WO2012049285A1

WO2012049285A1 - A method for the quantitative glycosylation analysis of proteins

Info

Publication number: WO2012049285A1
Application number: PCT/EP2011/067971
Authority: WO
Inventors: Kent Persson
Original assignee: Octapharma Ag
Priority date: 2010-10-14
Filing date: 2011-10-14
Publication date: 2012-04-19

Abstract

A method for the quantitative determination of glycosylation of a selected protein in solution comprising the steps: providing a conjugate of a nucleic acid or nucleic acid analogues, such as LNA and PNA hybrids, and a compound having an affinity to a carbohydrate moiety on glycosylated protein, such as a lectin binding to sialic acid attached to terminal galactose in (α-2,6), and (α-2,3), linkages; said conjugate is contacted with a protein which is suspected to be glycosylated and is called analyte-protein herein below; the conjugate is detected by a nucleotide hybridization depending technique which is employed for detecting the conjugate.

Description

A METHOD FOR THE QUANTITATIVE GLYCOSYLATION ANALYSIS OF PROTEINS

The present invention pertains to a method for quantitative glycoanalysis of proteins, a conjugate of a nucleic acid and a lectin.

BACKGROUND OF THE INVENTION Glycosylation is a form of co-translational and post-translational modification of a protein during its synthesis within the cell . Once the process is complete, the attached glycans serve a variety of structural and functional roles for the proteins. Cellular processes can regulate or alter the glycosylation of an analyte- protein. These modifications in protein glycosylation can be applied by the cell to cope with changes, or be the result of an infection or disease (Essentials of Glycobiology. Ajit Varki (ed.) (2nd ed.). Cold Spring Harbor Laboratories Press. ISBN 978-087969770-9).

In Biotech industry settings, the degree of glycosylation for a recombinantly produced protein can be affected by growth conditions of the cultured cell. Thus maintaining a healthy glycosylation degree is very important to preserve the structural and functional roles for the protein being produced. Increased understanding of these issues has led the FDA, EMEA and other regulators to tighten their rules on glycoprofiling throughout life cycle of the drug European Biopharmaceutical Review. Sprin 2006 ppl00-104. Glycans appear in different forms where proteins, lipids, or other organic molecules are attached during synthesis. One of these organic molecules is sialic acid. Sialic acid is a generic term for the N- or O-substituted derivatives of neuraminic acid, a monosaccharide with a nine-carbon backbone (1). It is also the name for the most common member of this group, N-acetylneuraminic acid (Neu5Ac or NANA). Sialic acids are found widely distributed in all animal tissues (Schauer R "Achievements and challenges of sialic acid research". Glycoconj. J. 2000, 17 (7-9) : 485-499. Sialic acids are most often found at the terminal position of glycans, and therefore are a likely point of contact for glycan-ligand interactions. The degree of sialylation for an analyte-protein can significantly influence its safety, efficacy and in-vivo half-life Schauer R. (2000). "Achievements and challenges of sialic acid research". Glycoconj. J. 17 (7-9) : 485-499. A full global glycan analysis of a protein is tedious and requires highly purified sample material. However, analyzing the sialylation pattern can be a very useful measure for consistency during manufacturing of recombinant protein products. WO-A-2008/007373 discloses a glycoanalysis for proteins.

US 2003/148335 Al relates generally to the field of target detection . In particular, the present inventions provides for methods and compositions for assaying a plurality of different non-nucleic acid targets or for assaying activities of a plurality of enzymes using, inter alia, oligonucleotide identification (ID) tags. The review of Wu Albert M., et al . in Glycoconjugate Journal vol. 26, no. 8, November 2009, pp 899-913 focuses on the specificity-based lectin classification and the methods of measuring lectin-carbohydrate interactions, which are used for determination of lectin specificity or for identification and characterization of glycoconjugates with lectins of known specificity. The most frequently used quantitative methods are shortly reviewed and the methods elaborated and used in our laboratories, based on biotinylated lectins, are described. These include the microtiter plate enzyme-linked lectinosorbent assay, lectinoblotting and lectin-glycosphingolipid interaction on thin-layer plates. Some chemical modifications of lectin ligands on the microtiter plates and blots (desialylation, Smith degradation, beta-elimination), which extend the applicability of these methods, are also described.

