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Dual Flow Chromatography for purification of proteins and plasmids

by Douglas T Gjerde

PhyNexus Dual Flow Chromatography

PhyNexus columns used with our proprietary Dual Flow Chromatography provide complete antibody binding for highly concentrated sample recovery from small samples and can be used in all major 8, 12 or 96 column robotic liquid handling systems.


PhyNexus is a life science company dedicated to producing tools to increase research and development productivity.  Dual Flow Chromatograph, developed and sold by PhyNexus to purify proteins and plasmids dramatically improves R&D productivity.

Chromatographic separations concepts and principles are used in dual flow chromatography, but in a novel manner using pipette tip columns. The chromatography functions with off-the-shelf liquid modified pipette tips in a 96-channel pipetting head.  The pipette tip columns can be used on any of the leading robotic liquid handling instruments.

The column bed is located at the end of the pipette tip.  The bed is contained by a top and bottom screen of woven mesh.  The column is available in 200 and 1000 µL pipette tip sizes and 5 different column bed sizes ranging from 5 µL to 160 µL.  Virtually any affinity resin type can be packed into the column format.

In Dual Flow Chromatography, the sample and mobile phase enter and exit at the column tip.  All flow is back and forth flow.  This includes sample loading, column washing and elution, and recovery of the desired material.  The columns are miniaturized and they operate in parallel up to 96 at a time with automation.

Biomolecules interact slowly with these affinity columns – due to their slow kinetics of capture.  Proteins purified from the columns are of high purity and an active and non-agglomerated product. This is very difficult to do since proteins denature or agglomerate very easily.  Plasmids (transfection grade) are also purified by the technology.

The time of interaction of biomolecules with the column may be insufficient and difficult to control (the flow is not reproducible).  The problem is exasperated with small columns. In dual flow chromatography, channeling does not matter, but the kinetics of interaction do.  There are multiple chances of interaction and it becomes impossible for interaction not to be complete at the end of the process.  Even separation beads along the walls of the column where channeling can occur capture sample.  With back and forth flow, it is almost impossible for interaction of the sample with the column not to be complete.

With dual flow chromatography on a pipette tip column: time of interaction is easily controlled, complete interaction of the analyses with the column can be achieved, sample is brought to all of the resin functional groups, there are multiple chances of interaction of sample molecules to the resin functional groups, and interaction time is sufficient to drive the equilibrium of sample and column interaction to completion.

Since in back and forth flow, column interaction is driven to completion or to equilibrium, separations are: Independent of flow rate, Independent of column diameter, Independent of how well the column is packed, Independent of amount of resin in the column, and Only dependent of column and mobile phase chemistry.

The chromatography is parallel, multivariable and scalable.   In one set of experiments, 96 sets of separation conditions were studied.  8 conditions each of capture, wash and elution conditions were varied with 4 different antibody proteins and captured on a Protein A affinity column.  Yield and agglomeration were measured.

The results show the importance of doing these experiments. The experiment shows that you can achieve high yield, but poor quality protein.  You can get high quality protein, but poor yield.  Only around 10 of the conditions studied gave both high yield and high protein quality.  Frankly, not all researchers will perform this study on a new protein or resin because it is so difficult.  With this technology, multi variant chromatography is very easy.

This technology has been adapted for all common robotic companies.  Virtually any robotic liquid handler is easily turned into a high throughput, 96 in parallel liquid chromatograph.


Why is dual flow chromatography different?

Well for one thing, there is No effect of slow capture kinetics; it’s irrelevant

There is no effect of how well the column is packed; it’s irrelevant

What does it allow us to do?

Miniaturize culture and sample (plate format used)

We can automate

We can process samples in parallel

Purifications conditions can scaled.  Scaling is important because conditions developed for a process are always scaled.