Thermo Scientific™ Nunc™ Polycarbonate Cell Culture Inserts in Multi-Well Plates

Standard culture membrane inserts are placed directly into the wells of a Multidish, and the feet at the bottom of the insert maintain a distance of about 1 mm between the membrane and the Multidish culture surface. The “carrier plate” culture membrane product adds a tray between the Multidish and the lid, with a set of hanger brackets at each well. These brackets engage one of three sets of tabs on each insert, to allow for selection between 3 different distances (approximately 1, 3, or 6 mm) between the membrane and the Multidish culture surface. This can allow for more flexibility in experiments, such as distance-dependent interaction between populations or media volume under cultures at the air-media interface.

All of the pre-packed POROS columns, regardless of column size or resin, have the same 10-32 female fittings on the column. Columns are compatible with Waters and Agilent HPLC systems. You will need 1/16 inch male fittings for the HPLC to attach the column.

All of the pre-packed columns, regardless column size or resin, have the same 10-32 female fittings on the column. They are all compatible with the AKTA instrument platform, however due to the column volume of the 2.1 mmD x 30 mmL columns (0.1 mL), these will not perform well in AKTA or Avant purification systems due to the relative size of the hold- up volume in these systems. The smallest column (PEEK or SS) that could feasibly be used in these systems is the 4.6mmD x 50mmL (0.8 mL) format.

UPLC is typically described as chromatography with sub-2 µm particles, which drives higher efficiency/better separation independent of operating flow rate. As particle size decreases, backpressure increases so the UPLC systems tend to have higher pressure ratings compared to HPLC systems. The flow rates on UPLC systems would be sufficient for POROS columns. Pump capabilities are typically 0.1-1.0 mL/min, so not as fast as a HPLC, but sufficient. Hold-up volumes on UPLC systems are lower than HPLCs, so this can help with sensitivity and efficiency. So from a pressure and flow standpoint there should be no challenge to operate a POROS CaptureSelect column on a UPLC system.

That being said, care should be taken running POROS columns on UPLC systems. Higher pressure safety interlock MUST be set at the rated column pressure, typically 180 bar. HPLC column hardware is not rated to operate at the static pressures possible and typical on UPLC systems. A clog or the like on a UPLC system could generate unsafe pressures on any HPLC-rated column before the default UPLC safety pressure interlock is tripped.

The physical stability of POROS resins is high. Customers have agitated, with overhead mixing and or bottom gassing for long periods of time (5-10 hours) and have performed hundreds of diaphragm pump passes during stall packs. Keep in mind that agitation with any device that creates grinding and sheer, such as with a magnetic stir bar, is not recommended for any type of resin.

POROS resins rehydrate quite well. Here is the procedure that we recommend:

1. Flow 5% ethanol over the column at ?350 cm/hour for 3 CVs, followed by water or 0.1 M sodium chloride at 300 cm/hour for 3 CVs.
2. If baseline noise is still present, flow 20% ethanol over the column at 300 cm/hour for 3 CVs, followed by water or 0.1 M sodium chloride at 300 cm/hour for 3 CVs.
3. If you have established column qualification tests and specifications, perform the tests after rewetting.

In general POROS resins have behaved better for large biomolecules such as viral particles, fusion proteins, and globular proteins, where shape (not size) matters due to its unique pore structure and large pore structure as compared to traditional resins. As the target molecule size increases, capacities obtained will decrease. The large pore structure of POROS resins, which allows for convective flow (and therefore enhanced diffusion) is especially well suited for the purification of large biomolecules. POROS resins offer the best blend of high capacity (associated with chromatography beads) and improved chromatography efficiency (typically associated with monoliths or membranes). The average pore size for POROS resins is 1,000-3,600 Angstrom (100-360 nm) depending on the base bead/chemistry being utilized. For example, the molecular weight of IgG is 150 kDa and the molecular radius is 55 Angstrom. The molecular weight of IgM is ~900 kDa (pentamer), and the molecular radius is 120 Angstrom. So both of these biomolecules can interact with the pore structure associated with POROS materials.

Resin lifetime depends on how the resin is used and the cleaning process that is employed. Therefore, each purification process will need to be evaluated specifically, especially if long lifetime is desired. Because binding can be different between resins, different cleaning schemes may be needed. An unoptimized cleaning process of any resin may yield <5 uses, and an optimized cleaning process can yield a hundred cycles or more. Discoloration of resin can occur for different reasons from process compounds such as metals like iron (Fe2+/3+) and copper (Cu2+), additives to the media (like vitamin B12 and folic acid), and elution solutions (like magnesium chloride, Mg2+). We can provide recommendations that have proved helpful for many customers to optimize the life of their POROS or CaptureSelect column.

POROS resins are mechanically rigid and incompressible and can be packed effectively in low-pressure glass columns and in high-pressure stainless steel columns. The lack of wall support with increasing column diameter has minimal impact on chromatography performance because the beads support themselves, allowing for flexible column packing approaches and consistent and robust results. Columns can be packed with traditional flow pack, axial compression, or pack-in-place/stall pack packing methods.

Increased titers and product demand have caused substantial bottlenecks in downstream processing for a range of biomolecules. POROS chromatography resins address these challenges with solutions that help maintain performance and add process flexibility. Due to the polymeric nature of the backbone and the way we attach the ligands, the beads have very robust physical and chemical stability. This allows for aggressive solutions to be used, if needed, to improve cleanability and thereby increase resin lifetime/reuse. Our ion exchange resins are the go-to resins in downstream monoclonal antibody and recombinant protein chromatography where capacity, resolution, and yield are critical. We are also working on a new line of hydrophobic interaction resins that may be of interest for future projects that will be a best-in-class product for capacity and resolution with an added benefit of being able to load under lower salt concentrations and faster flow rates. We are also a strong partner when it comes to quality and supply of our products.