Internal virus polarization model for virus retention by the Ultipor(®) VF Grade DV20 membrane.

Several recent studies have reported a decline in virus retention during virus challenge filtration experiments, although the mechanism(s) governing this phenomenon for different filters remains uncertain. Experiments were performed to evaluate the retention of PP7 and PR772 bacteriophage through Ultipor VF Grade DV20 virus filters during constant pressure filtration. While the larger PR772 phage was fully retained under all conditions, a 2-log decline in retention of the small PP7 phage was observed at high throughputs, even under conditions where there was no decline in filtrate flux. In addition, prefouling the membrane with an immunoglobulin G solution had no effect on phage retention. An internal polarization model was developed to describe the decline in phage retention arising from the accumulation of phage in the upper (reservoir) layer within the filter which increases the challenge to the lower (rejection) layer. Independent support for this internal polarization phenomenon was provided by confocal microscopy of fluorescently labeled phage within the membrane. The model was in good agreement with phage retention data over a wide range of phage titers, confirming that virus retention is throughput dependent and supporting current recommendations for virus retention validation studies. These results provide important insights into the factors governing virus retention by membrane filters and their dependence on the underlying structure of the virus filter membrane.

Revealing a positive charge patch on a recombinant monoclonal antibody by chemical labeling and mass spectrometry.

During purification process development and analytical characterization, a recombinant human monoclonal antibody, referred to as rmAb1, showed an anomalous charge heterogeneity profile by cation-exchange chromatography (CIEC), characterized by extremely high retention and poor resolution between charge variants. Mass spectrometry-based footprinting methodologies that include selective labeling of lysine with sulfosuccinimidyl acetate and arginie with p-hydroxyphenylglyoxal were developed to map the positive charges on the rmAb1 surface. On the basis of the average percentages of labeling obtained for the lysine and arginine residues by peptide mapping analysis, the positive charges were more distributed on the surface in the Fab region than in the Fc region of rmAb1. By a comparative study of in-solution and on-resin labeling reaction dynamics, seven positively charged residues were identified to bind to the cation-exchange resin and they were located in the variable domains. Among them, three lysine and one arginine residues appeared to cluster together on the surface to form a positive charge patch. When the charge patch residues were neutralized by chemical labeling, rmAb1 exhibited a more typical CIEC retention time, confirming that the charge patch was responsible for the atypical CIEC profile of rmAb1. To our knowledge, this work is the first report revealing the amino acid composition of a surface charge patch on therapeutic monoclonal antibodies.

Exploration of overloaded cation exchange chromatography for monoclonal antibody purification.

Cation exchange chromatography using conventional resins, having either diffusive or perfusive flow paths, operated in bind-elute mode has been commonly employed in monoclonal antibody (MAb) purification processes. In this study, the performance of diffusive and perfusive cation exchange resins (SP-Sepharose FF (SPSFF) and Poros 50HS) and a convective cation exchange membrane (Mustang S) and monolith (SO(3) Monolith) were compared. All matrices were utilized in an isocratic state under typical binding conditions with an antibody load of up to 1000 g/L of chromatographic matrix. The dynamic binding capacity of the cation exchange resins is typically below 100 g/L resin, so they were loaded beyond the point of anticipated MAb break through. All of the matrices performed similarly in that they effectively retained host cell protein and DNA during the loading and wash steps, while antibody flowed through each matrix after its dynamic binding capacity was reached. The matrices differed, though, in that conventional diffusive and perfusive chromatographic resins (SPSFF and Poros 50HS) demonstrated a higher binding capacity for high molecular weight species (HMW) than convective flow matrices (membrane and monolith); Poros 50HS displayed the highest HMW binding capacity. Further exploration of the conventional chromatographic resins in an isocratic overloaded mode demonstrated that the impurity binding capacity was well maintained on Poros 50HS, but not on SPSFF, when the operating flow rate was as high as 36 column volumes per hour. Host cell protein and HMW removal by Poros 50HS was affected by altering the loading conductivity. A higher percentage of host cell protein removal was achieved at a low conductivity of 3 mS/cm. HMW binding capacity was optimized at 5 mS/cm. Our data from runs on Poros 50HS resin also showed that leached protein A and cell culture additive such as gentamicin were able to be removed under the isocratic overloaded condition. Lastly, a MAb purification process employing protein A affinity chromatography, isocratic overloaded cation exchange chromatography using Poros 50HS and anion exchange chromatography using QSFF in flow through mode was compared with the MAb's commercial manufacturing process, which consisted of protein A affinity chromatography, cation exchange chromatography using SPSFF in bind-elute mode and anion exchange chromatography using QSFF in flow through mode. Comparable step yield and impurity clearance were obtained by the two processes.

Inline ultrafiltration.

