1

Validation of Free Flow Electrophoresis as a novel plasma and serum

processing and fractionation method in biobanking

Gwenaelle Gaillard1, Jean-Pierre Trezzi2, Fotini Betsou*2

1Biobanque de Picardie, avenue René Laennec, 80480 Saleux, France

2Integrated Biobank of Luxembourg, 6 rue Ernest Barble, L1210, Luxembourg

Short title: Free Flow Electrophoresis validation in biobanking

Keywords: proteomics, free flow electrophoresis, fractionation, validation, plasma, biobank

*Integrated Biobank of Luxembourg, 6 rue Ernest Barble, L1210 Luxembourg. Tel 003532744641, Fax

0035227446464, fay.betsou@ibbl.lu

Abstract

Free Flow Electrophoresis (FFE) is a fractionation method, based on isoelectric focusing (IEF). We

validate the reproducibility of the method and show that it can be applied by biobanks in order to

efficiently and reproducibly fractionate fluid biospecimens and facilitate downstream proteomic

applications. We also propose a simple method allowing to assess the reproducibility of each FFE run.

Introduction

Despite important advances in the use of genomics and transcriptomics for the identification of

clinically relevant biomarkers, circulating proteins or peptides remain the most sought-after

candidate biomarkers because of ease of blood sampling and biological meaningfulness. Their initial

identification in biological fluids requires proteomic analysis, usually performed on well annotated

fluid samples (e.g., serum, plasma) from biobanks.

One of the most important technical difficulties in this approach is linked to the dynamic range of

plasma proteins which is of the order of magnitude of 1010 (1). The most abundant protein in human

plasma is serum albumin which is present at 40-50 mg/ml while some cytokines and tissue leakage

proteins are present at levels approximating 10 pg/ml. To overcome this problem, two usual

strategies are followed: depletion of high-abundance proteins and/or fractionation. Depletion of

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albumin and IgG may increase sensitivity for detection of remaining proteins, but many proteins may

get irreversibly and non-specifically bound during the process. Previous fractionation of the biological

fluid is an alternative or can be combined to subsequent abundant-protein depletion. One of the

least explored sample fractionation methods is FFE.

Free Flow Electrophoresis, first described by Hannig (2, 3), is a prefractionation method enabling the

resolution and identification of low abundant proteins and the reduction of protein mixture

complexity. The method is performed with fractionation equipment that continuously streams a

sample into a carrier ampholine solution flowing between two plates, as a thin laminar film (0.3-

1mm). An electrical field is applied perpendicular to the direction of flow and proteins and peptides

are separated by IEF according to their pI values and >70 fractions are collected in a microplate. Each

fraction is separated by ~0.02-0.1 pH units depending on the pH gradient created. Using this method,

proteins with extreme physical-chemical properties can be detected. Free Flow Electrophoresis has

been used for pre-fractionation of samples because of two main advantages: improved sample

recovery due to absence of gel media and high sample loading capacity. Although FFE is not

automatable and samples can only be processed one by one, the typical turn-around time for one

sample fractionation is only 10-15 min. It has previously been shown that FFE is fit for purpose for

different downstream proteomic analyses, including both targeted ELISA (Enzyme-Linked

Immunosorbent Assay) and high throughput proteomic 2D-electrophoresis or mass spectrometry (4,

5, 6). However, in order for biobanks to be able to use FFE as a routine fluid biospecimen processing /

fractionation method, it is important to confirm and be able to assess the reproducibility of the

method in terms of pH gradient and consequently, protein contents in each fraction or pool of

fractions obtained.