US 2002/064779 Al relates to sensitive, rapid and convenient assays for detection and/or quantification of one or several analyte(s) in solution using so called proximity probes. The proximity probes comprise a binding moiety and a nucleic acid. The nucleic acid from one proximity probe is only capable of interaction with the nucleic acid from the other proximity probe when these are in close proximity, i .e. have bound to the analytes for which they are specific. The reference relates to methods and kits for proximity probing and are performed in sosolution without the need of a solid phase. WO 93/15229 A2 discloses a method for the amplified detection of an analyte, wherein amplification is achieved by replication of a target nucleic acid sequence which has been immobilized in response to analyte.

US 6514716 Bl discloses a method of determining terminal sialic acid residues in human transferrin according to a sandwich principle, which is characterized in that the sample fluid containing the human transferrin is incubated with a first receptor which binds specifically to human transferrin, the thus-formed complex is separated from the sample fluid and incubated with a second receptor which binds specifically to terminal sialic acid residues in human transferrin, the second receptor being bound or able to bind to a marker, and the complex made up of the first receptor, human transferrin and the second receptor is determined with the marker. According to one embodiment, the method allows the determination of sialic-acid-deficient human transferrin in body fluids.

SUMMARY OF THE INVENTION An object of the invention is to provide a sensitive high throughput quantitative method for glycosylation analysis on proteins, irrespectively of purity, facilitating limited sample material, avoiding the drawbacks of the presently applied methods.

This is accomplished by a method for the quantitative determination of glycosylation of a selected protein in solution comprising the steps:

- providing a conjugate of a nucleic acid or nucleic acid analogues, such as Locked Nucleic acid (LNA) and Protein Nucleic Acid (PNA) hybrids, and a compound having an affinity to a carbohydrate moiety on glycosylated protein, such as a lectin binding to sialic acid attached to terminal galactose in (oc-2,6), and (oc-2,3), linkages;

- said conjugate is contacted with a protein which is suspected to be glycosylated and is called analyte-protein herein below;

- the conjugate is detected by a nucleotide hybridization depending technique which is employed for detecting the conjugate. In one embodiment of the invention the nucleic acid is DNA and the modified DNA is a DNA having a backbone more resistant to hydrolytic attacks than the native DNA, such as PNA or LNA.

In another embodiment of the invention the analyte-protein is of patho- physiological relevance.

In a further embodiment of the invention the nucleic acid or modified nucleic acid in said conjugate consists of a nucleic acid of 10-500 nucleotides, in particular 50 to 180 nucleotides

In yet another embodiment of the invention the compound having an affinity to a carbohydrate moiety on glycosylated protein is selected from the group consisting of lectins having affinity to GIcNAc, Glc/Man, Mannose, Terminal GkNAc, Alpha Gal, Beta Gal, and GaVGalNAc.

In still another embodiment of the invention the nucleic acid or modified nucleic acid of the conjugate is conjugated to a lectin, by conventional protein DNA conjugation e.g. coupling of protein molecules on the nucleic acid through the 5 ^' -phosphate group, which binds specifically to moieties in glycosylation units of the selected protein. In particular, the nucleic acid sequence of the nucleic acid of the conjugate is Seq ID No. 1.

In a particular embodiment of the invention the hybridization dependent technique comprises a primer pair for nucleic acid amplification methods, or the hybridization dependent technique comprises a hybridization probe or oligo nucleotide, labelled or unlabeled with markers.

Subject matter of the present invention is also a conjugate of a sialic-moiety binding lectin protein with a nucleic acid or modified nucleic acid, in particular wherein the sialic-moiety binding lectin is conjugated with the nucleic acid or modified nucleic acid through coupling of protein molecules on the nucleic acid through the 5 ^' -phosphate group.

