 90 kDa chains) was used during competitive biopannings in the presence free 80-kDa chain protein, resulting selection several binders that showed dissociation constants (Kd) range 100-200 nM as determined biosensor analyses. One...

Abstract The effect of typical contaminants in inclusion body preparations such as DNA, ribosomal RNA, phospholipids, lipopolysaccharides, and other proteins on renaturation rate yield hen egg white lysozyme was investigated. Separate experiments were conducted which known amounts individual added to test their kinetics. On the basis a simplified model for kinetic competition between folding aggregation, it found that none above had an reaction, but some them significantly affected...

Abstract High‐throughput screening (HTS) of chromatography resins for identifying optimal protein purification conditions is becoming an integral part industrial process development. In this work, ceramic hydroxyapatite (cHA) 15 humanized monoclonal antibodies (mAbs) was examined by HTS. MAb binding, as quantified partition coefficient ( K p ), measured under 92 combinations sodium chloride, phosphate, and pH. Binding varied inversely with these variables all mAbs tested. However, the...

Formulation of protein biopharmaceuticals as highly concentrated liquids can improve the drug substance storage and supply chain, target product profile, allow greater flexibility in dosing methods. The Donnan effect cause a large offset pH from value established with diafiltration buffer during concentration charged proteins ultrafiltration membranes. For neutral formulations, will typically increase above for basic monoclonal antibodies decline below acidic Fc-fusion proteins. In this...

Affinity cosurfactants, consisting of hydrophilic ligands derivatized with hydrophobic tails, increase the efficiency selective protein recoveries using reversed micelles by extending operating range pH and salt concentration over which an extraction can be performed. Three different affinity cosurfactant-protein pairs have been used to demonstrate principles this extractive technique: (i) concanavalin A, a lectin, was extracted addition octyl glucoside; (ii) natural amphiphiles, such as...