Reproducibility of 2D FFE-IEF/RP-HPLC (Reversed-Phase High-Performance Liquid Chromatography)

system has previously been studied (7). In that study, reproducibility was assessed from the

downstream analysis point of view : RP-HPLC chromatograms, where Standard Deviation (SD) of

retention times and of microplate well numbers were calculated across three replicates for nine

peaks located between wells 13 and 87. The SD for uncorrected FFE well numbers was 0.83 (when

the FFE well numbers were adjusted by pH, the SD was 0.62). This corresponds to 0.05pH units within

FFE pools. However, this study was performed with standard cellular protein preparations. The

objectives of our study were to assess the reproducibility of FFE in human plasma samples, and to

identify internal quality control markers that can be used to define acceptance criteria for FFE

fractionation reproducibility. Therefore, we validated the reproducibility of FFE fractionation of

human serum and plasma, by showing that the position of proteins in a given range in a 96-well plate

can be reliably predicted, enabling post-fractionation protein analysis.

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Materials and Methods

FFE

FFE was applied as previously described (8, 9, 10).

Preparation of the FFE buffers

Deionised and overnight degassed water was used. For the Stripe test, which ensures homogeneity

of the applied electrical current, a specific dye (SPADNS; 2-(P-sulfophenylazo)-1,8-dihydroxy-3,6-

naphthalene disulfate) was diluted to 0.01% in distilled and degassed water. The three separation

buffers, the two stabilisation buffers and the electrode buffers were prepared according to the

manufacturers’ instructions for the QC2 test (BD protocol, IEF-GH 3-10, native). The 6M urea (Serva,

ref 24524) separation buffer was also prepared according to manufacturers’ instructions (BD

protocol, IEF 3-10, 6M urea, denaturating conditions). The separation buffer 1 was composed of

30,3g IEF Buffer 1, 69,7g distilled water and 54g urea, the separation buffer 2 contained 100g IEF

buffer 2, 100g distilled water and 108g urea and the separation buffer 3 contained 37g IEF buffer 3,

63g distilled water and 54g urea. The three IEF buffers, the SPADNS and the pI mix were included in

the BD kit (ref 441118). The pH and conductivities were measured after each buffer preparation. The

separation buffers contained glycerol, urea, hydroxyl prolyl methyl cellulose (HPMC), H2SO4, Prolyte1,

Prolyte 2, Prolyte 3, NaOH. The counterflow buffer contained glycerol and urea. The anodic and

cathodic circuit electrolytes were H2SO4 and NaOH respectively.

Plasma and serum samples

Plasma samples and serum samples from BD Vacutainer EDTA and SST collected whole blood

specimens respectively, were prepared from two different healthy donors. The blood was

centrifuged, one hour after collection, at 2000g for 10 min at room temperature, and 0.5 ml of serum

or plasma was aliquoted in 1.5ml microtubes and stored at -80°C. One aliquot for each separation

was thawed on the separation day and diluted 1 to 4 in Separation buffer 2 for the IEF 3-10, 6M urea,

denaturating protocol. Final protein concentration, measured by Biorad Bradford assay, was

approximately 15 mg/ml.

FFE performance test

The FFE was set up on each of five different separation days according to the manufacturer’s

instructions. The volume of the chamber, the media and sample pumps flow rates were measured. A

running Stripe test (QC1) and a running performance test with separation media (QC2, with pI mix)

were run. The absorbance of the microplates was then measured at 405nm to locate the SPADNS dye

and the pI markers, in addition to their visual inspection. Residence time in the separation chamber

was ~30min. Fractions of approximately 200 l were collected in 96-well polypropylene plates. BD

polypropylene 96-well microplates and BD polypropylene covers were used in order to avoid protein

adsorption.

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Validation of the method reproducibility: Five FFE separations in 6M urea, pH3-10

Five plasma aliquots from the same sample (plasma A) were used for the five separations to test the

technical reproducibility in denaturing conditions. One aliquot of a second sample (plasma B) was

used to test the biological reproducibility. The five separations were performed on five different days

with all buffers freshly prepared. The microplates containing the FFE fractions were stored at -80°C

for further analysis. Two serum aliquots were also used on two different days in the same separation

conditions to compare plasma to serum fractionation profiles. One plasma aliquot was run in 8M

urea denaturing conditions to test for robustness of the method to composition of the separation

buffer.