In one embodiment of the conjugate of the present invention the linker group is selected from the group consisting of 10-500 nucleotides, in particular the nucleotide sequence 5 ' -TCTCCTCATCTCACCTACCAAGCCTGCGCCTTGCTCCTTTGGTTCGAGTTGGTGATTGGTGAGGAG-3' .

Another subject matter of the present invention is the use of the conjugate of the invention in the method of the invention. Also subject matter of the invention is a kit for performing the method of the invention comprising the conjugate of the invention, primers hybridising with the nucleic acid part of the conjugate of the invention for performing a nucleic acid amplification reaction, optionally nucleotides, polymerases and buffers.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 : qPCR- and melt curve results derived from a dilution series made with the template DNA.

Figure 2 : Functional part of the analysis of the conjugate and its ability to selectively bind to sialic residues of the protein (FVIII).

Figure 3 : Amplification curves from the standard curve. Figure 4: Final results from an assay.

Figure 5 : A plot with log concentration of rFVIII standard curve (ng/ml) on the Y-axis and Ct on the X-axis.

Figure 6 : Standard curves from three separate runs.

Figure 7 : The mean sialylation degree in percent relative to the optimally sialylated Protein rFVIII.

DETAILED DESCRIPTION OF THE INVENTION

The method of the invention is a method for determination of the glycosylation of proteins comprising the steps:

- providing a conjugate of a nucleic acid or nucleic acid analogues, such as Locked Nucleic Acid (LNA) and Peptide Nucleic Acid (PNA) hybrids, and a compound having an affinity to a carbohydrate moiety on glycosylated protein, as lectin binding to sialic acid attached to terminal galactose in (oc- 2,6), and (oc-2,3), linkage or other lectins ;

- optionally removal of unbound protein;

- said conjugate is contacted with a protein which is suspected to be substituted with sugar moiety, such as sialic acid, and is called analyte- protein herein below;

- the conjugate is detected by a nucleotide hybridization assay such as a PCR based method, labeled or unlabeled nucleotide probes or oligo nucleotides. It may be useful that detected amount of glycosylation is amplified in an exponential way, using a suitable technique such as qPCR, facilitating the detection of glycosylation on single analyte-proteins and technically single molecule differences in glycosylation residues. It may be advantageous to use automated analytical systems, facilitating high through-put screening of many samples of the analyte-protein .

Lectins are sugar-binding proteins that are highly specific for their sugar moieties. They play a role in biological recognition phenomena involving cells and proteins. For example, some viruses use lectins to attach themselves to the cells of the host organism during infection . Lectins may be disabled by specific mono- and oligosaccharides, which bind to them and prevent their attachment to cell membranes.

In one embodiment of the present invention, the nucleic acid is DNA. In order to provide a backbone of the DNA which is e.g. more resistant to hydrolytic attacks than the native DNA, the backbone of the nucleic acid may contain one or more sODN linkages or peptide bonds leading to molecules known as PNA. Another advantage of the modification is the increase in the stability through increase of the melting point (Tm) of the complex formed after hybridisation .

The nature or origin of the analyte-protein which is to be analysed is in principle not limited. In particular the method of the invention is employed in the analysis of the glycosylation of an analyte-protein which has patho-physiological relevance. Also recombinantly produced proteins can have their glycosylation pattern analyzed, e.g. sialic acid-moieties, GIcNAc, and Glc/Man glycan structures etc.