Inline ultrafiltration (UF) can significantly increase the recoverable mass of biopharmaceutical products when pool tank volumes are limiting. Using relatively small commercially available ultrafiltration cassettes, a proof-of-concept study demonstrates that inline UF can significantly increase recoverable mass in an antibody purification process. With ever-increasing cell culture titers pushing product masses to higher levels, inline UF offers a relatively easy-to-implement and less disruptive alternative to installing larger pool tanks and enables more cost-effective production utilizing facilities designed for smaller bulk sizes.

Ion exchange chromatography of monoclonal antibodies: effect of resin ligand density on dynamic binding capacity.

Dynamic binding capacity (DBC) of a monoclonal antibody on agarose based strong cation exchange resins is determined as a function of resin ligand density, apparent pore size of the base matrix, and protein charge. The maximum DBC is found to be unaffected by resin ligand density, apparent pore size, or protein charge within the tested range. The critical conductivity (conductivity at maximum DBC) is seen to vary with ligand density. It is hypothesized that the maximum DBC is determined by the effective size of the proteins and the proximity to which they can approach one another. Once a certain minimum resin ligand density is supplied, additional ligand is not beneficial in terms of resin capacity. Additional ligand can provide flexibility in designing ion exchange resins for a particular application as the critical conductivity could be matched to the feedstock conductivity and it may also affect the selectivity.

Surface extenders and an optimal pore size promote high dynamic binding capacities of antibodies on cation exchange resins.

Increased recombinant protein expression yields and a large installed base of manufacturing facilities designed for smaller bulk sizes has led to the need for high capacity chromatographic resins. This work explores the impact of three pore sizes (with dextran distribution coefficients of 0.4, 0.53, and 0.64), dextran surface extender concentration (11-20mg/mL), and ligand density (77-138 micromol H+/mL resin) of cation exchange resins on the dynamic binding capacity of a therapeutic antibody. An intermediate optimal pore size was identified from three pore sizes examined. Increasing ligand density was shown to increase the critical ionic strength, while increasing dextran content increased dynamic binding capacity mainly at the optimal pore size and lower conductivities. Dynamic binding capacity as high as 200mg/mL was obtained at the optimum pore size and dextran content.

Modeling electrostatic exclusion effects during ion exchange chromatography of monoclonal antibodies.

Recent experimental studies have shown a reduction in dynamic- binding capacity for both monoclonal antibodies and antigen-binding fragments at very low conductivity, conditions that should generate the greatest electrostatic attraction. This behavior has been attributed to the steric and electrostatic exclusion of the charged protein from the entrance of the resin pores. This manuscript presents a quantitative mathematical description of this phenomenon. The protein partition coefficient was evaluated using models for the partitioning of a charged sphere into a charged cylindrical pore, with the pore size distribution evaluated by inverse size exclusion chromatography. The results were in very good agreement with experimental data for batch protein uptake and dynamic-binding capacity over a range of pH and conductivity. This theoretical framework provides important insights into the behavior of ion exchange chromatography for protein purification.

Application of high-performance tangential flow filtration (HPTFF) to the purification of a human pharmaceutical antibody fragment expressed in Escherichia coli.

High-performance tangential flow filtration (HPTFF) is shown to successfully enable concentration, purification and formulation in a single unit operation. This is illustrated with feedstreams comprising recombinant proteins expressed in Escherichia coli (E. coli). Using positively charged cellulosic membranes of 100 kDa molecular weight cut-off and operating under a selected range of buffer pH and ionic strength at a filtrate flux of 100 L m(-2) h(-1), a 10-fold removal of E. coli host cell proteins (HCP) was obtained with an overall process yield of 98%. The HPTFF performance was shown to be robust and reproducible. In addition, the novel charged membrane was regenerated and re-used seven times without loss of selectivity or throughput. When compared with a conventional purification scheme, the proposed process results in the elimination of one chromatographic step, a 12% yield improvement and a significant reduction in purification cost of goods.

Ion exchange chromatography of antibody fragments.

Effects of pH and conductivity on the ion exchange chromatographic purification of an antigen-binding antibody fragment (Fab) of pI 8.0 were investigated. Normal sulfopropyl (SP) group modified agarose particles (SP Sepharosetrade mark Fast Flow) and dextran modified particles (SP Sepharose XL) were studied. Chromatographic measurements including adsorption isotherms and dynamic breakthrough binding capacities, were complemented with laser scanning confocal microscopy. As expected static equilibrium and dynamic binding capacities were generally reduced by increasing mobile phase conductivity (1-25 mS/cm). However at pH 4 on SP Sepharose XL, Fab dynamic binding capacity increased from 130 to 160 (mg/mL media) as mobile phase conductivity changed from 1 to 5 mS/cm. Decreasing protein net charge by increasing pH from 4 to 5 at 1.3 mS/cm caused dynamic binding capacity to increase from 130 to 180 mg/mL. Confocal scanning laser microscopy studies indicate such increases were due to faster intra-particle mass transport and hence greater utilization of the media's available binding capacity. Such results are in agreement with recent studies related to ion exchange of whole antibody molecules under similar conditions.