Electrophoretic analysis of FFE fractions

Serum and plasma FFE microplate contents were electrophoretically analysed with the Agilent 2100

Bioanalyzer in order to detect marker peaks for assessment of FFE fractionation reproducibility. The

Agilent Protein 230 Kit (in denaturating conditions) was used for the electrophoresis of the fractions

according to the manufacturer’s instructions. Preliminary analyses of microplate fractions showed

that the most abundant protein, presumably corresponding to albumin, was detected in at least two

fractions in the fraction range 4F-5G. Therefore, for each of the five separations, the microplate

fractions 4F to 5G were analysed on the same chip. Collected electrophoretic data revealed six

markers, each of which corresponded to a specific molecular weight and isoelectric point. For each of

these protein markers, detected by Agilent electrophoresis in the fractions 4F to 5G, data including

the molecular weight in kDa, the relative concentrations in ng/µl and the percentage of total protein

were analyzed. The total relative concentrations of each fraction were also measured. Two different

calculations were made, with or without alignment to the fraction with the highest protein

concentration (presumably albumin).

Identification of internal QC protein markers

FFE separations of two plasma and two serum 96-well microplates were used for pooling and

Western blotting (Wb) analysis. For each plate, three pool series were prepared: Pool 1 (wells 4F, 4G,

4H, 5A), pool 2 (wells 5B, 5C, 5D 5E), pool 3 (wells 5D, 5E, 5F, 5G). Each pool was made up of identical

volumes of fractions from each individual well and stored at -20°C. Positive controls were prepared

of plasma and serum extracted from fresh blood tubes after centrifugation at 2000g for 10 min at

room temperature and supernatant dilution with PBS 1X (1/500 and 1/100 dilution for serum and

plasma respectively).

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using the

PhastSystemTM (GE Healthcare). Pooled samples and positive controls were diluted 1:5 in reducing

SDS-PAGE sample buffer (5 ml glycerol, 0.5 ml 2-mercaptoethanol, 1 g SDS, 0.38 g Tris/HCl, 0.25 mg

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Bromophenol Blue, 4.5 ml H2O, pH6.8). Samples were heated at 95°C for 2 min before

electrophoresis. One μl of each pooled sample was separated on 8-25% gradient SDS polyacrylamide

gels. Molecular weight markers used were the Pierce Chemiluminescent Blue Prestained Peroxidase-

labeled Protein Molecular Weight Marker. SDS-PAGE settings were 15°C gel bed temperature, 75 Vh

at 250V, 10 mA, 3 W.

Western blotting

Western blotting (Wb) was performed using the BioRad Trans-Blot SD Semi-Dry Transfer Cell.

Western Blot setup was 20 mA (constant), 20V for 25 minutes. Transfer buffer contained 3.94 g

Tris/HCl, 14.4 g glycine, 200 mL methanol per L, pH7.5. After electrotransfer, the Hybond ECL

nitrocellulose membrane was incubated with ECL (chemiluminescence) Blocking Agent (GE

Healthcare) for 1h. Primary antibodies used and corresponding dilutions in Blocking Reagent were

the following: anti-serum albumin, ABCAM, ab10241 (1/1000), anti-alpha-1-antitrypsin, ABCAM,

ab49088 (1/500), anti-ceruloplasmin, ABCAM, ab48614 (1/250), anti-apolipoprotein A-I, Cell

Signaling Technology, 3350S (1/500). Membranes were incubated overnight at 18-22 °C room

temperature (RT), three 10 minutes washing steps were made in TBS1x/Tween 0.05%. Secondary

antibodies used were anti-mouse IgG, Horseradish peroxidase-linked, NA931 (1/5000), and anti-

rabbit IgG, Horseradish peroxidase-linked, NA934 (1/25000). These were diluted in TBS1x/Tween

0.05% and incubated for 1h at RT, followed by three 10 minutes washing steps. Membranes were

finally incubated in detection reagent (ECL plus Western blotting detection System) for 5 minutes and

detection was performed using the ImageQuantTM LAS 4000 (GE Healthcare).