In particular the analyte-protein of patho-physiological relevance is selected from the group consisting of a blood clotting factor including fibrinogen, fibrin monomer, prothrombin, thrombin, FV, FVa, FX, FXa, FIX, FIXa, FVII, FVIIa, FVIII, FXI, FXIa, FXII, FXIIa, FXIII, FXIIIa, von Willebrand factor, ADAMTS13 and derivatives and muteins thereof, a transport proteins such as albumin, transferrin, ceruloplasmin, haptoglobin, hemoglobin, hemopexin, etc., protease inhibitors such as β-antithrombin, oc-antithrombin, oc2-macroglobulin, Cl-inhibitor, tissue factor pathway inhibitor (TFPI), heparin cofactor II, protein C inhibitor (PAI-3), Protein C, Protein S, Protein Z, etc., immunoglobulin's such as polyclonal antibodies (IgG), monoclonal antibodies, IgGl, IgG2, IgG3, IgG4, IgA, IgAl, IgA2, IgM, IgE, IgD, Bence Jones protein etc., cell related plasma proteins such as fibronectin, thromboglobulin, platelet factor4, etc., apolipoproteins such as apo A-I, apo A-II, apo E, complement factors such as Factor B, Factor D, Factor H, Factor I, C3b-Inactivator, properdin, C4-binding protein etc., growth factors like Platelet derived growth factor (PDGF), Epidermal growth factor (EGF), Transforming growth factor alfa (TGF-oc), Transforming growth factor beta (TGF-β), Fibroblast growth factor (FGF) and Hepatocyte growth factor, antiangionetic proteins such as latent-antithrombin and prelatent-antithrombin, etc., highly glycosylated proteins including alfa-l-acid glycoprotein, antichymotrypsin, inter- oc-trypsin inhibitor, α-2-HS glycoprotein, C-reactive protein, and other human blood proteins or human blood plasma proteins such as histidine-rich glycoprotein, mannan binding lectin, GC-globulin, plasminogen, oc-1 microglobulin, C-reactive protein, blood factors such as erythropoeitin, interferon, tumor factors, tPA, gCSF and derivatives and muteins thereof.

According to the invention in particular the lectin is selected from Sambucus nigra lectin binding preferentially to sialic acid attached to terminal galactose in (oc-2,6), and to a lesser degree, (oc-2,3), linkage. However the invention also includes other lectins with different affinities to other carbohydrate moieties to be used in glycoanalysis. Examples of other lectins and their affinities is found below.

GIcNAc The lectins in this group bind N-acetylglucosamine (GIcNAc) and its 4-linked oligomers with an affinity that increases with chain length of the latter. The carbohydrate-specificity of both lectins in this group do not differ, yet differences in their binding patterns are observed and probably stem from the non- carbohydrate portion of the samples.

Glc/Man

This group of lectins is a subgroup of the mannose binding lectins (see below), and are denoted Glc/Man binding lectins since they bind, in addition to mannose, also glucose. All of the lectins in this group bind to bi-antennary complex N-lined glycans with high affinity. In comparison to their affinity for bi-antennary structures, lectins Glc\Man(l) and (2) bind high mannose glycans with lower affinity, whereas lectin Glc\Man(3) will bind high mannose glycans with higher affinity.

Mannose This group consists of lectins that bind specifically to mannose. These lectins will bind high mannose structures and, with lower affinity, will recognize the core mannose of bi- antennary complex structures.

Terminal GkNAc

This lectin specifically recognizes terminal GIcNAc residues. Alpha Gal

These lectins bind terminal oc-galactose (a-Gal). Lectin Alpha-Gal(l) binds both oc- galactose and oc-GalNAc (oc-N-acetylgalactosamine) and may bind to both N and O-linked glycans. Lectin Alpha-Gal(3) binds mainly the Galili antigen (Galal-3Gal) found on N- linked antennae. Beta Gal

These lectins specifically bind terminal (non-sialylated) β-galactose residues. GaVGalNAc These lectins are specific for terminal galactose and N-acetyl-galactoseamine residues.

The different lectins within this group differ in their relative affinities for galactose and N-acetyl-galactoseamine. Lectins (2) and (5) from this group bind almost exclusively Gal; lectins (1), (3) and (4) bind almost exclusively GalNAc. The relative affinities for GalNAc / Gal for the remaining lectins in the group are ranked : (8)> (7)> (6).

Fucose Lectins from this group bind fucose residues in various linkages.

Lectin Fucose(6) binds preferentially to 1-2-linked fucose; Lectin Fucose(8) binds preferentially to 1-3 and 1-6 lined fucose; Lectins Fucose(12) and (13) bind preferentially to Fuel -4GIcNAc (Lewis A antigens).

