Brian D. Kelley

High-throughput screening of chromatographic separations: I. Method development and column modeling.

The development of purification processes for protein biopharmaceuticals is challenging due to compressed development timelines, long experimental times, and the need to survey a large parameter space. Typical methods for development of a chromatography step evaluate several dozen chromatographic column runs to optimize the conditions. An efficient batch-binding method of screening chromatographic purification conditions in a 96-well format with a robotic liquid-handling system is described and evaluated. The system dispenses slurries of chromatographic resins into filter plates, which are then equilibrated, loaded with protein, washed and eluted. This paper evaluates factors influencing the performance of this high-throughput screening technique, including the reproducibility of the aliquotted resin volume, the contact time of the solution and resin during mixing, and the volume of liquid carried over in the resin bed after centrifugal evacuation. These factors led to the optimization of a batch-binding technique utilizing either 50 or 100 microL of resin in each well, the selection of an industrially relevant incubation time of 20 min, and the quantitation of the hold-up volume, which was as much as one quarter of the total volume added to each well. The results from the batch-binding method compared favorably to chromatographic column separation steps for a cGMP protein purification process utilizing both hydrophobic interaction and anion-exchange steps. These high-throughput screening tools can be combined with additional studies on the kinetics and thermodynamics of protein-resin interactions to provide fundamental information which is useful for defining and optimizing chromatographic separations steps.

High-Throughput Screening of Chromatographic Separations: IV. Ion-Exchange

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 of IEX steps typically involves resin screening and selection of the pH and counterion concentrations of the load, wash, and elution steps. Time and material constraints associated with operating laboratory columns often preclude evaluating more than 20–50 conditions during early stages of process development. To overcome this limitation, a high‐throughput screening (HTS) system employing a robotic liquid handling system and 96‐well filterplates was used to evaluate various operating conditions for IEX steps for monoclonal antibody (mAb) purification. A screening study for an adsorptive cation‐exchange step evaluated eight different resins. Sodium chloride concentrations defining the operating boundaries of product binding and elution were established at four different pH levels for each resin. Adsorption isotherms were measured for 24 different pH and salt combinations for a single resin. An anion‐exchange flowthrough step was then examined, generating data on mAb adsorption for 48 different combinations of pH and counterion concentration for three different resins. The mAb partition coefficients were calculated and used to estimate the characteristic charge of the resin–protein interaction. Host cell protein and residual Protein A impurity levels were also measured, providing information on selectivity within this operating window. The HTS system shows promise for accelerating process development of IEX steps, enabling rapid acquisition of large datasets addressing the performance of the chromatography step under many different operating conditions. Biotechnol. Bioeng. 2008;100: 950–963.

High-Throughput Screening of Chromatographic Separations: II. Hydrophobic Interaction

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 to the resin screen, the solubility of the protein was assessed to determine the allowable HIC operating region by examining 384 combinations of pH, salt, and protein concentration. The resin screen then incorporated 480 batch‐binding and elution conditions with eight HIC resins in combination with six salts. The results from the screen were reproducible, and demonstrated quantitative recovery of the mAb and aggregate. The translation of the HTS batch‐binding data to lab‐scale chromatography columns was tested for four conditions spanning the range of product binding and selectivity. After accounting for the higher number of theoretical plates in the columns, the purity and recovery of the lab‐scale column runs agreed with the HTS results demonstrating the predictive power of the filterplate system. The HTS data were further analyzed by the calculation of pertinent thermodynamic parameters such as the partition coefficient, KP, and the separation factor, α. The separation factor was used to rank the purification capabilities of the resin and salt conditions explored. Biotechnol. Bioeng. 2008;100: 707–720.

Weak partitioning chromatography for anion exchange purification of monoclonal antibodies.

Weak partitioning chromatography (WPC) is an isocratic chromatographic protein separation method performed under mobile phase conditions where a significant amount of the product protein binds to the resin, well in excess of typical flowthrough operations. The more stringent load and wash conditions lead to improved removal of more tightly binding impurities, although at the cost of a reduction in step yield. The step yield can be restored by extending the column load and incorporating a short wash at the end of the load stage. The use of WPC with anion exchange resins enables a two‐column cGMP purification platform to be used for many different mAbs. The operating window for WPC can be easily established using high throughput batch‐binding screens. Under conditions that favor very strong product binding, competitive effects from product binding can give rise to a reduction in column loading capacity. Robust performance of WPC anion exchange chromatography has been demonstrated in multiple cGMP mAb purification processes. Excellent clearance of host cell proteins, leached Protein A, DNA, high molecular weight species, and model virus has been achieved. Biotechnol. Bioeng. 2008;101: 553–566.

High‐throughput screening of chromatographic separations: III. Monoclonal antibodies on ceramic hydroxyapatite

High-throughput screening (HTS) of chromatography resins for identifying optimal protein purification conditions is becoming an integral part of industrial process development. In this work, ceramic hydroxyapatite (cHA) chromatography of 15 humanized monoclonal antibodies (mAbs) was examined by HTS. MAb binding, as quantified by partition coefficient (K(p)), was measured under 92 combinations of sodium chloride, phosphate, and pH. Binding varied inversely with these variables for all mAbs tested. However, the magnitudes of binding among mAbs under identical conditions varied significantly, showing a >1.5 log range in K(p). Analysis of variance (ANOVA) techniques were used to describe the binding of each mAb as a function of the three screen variables. Linear models relating log K(p) to the pH, log[sodium chloride], and log[phosphate] fit the data for each antibody with 93-96% accuracy. From these models, characteristic charge values for the cation exchange and metal coordination components of the multi-modal mAb/cHA interaction varied twofold across the mAbs, reflecting inherent variability in the number of contacts between a particular mAb and the cHA surface. Furthermore, we reduced the number of test conditions required from 92 to 8 while maintaining an accurate representation of the full binding response surface. This eight-point modeling method accurately predicted the binding behavior of mAbs as well as mAb aggregates, a common impurity in crude mAb preparations. Using this eight-point modeling method, binding and selectivity information for mAb and aggregate can be obtained from less than two milligrams of protein, making the method attractive for early manufacturability assessments.