Results

FFE performance tests

The QC1 results for each separation showed three distinct peaks of approximately the same width,

14 to 17 wells, which correspond to successful Stripe tests according to the manufacturer’s guide

(Figure 1, Figure 2). The QC2 results also showed successful results, with seven distinct peaks

corresponding to markers of specific pI (data not shown). The gradient indicated by the distribution

of the pI markers of the QC2 tests, was approximately pH 5 for fractions 4F to 5G, which were

analysed with the Agilent system. The pH of all FFE buffers was stable across different preparations

and separation days with separation buffers 1, 2 and 3 having pH 4, 7 and 9 respectively (Figure 3).

Electrophoretic profiles of the FFE fractions

Total initial plasma protein concentration was approximately 60 to 80 g/L. Comparison between total

protein concentration across fractions 3A to 6G from plasma separated in 6M urea and 8M urea

separation buffer showed similar patterns (Figure 4). Bioanalyzer data from plasma and serum

separated in 6M urea buffer revealed six protein markers separated in three consecutive fraction

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pools (Figure 5). Pool 1 (fractions 4F to 5A) showed a high percentage (28 %) of a 23 kDa protein and

a lower percentage (8 %) of a 154 kDa protein. Mean measured protein concentrations, based on

comparison of protein peak areas with kit standards, were 18.2 and 5.3 ng/µl respectively (Figure 6A,

6F). Pool 2 (fractions 5B to 5E) analysis revealed a very high concentration (84 %) of a 64 kDa protein

and a very low concentration (1 %) of a 119 kDa protein. The respective concentrations were 569.3

and 6.5 ng/µl (Figure 6C, 6E). Pool 3 (fractions 5D to 5G) showed peaks corresponding to a 58 kDa (15

%) and a 93 kDa (12 %) proteins with respective concentrations of 12.5 and 16.9 ng/µl (Figure 6B,

6D).

Therefore, six candidate internal fractionation quality control protein markers were identified at 23,

58, 64, 93, 119, 154kD. Figure 7 shows the concentrations and percentages of total protein in each of

the three consecutive fraction pools from either plasma or serum. Results are shown in microwells

non-adjusted by pH (and consequently by highest protein concentration fraction), because this

approach was more relevant to validation purposes. When microwells were adjusted by pH, the

uncertainty was lower (results not shown). ExPaSy database searches, limited to the Homo sapiens

plasma protein map, and corresponding to each molecular weight, as detected by Agilent Bioanalyzer

electrophoresis, and corresponding pI ranges, as detected by the FFE separation, showed one to

seven possible protein matches, corresponding to each molecular weight. For each molecular weight,

we selected the protein corresponding to the highest concentration in plasma, as being the most

probable match. These selected protein markers were apolipoprotein A-I (23 kDa, pI~5, expected

concentration in plasma 900-2100 mg/L), alpha-1-antitrypsin (58 kDa, pI~4.7, expected concentration

on plasma 1900-3500 mg/L), serum albumin (67 kDa, pI~5.8, expected concentration in plasma

35000-55000 mg/L), secretory immunoglobulin chain alpha (93 kDa, pI~5.3, expected concentration

in plasma 900-4500 mg/L), ceruloplasmin (119kDa and 126 kDa, pI~5.1, expected concentration in

plasma 150-600 mg/L).

