Optimization of Protein Purification Using Micro-Scale Separation ColumnsJeremy Lambert, Murray Anderson, Doug Gjerde, Lee Hoang, PhyNexus, Inc., San Jose, CA

Biophysical and functional characterization of therapeutic candidates require that proteins are well purified and enriched once they have been expressed. The process for adequately preparing protein requires that sufficient quantities of material be scaled up and then processed in a time-consuming and serial manner using expensive chromatography equipment. Consequently, functional and analytical assays continue to be performed considerably downstream from the initial selection screens, thus relegating the high-value information they provide to the latter stages of drug discovery and development, depriving researchers of the critical information they need to make increasingly informed decisions earlier in their processes.

Recent advances in the area of miniaturized high-throughput tools for purification, enrichment and desalting of proteins eliminate bottlenecks associated with traditional protein purification techniques. By performing high-performance functional protein separations on small samples in parallel, it is now possible to obtain more relevant data in a completely automated format – thus making substantial improvements in return-on-investment by dramatically increasing the decision-making power available at the earliest stages in the antibody discovery and development process.

Investigation of protein separations in micro-scale chromatography columns is presented along with optimized conditions enabling functional and analytical characterization of therapeutic proteins purified by this unique format.

PhyTip column technology has been designed to provide high-performance protein purification in a format that allows for complete automation while maintaining a high level of control over the separation process. The high capacity disposable micro-columns are confined within the body of plastic pipette tips by encasing the resin between two inert screens situated at the ends of the tip (Fig 1). The unique design contributes virtually no dead volume to the column, resulting in extremely efficient processing of small sample volumes. In addition, the low backpressure of the columns facilitates processing using a low-cost automated pipetting system.

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Presented at IBC PepTalk Meeting, January 9-12, 2007, Coronado, CA

Figure 1. PhyTip affinity columns in 1000 µL tip size. Shown in the photo are columns with 10, 20, 40, 80, 160 µL affinity resin. The columns are prepared with a glycerol storage solution to maintain hydration of the agarose resin (A). Samples are introduced to the column by pipetting from microtiter plates (B).

A B

Samples and buffers are presented to the columns using SBS-standard 96-well plates. Flow-through, wash and elution fractions are retained in the original discrete wells throughout the process allowing for simple sample tracking and process monitoring. No material is discarded to bulk waste using the PhyTip column format (Fig 3).

Figure 3. Load, wash and elution fractions are retained in the original wells throughout the purification process. The columns are transferred between plates using an automated pipetting system.

Figure 4. Automated processing of PhyTip columns is achieved using the MEA Personal Purification System. The MEA is a compact bench top pipetting system providing options for purification using PhyTip columns of both 200 µL and 1000 µL tip sizes. The MEA is capable of processing 1-96 samples per run using five positions for sample and buffer plates and two positions for PhyTip columns and standard pipetting tips. Sample volumes ranging from 0.1 to 50 mL can be processed with the platform.

PhyTip® Columns: Design and Operation

Introduction

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Column Properties

Figure 5a. Flow rate and linear velocity of PhyTip columns (bed sizes 5-320 µL).

Figure 5b. Residence time as function of flow rate for a single intake cycle (left) and 4 intake/expel cycles (right). The PhyTip column format provides the ability to increase the residence time with repeated passes through the column.

Affinity Purification Columns

Example 1. Human IgG Purification with Protein A Columns

Figure 6a. Capture efficiency of hIgG as a function of residence time (5, 10, 40, 160 µL column).

Figure 6b. Elution efficiency as a function of buffer volume (5-160 µL bed size).

Figure 6c. Maximum yield as a function of column size using optimized binding and elution conditions.

Example 2. 6X-His tagged recombinant protein purification with Ni-IMAC Columns

Figure 7a. Capture efficiency of 6XHis-Ubiqutin (5 µL column).

Figure 7b. Elution efficiency of 6XHis-ubiqutin as a function of elution buffer concentration.

Figure 7c. Purity of 6XHis-ubiqutin as a function of imidazole in wash buffer.

Ion Exchange Columns

Figure 8a. Recovery of BSA from DEAE (left) and lysozyme from CM (right) ion exchange columns. A matrix of binding buffers of varying pH and elution buffers of increasing salt concentration allow for rapid process optimization using micro scale columns.

Figure 8b. Further process optimization of lysozyme binding conditions with cation exchange columns: binding efficiency as a function of residence time and capture cycles at pH 6.0.

Figure 8c. Separation of lysozyme and BSA with cation exchange columns. Protein recovery from a solution of lysozyme alone and a mixture of equal amounts of lysozyme and BSA.

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Micro-Scale Gel Filtration

Operation of gel filtration columns differs from the process described in previous sections. The columns are first equilibrated with the buffer of choice by passing the solution back and forth through the packed bed (A). The conditioned column is placed into a holder positioned above a 96-well collection plate and protein samples are loaded onto the top of the column using a transfer pipette tip (B) and pushed into the bed using the pipettor (B). A volume of chaser liquid is loaded onto the top of the column (D) and protein is recovered in the collection plate (E).

Figure 9. Micro-scale gel filtration columns (80,160,600 µL) and chromatography process.

Table 1 lists optimal sample and chaser volume conditions for maximizing buffer exchange and protein recovery (95-97% salt removal, 60-80% protein recovery). PhyTip gel filtration columns have been used for protein purification ranging for functional cell-based assays and analytical capillary electrophoresis assays.1

Column Size Sample Vol. (µL) Chaser Vol. (µL)80µL 20 3080µL 35 15160µL 40 80160µL 50 70160µL 60 60160µL 70 50160µL 80 40160µL 90 30600µL 100 450600µL 350 200

Table 1. Sample and chaser volumes for PhyTip gel filtration columns.

Figure 10. A mixture of DNP-glutamate (yellow color, MW 313) and myoglobin (brown color, MW 16,7000) was applied to a column containing 600 µL gel filtration resin chosen to retain molecules with a MW of 5,000 or less. A series of elution fractions were collected in separate wells within the collection plate.

Tips from left:

1. 600 µL column2. 200 µL sample load3-6. 4x100 µL chaser7. 400 µL chaser

Tubes from left:

1. Starting sample 2. 200 µL fraction 3-6. 100µL fractions7. 400µL fraction

• Micro-scale purification columns provide high-performance separation from small sample volumes eliminating therequirement to scale-up sample preparation procedures, resulting in reduced sample consumption and time associated with previous purification methods.

• The availability of multiple column bed sizes provides a range of capacities suitable for delivering purified protein

samples suitable for a wide range of downstream applications including analytical, functional, and structural studies. The automated process can be set up to purify proteins from sample sizes ranging from 100 µL to 50 mL.

• PhyTip 5k gel filtration columns allow for automated desalting of micro-scale volumes of protein, providing efficient salt removal and protein recovery for applications requiring buffer exchange.

Summary

References

1. James McElroy, Lynn Gennaro, Oscar Solas-Solano, “Automation of Sample Preparation with a Robotic Platform Purification System.” CASSS CEPharm 2006 Meeting, Jersey City, NJ, Oct. 2-5, 2006.

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