These lectins generally do not bind the core fucose of N-linked oligosaccharides on intact glycoproteins due to steric hindrance.

It should be understood that these examples for methods and assays for analyzing the glycosylation patterns in a sample, are provided for the purposes of example only and are not intended to be limiting by any means, as any other suitable method and/or assay could optionally be used with the present invention. The principles and operation of the present invention may be better understood with reference to the accompanying description, as well as the following example.

According to the invention the nucleic acid or nucleic acid analogues in said conjugate should be designed according to well established principles for qPCR based assays (covering length, base combinations and the sequence), preferable consisting of about 50-180 nucleotides in length. Additionally, the invention also covers nucleic acid or nucleic acid analogues in said conjugate lengths between about 10-500 nucleotides.

In particular, according to the invention the nucleic acid or modified nucleic acid of the conjugate is conjugated by standard methods used for general protein DNA conjugation e.g. coupling of protein molecules on the nucleic acid through the 5 ^' -end phosphate group. Such methods are well known to the skilled person and for example is described in Lind, K and Kubista, M., Development and evaluation of three real-time immuno-PCR assemblages for quatification of PSA. J. Immunol. Methods 2005, 304, 107-116, Lind, K and Norberg, J. , Immuno- qPCR detection of the tandem a ffinitypurifi cation (TAP)-tag as a sensitive and accurate toolsuitable for large-scale protein quantification. Proteomics, 2007, 7(24), 4414-4423)

A typical example of the DNA acid sequence which can be used in the method of the invention is the DNA of Seq ID No. 1.

5' -TCTCCTCATCTCACCTACCAAGCCTGCGCCTTGCTCCTTTGGTTCGAGTTGGTGATTGGTGAGGAG-3'

However, any sequence combination of the nucleotide bases or their analogues, adenine, cytosine, guanine, and thymine, adhering to the well established principles for qPCR based assays can be used.

According to the invention hybridization is carried out with a primer pair, which binds at specific sites to the nucleic acid part of the conjugate and is used in nucleic acid amplification methods. The respective nucleic acid part of the conjugate can hence be amplified and made visible/detectable/quantified by the conventional analytical amplification methods, such as PCR or qPCR based assay in particular.

In another embodiment of the invention, hybridization is carried out with a nucleic acid fragment or oligonucleotides added to the mixture of analyte-protein and conjugate. The hybridizing nucleic acid fragment or oligonucleotides may be labeled with markers whith intrinsic and/or inducible signalling.

Intrinsic signalling means e.g. radioactively labeled markers, whereas inducible signalling is represented e.g. by fluorescent dyes, chemiluminescent markers, enzymes involved in dye formation, etc. Examples of fluorescent dyes that can be employed are: Cy3; Cy5 and Texas Red.

Generally, the specific methods of hybridizing nucleic acids are known to the skilled person. Many different variations of nucleotide hybridization techniques exist, however, they all have in common the denaturing step for the template sequence (here the conjugated DNA to the ligand) then followed by nucleotide hybridization to the single stranded template sequence using labeled or unlabeled nucleotide fragments or oligonucleotides. If the template sequence is single stranded naturally the denaturation is omitted and hence the hybridization step can be employed directly. Once base pairing is achieved the hybrid between the template DNA and nucleotide fragments/oligonucleotides can be visualized by fluorescent markers (if labelled), used in ligation assays, or followed by an additional hybridizing step where the added nucleotide fragments or oligonucleotides depend on a different template sequence attached for the actual quantitation/detection.

