Size exclusion questions

[ScottHorn]

Hi,
I work in a lab that deals primarily with antibodies and antibody-fluorochrome conjugates. We're in the process of building a protein purification system. We're going with the modular route as opposed to buying a complete system (cheaper and easier to customize). We're also packing our own columns as opposed to buying prepacked. This has led to some questions. Since I have quite a few I'll put them in a list.

1. I've used superdex 200 in the past, and like the resolution. The problem is that some of our conjugates are around 800kD or larger, so with superdex those elute as one giant peak before everything else. I'm considering sephacryl 300 (fractionates up to 1500kD). Using the same column will I see a substantial loss of resolution with sephacryl as opposed to superdex?

2. Are there newer size exclusion media that perform better than sephacryl and superdex in this situation? (globular proteins between 150-1000kD, 10-500 mg scale) What about toyopearl, superose, etc?

3. When packing sephacryl into a column, I understand that one must use a packing reservoir to pack the column in order to avoid having to add media multiple times as the bed settles. What are the downsides of adding media, packing, adding more media, and packing again without using the reservoir?

4. Has anyone used the "Clarity" chromatography software package? Any complaints or comments on it, with regard to size exclusion chromatography?

Thanks for any advice you can share. I really enjoy playing with all my new "toys", but I want to avoid learning through trial and error where I can.

Scott Horn

[HW Mueller]

A search of this forum and catalogs (of course including the internet) should show very nicely what modern SEC can do. I sort of went in the other direction, separating Fab from Mab using a high resolution silica based column (from Tosoh, don´t have the name in the head, but it is mentioned in this forum many times).
I wonder whether you already have some experience with conjugates, no problem with dissociation on the chromatography timescale?

[ScottHorn]

I've been to plenty of manufacturers wesbites, and searched the archives of this forum, but I'm having trouble finding some of the answers I seek. Mainly I'm curious if anyone has used the media I currently use as well as the newer media, and can offer a comparison of their performance when it comes to the types of molecules I want to separate.
As for your question about the conjugates, I've been conjugating everything you can imagine to lots of different antibodies for about three years now. For most of our run-of-the-mill conjugations we covalently bond the fluorescent molecule (be it a 240kd protein, or a small 400mw molecule) to the antibody. We have product that we've observed to be stable for up to 4 years, so the timeframes involved in chromatography are no problem. The antibodies (usually the mAbs) tend to start falling apart or aggregating long before the linkers fall apart.

[Fred Tepper]

At PREP 09 We will be reporting on a new LC media based on nano alumina fibers in a non-woven structure. The nano alumina (AlOOH) is the same as approved by FDA for use as vaccine adjuvants. It can separate live virus from each other by chromatography at a pressure about 1 bar and at a rather high flux. We have done no work with antibodies.

The nano alumina is tethered to a microglass scaffold. The zeta potential is +50 mv at pH 7. Because the nano alumina is in the form of 2 nm diameter fibers, the charge density is rather high. Virtually all of these fibers are exposed to the moving phase as compared to the claimed surface area of porous beads. The attached AlOOH fibrilles can be functionalized via a number of routes.

Because it is a non-woven structure, packing of spheres is not necessary. And because this is a spin-off of a maturing technology in filtration, its cost is projected to be far below that of a packed HPLC column. Once developed for a process, the likelihood of low cost disposable columns is likely.

[ScottHorn]

So is this a type of size exclusion media? What kind of fractionation range does it have? This sounds more like an advertisement than an answer to the question I posed. It sounds like an interesting technology, but I'm not sure if it's what I'm looking for.

[Fred Tepper]

The media does not use size exclusion, except for very large bacteria. It has a 2 micron pore size. It separates because the media is charged and it is capable of filtering out even virus,, and at a high flux. While still in it infancy, we are exercising this filter media for chromatographic separation, focusing on separating virus from each other. It may be amenable to separating antibodies. The active component is AlOOH, that is the same used as an adjuvant that carries antigens in vaccines.