• PhyTip gel filtration columns operate as small scale, chromatographic separation columns.
  • They are the only high throughput, automated, chromatography desalting solution.
  • Similar to the PhyTip affinity columns, the resin of choice is positioned between two inert screens that maintain the resin bed at the base of the column.
  • Unlike the affinity columns, the sample is passed unidirectionally through the column by gravity flow.

High Performance:

  • Remove >95% of salts with >80% yield of protein
  • Retain protein functionality
  • <10% CV for volume and concentration between final samples
  • Ready for downstream assay

Automated desalting/buffer exchange of functional proteins:

  • Fully automated process with MEA Personal Purification System
  • 12 or 96 samples desalted/buffer exchanged in ~30 minutes.

High throughput format:

  • Available in 96 sample format for high throughput processing

Description

For biopharmaceutical companies whose focus is on antibody and recombinant protein molecules, there is an increasing desire to perform more screens of potential leads where the data generated are more physiologically relevant to the issues of potency, toxicity, and other factors. These assay formats all require that the antibodies and recombinant proteins are well purified, enriched and functional. By utilizing high-performance micro scale functional protein separations, it is now possible to obtain more physiologically relevant data at the high-content screening step and thus make the decision-making power available at the earliest stages in the discovery process. PhyNexus PhyTip column technology has been developed for high-through-put preparation of antibodies and recombinant proteins to facilitate the process of preparing large numbers of potential leads that are ready for functional assays without the need for scale-up.

In many cases differing elution conditions are required for various downstream functional assays. Furthermore, the final purification product may require an additional preparation step to either reduce toxic buffer salts or to completely exchange buffer conditions. This latter step can now be achieved through fully automated desalting and buffer exchange with PhyTip 5K gel filtration columns used in conjunction with the MEA Personal Purification System. These unique columns allow for high recovery of the functional antibody of interest while removing greater than 95% of salts.

Application

Sample Separation Process:

  1. Column Equilibration: PhyTip gel filtration columns are packaged in a shipping fluid designed to maintain proper hydration of the size exclusion resin. The shipping fluid is replaced with the buffer or solvent of choice by equilibrating the column by soaking the box in the solvent overnight.
  2. Sample Loading: The sample is applied to the top of the column using a transfer pipette tip. The sample enters the column by gravity.
  3. Elute: A chaser volume is added to the top of the column following sample loading to allow the sample to fully enter and exit the column. Discrete chromatographic fractions can be collected as they are eluted from the column.

PhyTip 5K Gel Filtration Columns

The design of the patent pending 5K gel filtration columns is based upon a unique manufacturing process where pipette tips are filled with gel filtration resin. Thin screens are placed above and below the resin bed that retain the gel filtration media within the column structure, while allowing the passage of liquids. The top frit is a larger liquid interface barrier that helps facilitate the transfer of liquid to the top of the column. PhyTip 5K gel filtration columns retain small molecules while molecules over 5 KDa will pass through the gel filtration media.

Desalting or Buffer Exchange Process

Using the MEA Personal Purification System, a row of either 200µL or resin bed PhyTip 5K gel filtration columns are conditioned in 1mL of buffer. After the columns are conditioned, samples are added and processed followed by differing volumes of chaser buffer addition and processing. The samples can be collected in fractions to determine the optimal sample recovery and salt removal conditions. Using the MEA Personal Purification System, each row of 12 samples requires 30 minutes to process while the entire plate of 96 samples can be processed in 2.5 hours.

Data
The process of desalting or buffer exchange using the PhyTip 5K desalting columns and the MEA Personal Purification System have been carefully optimized to ensure both maximum recovery of the protein of interest and removal of salt.
Documents
Robotics Systems Compatibility

High-throughput Gel Filtration PhyTip Columns are available for these robotic systems.

Part Numbers

High-throughput Gel Filtration PhyTip Columns