Subject matter of the present invention is also a conjugate of a nucleic acid or nucleic acid analogues, such as LNA and PNA hybrids, and a compound, such as a sialic-moiety binding lectin, which compound having an affinity to a carbohydrate moiety on a glycosylated protein for example a sialic acid attached to a terminal galactose (in oc-2,6 and oc-2,3 linkage), lectin protein with a nucleic acid or modified nucleic acid. In particular the sialic-moiety -moiety binding lectin is conjugated with the nucleic acid or nucleic acid analogues. Different strategies can be applied when using chemical reagents to effect the conjugation of nucleic acids to proteins. A suitable DNA-protein conjugate protocol can be achived by using a DNA-strand with amino-modification at the 5'end to be covalently linked to a protein using the heterobifunctional cross-linking agent succinimidyl-4-(N- maleimidomethyl) cyclohexane-l-carboxylate, SMCC where the protein is activated by thiol groups (Lind, K and Kubista, M ., Development and evaluation of three real-time immuno-PCR assemblages for quatification of PSA. J. Immunol . Methods 2005, 304, 107-116).

Further, a subject matter of the invention is a kit for performing the method of the invention comprising the one or several conjugate(-s) of the invention, selected antibody (for selected protein) for immuno capture prior to conjugate binding, primer pair hybridizing with the nucleic acid of the conjugate(-s) for performing a nucleic acid amplification reaction, optionally nucleotides, thermostable DNA polymerase and buffers as well as suitable plastic tubes and 96-well plates and optimized protocol for glyoprofile analysis of the selected protein.

In the following, the invention is further described by the example of assaying sialylation of a protein. The invention uses a novel conjugate in a novel application to determine the glycosylation degree in particular the sialylation degree of any protein of interest. The protein of interest may be in any purification degree i.e. highly purified or in a crude sample mixture i .e. unpurified, mixed together with other proteins and the like. The degree of sialylation for a protein is most easily determined by comparing the acquired data to a standard preferably derived from the protein of interest itself.

As indicated the invention utilizes in a specific embodiment of the conjugate lectin from Elderberry bark (Sambucco Nigra), which is 150,000 molecular weight tetrameric glycoprotein. Sambucus nigra lectin binds preferentially to sialic acid attached to terminal galactose in (a-2,6), and to a lesser degree, (a- 2,3), linkage.

To determine the glycosylation degree, in particular the sialylation degree of an analyte-protein the invention applies a specific antibody or a similar agent to capture the analyte-protein from any type of sample material . This capture is e. g. carried out by coating a well, a tube or a resin with the antibody or other ligand specific for the protein of interest. After coating, the protein mixture, containing known amounts of protein of interest, is applied, once the protein of interest is captured unwanted proteins can be washed away. After the removal of the impurities, the captured protein is either first eluted, or directly subjected to incubation with the lectin-DNA conjugate where the lectin moiety of the conjugate will bind to sialic residues on the protein of interest. After incubation with the conjugate excess, ligands are washed away and the proteins with bound lectin-NA (nucleic acid) conjugates are subjected to a qPCR where specific primers will exponentially amplify the template DNA found on the lectin-NA conjugate.

In case the NA (nucleic acid) was DNA, the increase of amplified products can be detected by SYBR Green or similar DNA intercalation/binding agents or by probe associated detection e.g. TaqMan, Beacons Scorpions etc. Independent of detection chemistry, the increase of emitted light for a sample, derived from of specific primer amplification, is directly proportional to the amount of conjugates e.g. bound to sialic residues on the analyte-protein of interest. The higher the amount of e.g. sialic residues on the analyte-protein of interest the earlier is passed the threshold value in the qPCR analysis. When the analysis is finished, the data derived from the samples are compared with data from the standard curve. The standard curve is derived from the purified protein of interest which is analyzed in parallel on the same 96-well plate. In particular, differently sialylated analyte-proteins can be used as standard but a dilution series (different amount) of a carefully characterized analyte-protein can also be used as a siayl group standard. Correctly applied this dilution series works well since different amount of the protein will give different amount for sialic groups

The invention will be described in greater detail by the following non-limiting examples. EXAMPLES

The method of the invention is performed as assay comprising different steps that can be tested and optimized individually, depending on the status of the sample and the nature of the analyte-protein. Final reagents included lectin (Elderberry Bark) and highly purified recombinant FVIII (according to the process described, WO-A-2009/156430) with optimal sialylation degree with a known concentration used as standard. A recombinant cultivation sample (according to the process described in WO-A-12009/156430) without rFVIII but did contain all other background proteins was used to simulate a crude protein mixture. Recombinant FVIII samples with unknown degree of sialylation were also analyzed.

