Experimental characterization of next-generation expanded-bed adsorbents for capture of a recombinant protein expressed in high-cell-density yeast fermentation.

Expanded-bed adsorption (EBA) can be particularly useful in protein recovery from high-cell-density fermentation broth where conventional methods for harvest and clarification, such as continuous centrifugation and depth filtration, demand long processing times and are associated with high costs. In this work, the use of next-generation high-particle-density EBA adsorbents, including two mixed-mode resins, for the direct capture of a recombinant protein expressed in yeast at high cell densities is evaluated. Using classical experimental approaches and under different conditions (pH, salt, etc.), Langmuir isotherm parameters for these resins are obtained along with pore diffusivity values. Additional batch adsorption studies with Fastline® MabDirect, the resin that demonstrated the highest static binding capacity for the recombinant protein of interest under the conditions evaluated in this study, indicate competitive binding of nontarget proteins and approximately a 30% reduction in equilibrium binding capacity to 50 mg/mL settled bed in the presence of a 5%-10% cell concentration. Packed-bed (PB) dynamic binding capacities for the MabDirect resin (25-40 mg/mL PB) were significantly higher than for the Fastline® HSA resin and for the MabDirect MM resin in expanded-bed mode (5-10 mg/mL settled bed). Bed expansion behavior for the mMabDirect MM resin along with process yield and eluate purity are identified as a function of linear velocity and cell density, demonstrating process feasibility for pilot scale use.

Virus filtration of high-concentration monoclonal antibody solutions.

The ability to process high-concentration monoclonal antibody solutions (> 10 g/L) through small-pore membranes typically used for virus removal can improve current antibody purification processes by eliminating the need for feed stream dilution, and by reducing filter area, cycle-time, and costs. In this work, we present the screening of virus filters of varying configurations and materials of construction using MAb solutions with a concentration range of 4-20 g/L. For our MAbs of interest-two different humanized IgG1s-flux decay was not observed up to a filter loading of 200 L/m(2) with a regenerated cellulose hollow fiber virus removal filter. In contrast, PVDF and PES flat sheet disc membranes were plugged by solutions of these same MAbs with concentrations >4 g/L well before 50 L/m(2). These results were obtained with purified feed streams containing <2% aggregates, as measured by size exclusion chromatography, where the majority of the aggregate likely was composed of dimers. Differences in filtration flux performance between the two MAbs under similar operating conditions indicate the sensitivity of the system to small differences in protein structure, presumably due to the impact of these differences on nonspecific interactions between the protein and the membrane; these differences cannot be anticipated based on protein pI alone. Virus clearance data with two model viruses (XMuLV and MMV) confirm the ability of hollow fiber membranes with 19 +/- 2 nm pore size to achieve at least 3-4 LRV, independent of MAb concentration, over the range examined.

Maximizing productivity of chromatography steps for purification of monoclonal antibodies.

The large scale production of monoclonal antibodies presents a challenge to design efficient and cost effective downstream purification processes. We explored a two stage resin screening approach to identify the best candidates to be utilized for the platform purification of monoclonal antibodies. The study focused on commercially available affinity resins including Protein A, mimetic and mixed-mode interaction resins as well as ion exchangers used in polishing steps. An initial screening using pure proteins was followed by a final screening where selected resins were utilized for the purification of MAbs in complex mixtures. Initial screenings aimed to measure the theoretical upper limit for dynamic binding capacity (DBC) at 1% breakthrough and productivity. We confirmed that DBC of affinity, mimetic and mixed-mode resins was a strong function of the linear velocity used for loading. Productivities >27 g/(L-h), were obtained for rProtein A FF, Mabselect and Prosep rA Ultra at 2 min residence time. For the cation exchangers, we identified UNOsphere S and Fractogel SO(3) as the best candidates for our purification based on DBC. For anion exchangers operated in flowthrough mode, Q Sepharose XL and UNOsphere Q were selected from the initial screening based on DBC and resolution of IgG from BSA. Finally, a three step purification scheme was implemented using the selected affinity and ion exchangers for the purification of IgG from complex feedstocks. We found that Mabselect followed by UNOsphere Q and UNOsphere S provided the best purification scheme for our applications based on productivity.

Normal phase high-performance liquid chromatography of pneumocandins: in situ modification of silica with L-proline to separate structural analogues.

Lipopeptides such as pneumocandin B(0) are often produced by fermentation processes. Many compounds with similar structures (structural analogues), and hence similar physiochemical properties, are coproduced in the fermentation. We employed high performance liquid chromatography using silica gel as the stationary phase and a ternary ethyl acetate/MeOH/water mobile phase to separate pneumocandin B(0) from these structural analogues. Despite extensive efforts to optimize this system, two key structural analogues, pneumocandin E(0) and pneumocandin B(5), continued to be poorly resolved from the main product peak (pneumocandin B(0)). As a result, feed load was restricted and productivity was limited. In situ modification of the silica gel stationary phase with l-proline or other amino acids significantly enhances the resolution of the two key structural analogues from the compound of interest, enabling a two-fold increase in productivity. Results of a systematic study showed that the amine group in l-proline and other amino acids plays a key role in the modification of the surface of the silica gel to mediate the selectivity enhancement.

Preparation and evaluation of novel stationary phases for improved chromatographic purification of pneumocandin B0.

Preparation and evaluation of a number of stationary phases for improved chromatographic purification of pneumocandin B0, a key intermediate in the synthesis of the antifungal agent, Cancidas, has led to the identification of several materials with potential for improved performance.