The document introduces the MEA 2 Personal Purification System, which utilizes high-throughput robotics and PhyTip columns for automated sample preparation and purification. Key features of the PhyTip columns include flexible resin bed volumes, back-and-forth flow for increased throughput, and placement of resin and screens for low dead volume and high concentration elution. The MEA 2 system is equipped for automated processing of PhyTip columns, accommodating tips, reservoirs, and plates with a simple interface compared to FPLC systems. It allows for methods development, screening, scale-up, and various downstream applications such as crystallization and glycan analysis.

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The topic of today’s webinar is to introduce the MEA 2 Personal Purification
System. This is used exclusivley with PhyTip columns for complete purification
and sample prep solutions.

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4. Analytical tools require small amounts of sample. However, the samples must
be pure. PhyNexus has identified a major bottleneck in sample preparation. The
solution was to miniaturize, process in parallel and automate.
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5. Protein purification is not automated for any significant throughput. High
throughput systems lack performance.
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Utilize high throughput robotics by incorporating chemistry.

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The PhyTip column is the core technology.
Flexible: tip volume, resin bed volume.
The back-and-forth flow is cornerstone of technology. Unique process for
increased throughput.
Placement of resin and low dead volume screens for high performance capture,
efficient wash and high concentration elution.

8. PhyNexus is committed to installing solutions. The variety of resin bed sizes
gives the ability to fine tune yield and final concentration.This variety of resin
bed volumes is for specific requirements. PhyNexus installs solutions based on
downstream needs and performance requirements.
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9. Equilibrium binding for scale-up
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10. Efficient wash. Low dead volume, large dilution of interstitial space.
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11. Placement of resin and screens means low dead volume and high
concentration elution. Resin is mostly water, gentle for proteins and required to
maintain activity.
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Flexible technology for many applications. High performance due to column
design.
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13. Compatible robotic systems.
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14. System dedicated to processing PhyTip columns. Easy to use, does not require
specialized FTE. Versatile. Small footprint. Low maintenance. Just as reliable
and robust as large systems, but less intimidating.
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16. The standard MEA 2 is equipped with a 1200 and 200 µL 12-channel head,
software and computer. The MEA is equipped with a chiller and buffer reservoir
holder.
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17. The MEA deck accommodates tips, reservoirs and plates.
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20. IEX is possible by using step gradient elutions. Find conditions for large
isotherms. The protein is either on the resin or it’s off the resin.
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21. Conditions screening. In one experiment, study 288 conditions. Only possible
way of feasibly performing DOE studies.
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Method development. Chromatographic separations.

23. Scale-up. Predictable results because of equilibrium binding. Screen conditions
and use results to design scale-up purification.
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24. Merck was able to reproduce their two production scale columns using an 80 uL
and a 40 uL PhyTip column.
In this way, hundreds of fermentation conditions can be tested and the results
are directly scale-able.
These data are published.
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25. 1000-fold smaller column. Get scaleable results and comparable purity.
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26. Study many buffers at once. Screen for both conditions of high yield and high
activity. These experiments are not possible if running one condition at a time.
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28. The MEA uses a 12-channel system. It is very straight forward compared to
FPLC systems.
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29. Scale-able because of linear relationship between recovery and resin bed
volume. This is because of equilibrium binding.
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30. Utilize specialized labware to process large volumes. The MEA 2 is capable of
processing 80 mL samples. In practice, most high volume users process 10-60
mL.
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31. High volume purification is comparable to gravity with the advantage of
automation and parallel processing. Different affinity resins are displayed here.
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33. Obtaining crystals that diffract is not trivial. The protein must be soluble,
homogenous and high concentration. Proteins are also engineered to crystallize
be removing hydrophobic residues on the surface and fixing flexible regions.
Screening was demonstrated, here, using the MEA. These data are from SGC.
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34. Same approach was taken at Celgene.
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PhyTip columns have the advantage of high performance. This is needed to
utilize the most effective downstream analysis such as SDS-PAGE. For
construct screening, the data have to be relatively high concentration to be
useful.

36. High performance is demonstrated by comparison with spin columns. The
capability of loading a small resin bed more and eluting in a smaller volumes
means higher concentration samples. Compared with spin columns, the 20 µL
Phytip columns are able to bind as much protein as a 100 µL spin column. The
real advantage is the low volume elution, which keeps the pure protein more
concentrated. The 5 µL PhyTip column does not have enough capacity, but the
tradeoff is higher pure protein concentrations complared to the 20 µL Phytip
column.
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37. Reproducibility between tip to tip. CV values of 10 or less.
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Gel filtration chromatography through gravity flow. Fractionation shown here is
an example of capabilities.

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Buffer exchange for dye labeling and excess dye removal. CE-LIF application
benefits from smaller volumes and higher throughput automation.

42. Sample prep such as glycan analysis is fully automated.
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43. Using PhyTip columns packed with normal phase resin, thederivatized glycans
are enriched.
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44. An example of glycan analysis using the HPCL.
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Current HTP plasmid approaches require sample supernatent be transferred ro
plates or vacuums.

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Using PhyTip columns, the crude sample san be processed without clearing the
supernatant, first.

48. DNA quality is copatible with sequencing, agarose gel analysis and transfection.
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49. DNA used for expression screening.
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52. PhyTip columns are used at every stage of the drug development process.
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