Part 1. Template DNA and Primer Pair Sequences

A DNA oligo nucleotide with complementary primers were purchased. The PCR assay was tested to determine efficiency and to confirm absence of primer dimmer formation. A dilution series was made with the template (synthesized DNA strand) and performed according to instructions in the method. The PCR system was tested with FastStart Universal SYBR Green Master (Roche, cat.no 04 913850 001) to determine efficiency and no primer dimer formation, see figure la and figure lb. for results.

Primer pair sequence. Primer name Sequence Product length (bp) Product Tm (°C) die fw: 5'-CTC CTC ATC TCA CCT ACC AA-3' die rev: 5'-CTC CTC ACC AAT CAC CAA CT-3' Sequence of conjugated synthetic dsDNA 5' -TCTCCTCATCTCACCTACCAAGCCTGCGCCTTGCTCCTTTGGTTCGAGTTGGTGATTGGTGAGGAG-3'

Figure la depicts a dilution series with the designed PCR assay. The results show an efficiency of 86%.

Figure lb shows the melting curve from the dilution series. One even peak indicates that only one product is amplified and no primer dimers are formed. The melting temperature (Tm) for the product was calculated to 78.5°C.

Part 2. Confirmation of conjugation between lectin and DNA

Lectin (Vector Laboratories Inc. Elderberry Bark Lectin, cat. no L-1300) was conjugated to the DNA strand (Lind, K and Kubista, M ., Development and evaluation of three real-time immuno-PCR assemblages for quatification of PSA. J. Immunol . Methods 2005, 304, 107-116). A test where rFVIII was coated directly in the wells was made to confirm that the conjugation between lectin and DNA had succeeded and that different amount of sialic residues responded with corresponding Threshold Cycle values.

Figure 2: Functional part of the analysis of the conjugate itself and its ability to selectively bind to sialic residues of the protein (FVIII) : A 96-well plate was coated with recombinantly produced and highly purified FVIII at a concentration of 4000, 1000 and 250 ng/ml (Standard) and unpurified FVIII of unknown concentration (Unknown). Two HPLC-fractions from the DNA-lectin conjugation were tested, both fractions did show similar results. For successful conjugation different amounts of FVIII are expected to give different Threshold Cycle (Ct) values the more FVIII (more sialic residues) the lower the Threshold Cycle (Y- axis). On the X-axis have the log concentrations been plotted (250 ng/ml, 1000 ng/m I and 4000 ng/ml). The results shows that the lectin of conjugate is binding to sialic residues in the glycosylation units of FVIII and that the attached DNA allows for PCR-amplification and subsequent qPCR-detection. The results show that the conjugation has been successful.

Part 3. Final PLC (DNA-lectin-coniuqate)-qPCR sialylation assay

Different anti human FVIII antibodies have been tested to find the optimal FVIII capture ligand. Several different blocking buffers, sample dilution buffers and conjugate dilution buffers were evaluated. Also washing steps and conjugate dilutions have been evaluated. In figure 3 the results of the assay is shown. After the DLC-qPCR run has finished, data is analyzed by looking at the amplification curves and set the threshold in the exponential phase, where the curves are parallel in logarithmic scale. The melting curve data should look like in figure IB with one even peak with a Tm around 78.5°C. Due to FVIII stick/adherent nature the FVIII the standard curve is somewhat S-shaped which means that it does not fit a straight line, see figure 4. To analyze the unknown samples the Ct values from the DLC-qPCR run are imported into an Excel sheet. Mean Ct values from the standard point are plotted against log concentration, then a polynomial 3 order trendline is added, see figure 5. Sialylation analyses of the unknown samples are made using the equation from the standard curve. To calculate a difference in sialylation degree between the standard point and the unknown sample, see the equations below in the statistics section. Figure 3: Typical amplification curves from the standard curve from the DLC- qPCR standard curve. A) All samples standard sample replicates visualized, blue curves come from the highest standard point (3.200ng/ml FVIII) and orange curves are the No protein controls. B) Amplification curves from the standard curve where replicates are treated collectively. Blue curve comes from the highest standard point (3.200ng/ml protein X) and orange curve is the No protein control.

