IEF-FFE
The first technique relies on a continuous separation of samples with respect to the isoelectric point of each component. To this end a pH gradient is generated by commercial ampholytes, which was used for preparative separation of monoclonal antibodies up to 100 mg. The throughput is 3 mg/h with a resolution of < 0.02 Δ-pH.
Figure 1: IEF-FFE separation of crude mAB sample on pH with an ultraflat pH-gradient from 8 to 8.5. The fractions within the green area were applied to an IEF gel (Figure 2).
Figure 2: IEF-PAGE (pH 6-10) of the crude sample (S) and selected fractions of IEF-FFE separated mAB sample. M: Serva IEF Marker.
Figure 3: Re-FFE-electrophoresis was performed with fraction 56 to verify the stability of the separation process.
ProLytes-FFE
The second technique, matching handling, resolution and throughput of the IEF-FFE, makes use of a proprietary mixture of acids and bases to generate the pH-gradient needed for separation. Because no ampholytes get in contact with the samples, clinical application of the separated isoforms is possible.
Figure 4: Relative abundance (black line) of the separated mAB sample and pH profile (red line). The fractions within the green area were applied to an IEF gel (Figure 5).
Figure 5: IEF-PAGE (pH 6-10) of the crude sample (S) and selected fractions of ProLytes-FFE separated mAB sample. M: Serva IEF Marker.
IZE-FFE
The third technique (IZE-FFE) relies on differences of electromigration in a stepwise pH-gradient, formed by the use of different acid and base containing buffers over the width of the separation chamber. Because the proteins are not separated at their isoelectric point, this technique minimizes protein-protein interactions and its short interval time of 10 minutes and throughput of up to 100 µg protein per interval, allows for rapid analysis or preparation of samples by continuous collection of fractions. Direct MS measurements are possible, due to the lack of ampholytes and polymers.
Figure 6: UV-profile (black line) of the separated mAB sample and pH profile (red line). The fractions within the green area were applied to the IEF gel (Figure 7).
Figure 7: IEF-PAGE of the crude sample (S) and selected fractions of IZE FFE separated mAB sample.
Choice of the pH gradient
The Free Flow IEF separations can be performed at a huge variety of pH gradients.
The 
standard gradient is ranging from pH 3 to pH 10 with a ΔpI of 0.1 between the single fractions.
However, below we show a range of flattened gradients to deliver high resolution separations for the pH extremes in the very acidic (pH 3-4) and alkaline (pH 8-10) region as well for the neutral region.
Values for ΔpI of 0.015 between the single fractions are possible.
The pH gradient is highly adjustable to the specific separation needs.
1. IEF Separation of acidic proteins
A protein mixture was separated by Free Flow IEF at a pH gradient ranging from pH 3 to pH 5. The fractions were subsequently applied to IEF-PAGE.
The dotted line indicates the pH gradient of the FFE run, the bars show the optical density of separated pI marker dyes.
Fractions of the blue marked region were applied to IEF-PAGE.
S/M=crude sample
#=FFE fraction
2. IEF Separation of alkaline proteins
Isoforms of Elastase (pI 10.4) were separated in a flat alakline pH gradient ranging from pH 9 – pH 11. Free Flow Electrophoresis IEF mode was run at 1800 V with a transition time of 5.5 Minutes.
The dotted line indicates the pH gradient of the FFE run, the bars show the optical density of separated pI marker dyes.
Fractions of the blue marked region were applied to IEF-PAGE.
S=crude sample
M=pI marker proteins
#=FFE fraction
3. IEF Separation of a monoclonal Antibody (mAB)
Isoforms of a monoclonal Antibody were separated under native conditions in the pH range of pH 7.8 to pH 9.3
The dotted line indicates the pH gradient of the FFE run, the bars show the optical density of separated pI marker dyes. These dyes are just used for our internal quality control.
Fractions of the blue marked region were subsequently applied to IEF-PAGE.
S=crude sample
M=pI marker proteins
#=FFE fraction
4. Novel iZE separation of monoclonal antibody isoforms
Isoforms of a monoclonal Antibody was separated just by its charge heterogenity on interval zone electrophoresis (iZE) mode.
The buffers used for interval zone don’t contain any polymer or ampholyte. The mixture just contains well defined chemicals (one acid, one base and mannitol). Thus, this technique is compatible for clinical and pre-clinical applications.
The separation was conducted at 1500 V with a transit time of 8 Minutes.
The dotted line indicates the pH gradient of the FFE run, the bars show the optical density of separated pI marker dyes. These dyes are just used for our internal quality control.
Fractions of the blue marked region were subsequently applied to IEF-PAGE.
S=crude sample
M=pI marker proteins
#=FFE fraction
5. Comparison of IEF and iZE separation of monoclonal Antibody isoforms
Depending on the desired downstream analytics, we offer different modes of separation:
- buffers for IEF separation which contain commercial ampholytes
- buffers for IEF separations which contaidefined chemicals (Prolytes)
- iZE buffers which contain just one anion, one cation and mannitol
The results of the different separations are shown below on a native separation of monoclonal antibody isoforms.
M=protein marker
S=crude sample
#=FFE fraction
The separation time was 8 Minutes on all separation profiles. IEF separation were run on a pH 6 – pH 8 gradient.
The separated sample was applied directly to IEF-PAGE and silver stained.
IEF buffers contain 25% glycerol and 0.2% HPMC (hydroxypropylmethylcellulose) to suppress the imminent electroendosmotic flow. However, this limits the possibility of post-FFE sample concentration.
IEF separations can be conducted either in buffers containing commercial ampholytes or with proprietary Prolyte buffers.
Prolytes contain a mixture of 15 well defined acids and bases to form the pH-gradient.
iZE buffers just contain mannitol which can easily removed and enables highest concetration factors, if needed. Furthermore these buffers contain only a few well defined ingredients which are compatible with crystallography and (pre)clinical applications
All shown separations are on file at FFE Service by SERVA.