Identification of FFE internal quality control markers

Western Blot results (Figure 8) confirmed the initial hypothesis on the identity of the potential FFE

internal quality control markers except secretory immunoglobulin chain alpha. Albumin was well

separated, showing bands in pool 2 and 3, in both plasma and serum samples. These pools have two

fractions in common. Pool 3 contained monomeric alpha-1 antitrypsin. Higher molecular weight

alpha-1 antitrypsin bands may be due to serine proteinases binding. A free alpha-1 antitrypsin

gradient was observed comparing the 3 pools for both plasma and serum samples. Pool 1 contained

most self-aggregated alpha-1 antitrypsin whereas pool 2 and especially pool 3 contained less self-

aggregated alpha-1 antitrypsin. Pool 3 showed very efficiently separated monomeric alpha-1

antitrypsin. Pool 1 contained free, monomeric apolipoprotein A-I. At low concentrations,

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apolipoprotein A-I can be found in a monomeric state whereas at higher concentrations it tends to

aggregate, giving rise to higher molecular weight complexes (11). Pool 1 contains the most free

apolipoprotein whereas pools 2 and 3 show minor apolipoprotein A-I detection in either plasma or

serum samples. Ceruloplasmin was mainly detected in two forms: abundant glycosylated (154 kDa)

and low-concentration non-glycosylated (119 kDa) form. It was detected in higher concentration in

pool 1 (154 kDa) and lower concentration in pool 2 (119 kDa).

Based on the above results, a quality control assay for serum/plasma FFE fractionation

reproducibility can thus be based on Western blotting detection of apolipoprotein A-I and/or

ceruloplasmin in microwell pool 4F-5A, albumin in microwell pool 5B-5E and alpha-1 antitrypsin in

pool 5D-5G.

Discussion

FFE is matrix-free electrophoresis; the molecules are separated according to their charge and

electrophoretic mobility in a continuous laminar flow of buffer solutions in an electric field. The

laminar flow is collected in 96 capillaries at the end of the separation chamber and allows continuous

fractionation in collection plates.

Microwell fractions were analyzed based on our hypothesis that the fractions 4F to 5G can be

grouped into three pools, each containing two of the six reproducible markers indicated by Agilent

Bioanalyzer (Figure 5). Western Blot results confirmed our hypothesis.

Variation in the molecular weights of the markers may be attributed to different post-translational

modifications and/or different pre-processed status of the same protein.

All of the proposed internal quality control markers were reproducibly detected in their

corresponding fraction pools, with the exception of the 58 and 93 kDa markers which were absent in

the 20090805 microplate. This might be due to a relatively higher flow rate of the media pump on

the day of this fractionation (Figure 9).

Whereas one single ~60kDa form was detected for albumin, and found predominantly in pool 2,

different MW forms than the expected ones were observed for the other proteins. Protein pre-

processed forms may explain these different molecular weight (MW) forms observed on Western

blots.

Apolipoprotein A-I may aggregate with other lipid components of HDL (11, 12). Pool 1 contained the

expected, lower MW apolipoprotein A-I in higher concentrations than pool 2 and 3. This might be

due to free apolipoprotein A-I being eluted into pool 1 wells whereas lipid-aggregated apoA-I is

eluted into other pools. This finding proves that plasma and serum fractionation by FFE is efficient

and could, for example, be used for apolipoprotein A-I purification.

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In ceruloplasmin Western blots, a band over 220 kDa was observed. This might be due to

ceruloplasmin (122 kDa) binding to myeloperoxidase (MPO) (140 kDa) (13). Furthermore,

ceruloplasmin auto-aggregation has previously been observed (14). Pool 1 contained a 150kDa and

the >220kDa forms, pool 2 contained the 150kDa and a lower 60kDa form while pool 3 contained

only the lower 60kDa form. Ceruloplasmin contains a number of potential glycosylation sites and this

may explain the higher molecular weight form of 150kDa. The lower MW form may correspond to

ceruloplasmin fragments.

