ProteinSimple eBook - 11

Simplifying Protein Analysis * Mastering Charge Heterogeneity Analysis of Therapeutic Proteins

the protein of interest, carrier ampholytes, and pI markers.
When this sample mixture is injected, it fills the entire capillary cartridge,
where separation takes place. Samples are focused by applying voltage across the
cartridge, and during the focusing step, a pH gradient forms across the capillary.
The pI markers and protein of interest migrate through the capillary until they
reach a pH value where their net charge is zero-this is their isoelectric point.
The iCE3 then uses whole-column imaging detection at 280 nm to capture the
separation within the capillary. Finally, the capillary is washed to ready it for the
next sample injection. The full process from sample injection through final wash
takes place in 10-12 minutes.
Method Development
A successfully defined and optimized iCE icIEF method gives you a highly
reproducible peak profile and satisfactory peak resolution for the targeted
application. This is quickly done on the iCE3 as only a few parameters need to be
optimized by following a simple workflow as outlined in Figure 1. The first step
in method development is to screen new compounds with a generic method
employing a Pharmalyte 3-10 pH gradient as shown in Figure 2A. For many
molecules, methods with this broad pH range provide sufficient performance
and do not require further development. For more challenging molecules with
complex peak profiles and/or limited solubility, method optimization can be
accomplished using the following simple strategies.
In icIEF, proteins simultaneously lose surface charge while being focused
11

| GENengnews.com

Step 1:

Run System Test Mix

Fail

Call ProteinSimple
Tech Support

Pass

Step 2: Run sample with generic
pH 3-10 method
Yes
Current < 40 mA?

Desalt sample

Yes
0.6 > signal > 0.05?

Adjust protein
concentration

Add urea

Yes
Profile reproducible?
Yes
Resolution sufficient?

Yes
Method development
is complete

Figure 1. Method optimization workflow

Optimize resolution with
narrow range ampholytes

http://www.GENengnews.com

ProteinSimple eBook

Table of Contents for the Digital Edition of ProteinSimple eBook