GenWay is a world leader in the immuno-fractionation products for proteomic
partitioning in the field of biomarker discovery. Our original line of products,
successfully marketed and distributed by Beckman-Coulter, was the first step in
providing scientists with the quality proteomic partitioning products for removal of
highly-abundant proteins (HAPs) from the plasma, serum, urine and CSF fluids of
primates as well as rodent species.

Twelve most abundant proteins comprise 96% of the plasma protein mass. Only
4 % of the plasma proteins are medium-abundant or low-abundant. Almost all the
biomarkers reside among the low-abundant fraction (Fig.1).

Figure 1. Relative abundance of plasma proteins. Relative ratios are given in
percentage of each protein fraction mass to the total plasma protein mass.

Beckman’s ProteomeLab IgY12 system removes top 12 HAP reproducibly in a
highly-specific manner, reducing dynamic range of plasma protein concentration by 1-2
logs (Fig.2-3). These products paved the way for discovery and validation of biomarkers
(see ref. 1-2), which primarily reside in the low-abundant fraction of plasma proteins
(LAPs).

Figure 2. Specific removal of HAP target proteins by IgY-microbeads.

Figure 3. 2D-gel of plasma sample proteins before and after IgY12-mediated
processing.

Current immunodepletion technology permits rapid processing of plasma samples.
However, it still requires significant labor input and has significant throughput
limitations. Present plasma biomarker discovery increasingly demands high throughput
plasma sample processing. To discover clinically-relevant biomarkers, throughput has to
increase to a level of 100 sample processing per day. One of the most important ratelimiting
steps in sample processing is depletion of HAPs. This challenge was successfully
solved by GenWay (Fig.4).

Figure 4. Seppro ® Tip design.

The solution is the SepproTip. This product allows simultaneous processing of 12
samples at a time in a completely automated fashion. Up to 100 plasma or serum samples
can be processed daily using the system. The system consists of 12-GC-Pro automation
system (link to the movie), disposable pre-filled cartridges, and SepproTip.

SepproTip may contain (Table I) IgY12 (human and primate HAP removal), IgY-R7 (rat
HAP removal), IgY-M7 (mouse HAP removal), IgY-Rubisco (Rubisco removal from
plant extract samples) and SuperMix (partitioning of MAP and LAP fractions for digging
deeper into the proteome).
.

The upper ring (Fig.4) moves up and down and moves plasma sample, washing solution
or regenerating solution through the beads to deplete the plasma, serum, or CSF samples
and subsequently regenerate the tip (Fig.5).

Figure 5. Automated sample processing.
The usual plasma sample size is 20 ul. Diluted to 500 ul, it is inserted into the 12GC Pro
instrument (Fig.6) together with sterilely pre-filled cartridges for automatic sample
processing (link to the movie). With a single press of a button the samples get processed
and tips get regenerated in completely automated fashion, which includes wash, stripping,
and regeneration. The processing time varies between 55 mins. and 65 mins. The system
can process 12 samples at a time, which put the throughput capability at 100 to 150
plasma or serum samples per day.

Figure 6. Sample Processing. A. Picture of the SepproTips during plasma samples
processing. B. Quality of the plasma sample processing.

In addition to high throughput, the HAP removal is highly efficient (Fig.6, right panel).
Flow-through fraction does not contain any or very little of HAP proteins. Each tip can be
used up to 100 times. Present instrument model permits 30 time use of each tip. However,
the microbead content of the tip can be used 100 times. There is also a very high
reproducibility between the runs (Fig.7).

Figure 7. Run-to-run variation in plasma depletion efficiency.

As you can see, there is very little variation between HAP depletion efficiency between
Run#1 and Run#10. Starting Run#35 there is decreasing depletion efficiency since the
effective IgY microbead immunodepletion use is limited to 100 runs
.
Overall, the system is extremely robust, allows high-throughput samples processing, and
is perfect for large number of samples projects, including clinical environment, which
requires high degree of automation due to the research personnel time constrains.

Finally, the instrument can be used not only for plasma/serum/CSF sample processing but
also for nucleic acid automated sample processing and Ni-affinity purification using Nitip
kit (link to His-tag kit).


1Qian WJ, Jacobs JM, Liu T, Camp DG 2nd, Smith RD. (2006). Advances and challenges
in liquid chromatography-mass spectrometry-based proteomics profiling for clinical
applications. Mol Cell Proteomics 5, 1727.

2Liu T, Qian WJ, Mottaz HM, Gritsenko MA, Norbeck AD, Moore RJ, Purvine SO, Camp
DG 2nd, Smith RD. (2006). Evaluation of multiprotein immunoaffinity subtraction for
plasma proteomics and candidate biomarker discovery using mass spectrometry. Mol
Cell Proteomics 5, 2167.

SepproTip Systrem

12 Proteins Comprise ~96% of
the Protein Mass in Plasma

Table I. Various SepproTip content