Alpha-1-antitrypsin was expected to be found in higher concentrations in pool 3 than in pool 1 and 2

but this was not the case. Monomeric alpha-1-antitrypsin was detected in pool 3 but higher MW

forms were also detected in pools 1 and 2. Residual binding of alpha-1 antitrypsin (52 kDa) to serine

proteinases (66-75 kDa) (15) may be at the origin of the higher alpha-1 antitrypsin molecular weight

bands, observed in pool 1 and 2.

Free-flow electrophoresis shows powerful pre-fractionation capacities enabling fast and accurate

analysis of a given protein mixture from a biological fluid specimen, compared to chromatography or

isotachophoresis. We showed that the FFE fractions are obtained following a reproducible separation

scheme. Fractionation reproducibility could be confirmed, not at a single fraction level, but at a four-

fraction pool level. We propose a simple internal QC method to assess reproducibility of

fractionation; this is Western Blotting detection of apolipoprotein A-I and/or ceruloplasmin in

fraction pool 4F-5A, albumin in fraction pool 5B-5E and alpha-1 antitrypsin in fraction pool 5D-5G.

Confirmation of the reproducibility of the fractionation pattern, allows the biobank to sub-collect,

store and distribute specific FFE fractions or fraction pools.

Acknowledgements

We are grateful to the Conseil Régional de Picardie for supporting this work.

References

1. Anderson NL, Polanski M, Pieper R, Gatlin T, Tirumalai RS, Conrads TP, Veenstra TD, Adkins

JN, Pounds JG, Fagan R, Lobley A. The human plasma proteome: a nonredundant list

developed by combination of four separate sources. Mol Cell Proteomics 2004;3:311-326.

2. Hannig K. Die trägerfreie kontinuierliche Elektrophorese und ihre Anwendung. Fresenius

Zeitscr Anal Chem 1961;181:244-274.

3. Hannig K, Heidrich HG. Free Flow Electrophoresis. 1990 GIT Verlag GmbH: Darmstadt,

Germany.

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4. Hoffmann P, Ji H, Moritz RL, Connolly LM, Frecklington DF, Layton MJ, Eddes JS, Simpson RJ.

Continuous free flow electrophoresis separation of cytosolic proteins from the human colon

carcinoma cell line LIM 1215: a non two-dimensional gel electrophoresis-based proteome

analysis strategy. Proteomics 2001;1:807-18.

5. Cho SY, Lee EY, Lee JS, Kim HY, Park JM, Kwon MS, Park YK, Lee HJ, Kang MJ, Kim JY, Yoo JS,

Park DJ, Cho JW, Kim HS, Paik YK. Efficient prefractionation of low-abundance proteins in

human plasma and construction of a two-dimensional map. Proteomics 2005;5:3386-3396.

6. Moritz RL, Clippingdale AB, Kapp EA, Eddes JS, Ji H, Gilbert S, Connolly LM, Simpson RJ.

Application of 2-D free-flow electrophoresis/RP-HPLC for proteomic analysis of human

plasma depleted of multi high-abundance proteins. Proteomics 2005;5:3402-3414.

7. Moritz RL, Ji H, Schütz F, Connolly LM, Kapp EA, Speed TP, Simpson RJ. A proteome strategy

for fractionating proteins and peptides using continuous free-flow electrophoresis coupled

off-line to reversed-phase high-performance liquid chromatography. Anal Chem

2004;76:4811-4824.

8. Weber G, Bocek P. Recent developments in preparative free flow isoelectric focusing.

Electrophoresis 1998;19:1649-1653.

9. Moritz RL, Simpson RJ. Liquid-based free-flow electrophoresis-reversed-phase HPLC: a

proteomic tool. Nature Methods 2005;2:863-873.

10. Wildgruber R,Yi J, Nissum M, Eckerskorn C, Weber G. Free-Flow Electrophoresis system for

plasma proteomic applications. Methods Mol Biol 2008;424:287-300.

11. Brouillette CG, Anantharamaiah GM, Engler JA, Borhani DW. Structural models of human

apolipoprotein A-I: a critical analysis and review. Biochim Biophys Acta 2001;1531:4-46.

12. Ajees AA, Anantharamaiah GM, Mishra VK, Hussain MM, Murthy HM. Crystal structure of

human apolipoprotein A-I: insights into its protective effect against cardiovascular diseases.

Proc Natl Acad Sci U S A 2006;103:2126-31.

13. Segelmark M, Persson B, Hellmark T, Wieslander J. Binding and inhibition of myeloperoxidase

(MPO): a major function of ceruloplasmin? Clin Exp Immunol 1997;108:167-74.

14. Kang JH. Modification and Inactivation of Human Ceruloplasmin by Oxidized DOPA. Bull

Korean Chem Soc 2004;25:625-628.

15. Kalsheker N. Alpha,-Antitrypsin: Structure, Function and Molecular Biology of the Gene.

Bioscience Reports 1989;9:2129-138.

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Figure 1

A. The FFE apparatus with horizontal separation chamber. B. Stripe test (QC1) showing straight flow

of the pH gradient.

A B

11

Figure 2

QC1, Stripe test on six different fractionation days.

Figure 3

Plot showing stability of the different FFE buffer pH on different days. BD Ref corresponds to values

obtained upon IQ/OQ of the equipment.

12

Figure 4

Plasma separation in 6M and 8M urea separation buffer. Total protein distribution across fractions

shows the same pattern.

Figure 5

Validation hypothesis based on 6 protein markers separated in 3 different microwell pools

4F 4G 4H 5A 5B 5C 5D 5E 5F 5G

23 kDa = pI~5 154kDa= pI~5,2

64 kDa = pI~5,6 119kDa = pI~5,1

58kDa = pI~5,3 93kDa = pI~5,3

13

Figure 6

Total protein concentrations (A) and distribution of each of the internal QC protein markers in

microwells 4F to 5G (B, C, D, E, F). Average and standard deviation of concentration in ng/l is shown

for each of the five plasma A separations. Concentration in ng/l is shown for plasma B separation.

Well numbers were not adjusted by highest protein concentration fraction.

A

14

B

C

15

D

E

16

F

G

17

Figure 7

Concentration and total percentage of internal QC proteins in each of the three target pools, in

plasma (A) and in serum (B)

A

4F 4G 4H 5A 5B 5C 5D 5E 5F 5G

18

4F 4G 4H 5A 5B 5C 5D 5E 5F 5G

4F 4G 4H 5A 5B 5C 5D 5E 5F 5G

B

4F 4G 4H 5A 5B 5C 5D 5E 5F 5G

19

4F 4G 4H 5A 5B 5C 5D 5E 5F 5G

4F 4G 4H 5A 5B 5C 5D 5E 5F 5G

20

Figure 8

Representative W. blots of albumin (A), alpha-1-antitrypsin (B), apolipoprotein A-I (C) and

ceruloplasmin (D) in plasma and serum FFE pools. 1, plasma pool of fractions 4F to 5A ; 2, plasma

pool of fractions 5B to 5E; 3, plasma pool of fractions 5D to 5G; 4, plasma positive control; 5, serum

pool of fractions 4F to 5A; 6, serum pool of fractions 5B to 5E; 7, serum pool of fractions 5D to 5G; 8,

serum positive control.

A

B

220 k

104 k

76 k

45 k

1 2 3 4 5 6 7 8

220 k

104 k

76 k

45 k

1 2 3 4 5 6 7 8

21

C

D

1 2 3 4 5 6 7 8

220 k

104 k

76 k

45 k

104 k

76 k

45 k

33 k

18 k

1 2 3 4 5 6 7 8

22

Figure 9

Effective flow rates of sample pump (l/h, right ordinate), media pump (ml/h) and separation

chamber volume (ml, left ordinate), measured on every one of the five plasma fractionation days.