Figure 4: Final results from the assay. The standard points are marked as squares and the no protein controls are marked as triangles A log standard curve with two fold dilutions of rFVIII from 3.200ng/ml - 50ng/ml. The noProt controls and the control sample (the sample with background proteins and sample matrix but does not contain rFVIII) had the same or later Ct-values as the last standard point. An unknown sample containing rFVIIIshows good replicates. The standard curve is somewhat S-shaped and does not fit a straight line.

Figure 5: A plot with log concentration of rFVIII standard curve (ng/ml) on the Y-axis and Ct on the X-axis. A polynomial 3 order trend line has been added. Calculate the concentrations of the unknown samples by using the equation from the standard curve. To calculate a fold difference in sialylation degree between the standard point and the unknown the equations found in Part 3 of the Examples were applied.

Part 4. Validation, inter-assav variability and sialylation data results Validation of the optimized DLC-qPCR assay was performed by repeatedly running the protocol with the standard curve. In figure 6 three standard curves from three separate runs is shown. The curves have very similar shape and Ct values which indicate good reproducibility.

To assess the inter-assay variability of the method three quantifications were performed on two different samples. Three separate DLC-qPCR runs with the standard curves and the two samples were performed. Table 1 shows the results from the three quantifications. The fold difference of the sialylation degree of rFVIII in the two unknown samples compared with the sialylation degree of the optimally sialylated rFVIII is calculated by subtracting the quantity of rFVIII in the unknown samples with the quantity of the standard point. The quantities were calculated by using the equation from the standard curve. In figure 7 the data from table 1 has been plotted with 95% confidence interval as error bars in a diagram.

Table 1. Results from two unknown samples from three separate runs. Mean relative glycosylation and CI (mean) are calculated based on log relative glycosylation values because the values are not evenly distributed in linear scale. Figure 6: Standard curves from three separate runs are here plotted with log concentration (ng/ml) in the Y-axis and Ct-values on the X-axis. All three curves have very similar shape and Ct-values, which indicate good reproducibility.

Figure 7: The mean sialylation degree (in percent relative to the optimally sialylated Protein X, here as 100%) for each test and the mean of the three tests (all) have here been plotted with 95% confidence interval as error bars. The confidence interval for "all" is calculated based on the standard error of the mean (SEM) and with log relative glycosylation values (see equation 2 and 3 in appendix 2). This indicates the spread which will arise if the same sample is run over again. The confidence interval for test 1-3 is also calculated based on the standard error of the mean but with SEM ratio of concentration in unknown sample - concentration in standard sample (see equation 4 and 5 in appendix 2). This indicates the technical spread "how good the assay replicate have been". More replicates will give a lower SEM (closer to the real mean) and also a smaller confidence interval . It is recommended to run eight replicates for the unknown samples. Statistics used for calculations in table 1 and figure 7.

Equation 1.

Amean = meanT - meanR

T=tarqet R= reference

Equation 2.

SEM = SD/Vn

SEM = Standard error of the mean. SD= Standard deviation. n= number of replicates

Equation 3.

CI= mean ± ft * SEMI

CI= confidence interval . t= critical T-value. SEM = Standard error of the mean Equation 4.

SEMA = V SEMT2 + SEMR2

Equation 5.

CIA = Amean ± ft * SEMA1

Claims

1. A method for the quantitative determination of glycosylation of a selected protein in solution comprising the steps: providing a conjugate of a nucleic acid or nucleic acid analogues, such as LNA and PNA hybrids, and a compound having an affinity to a carbohydrate moiety on glycosylated protein, such as a lectin binding to sialic acid attached to terminal galactose in (oc-2,6), and (oc-2,3), linkages;

said conjugate is contacted with a protein which is suspected to be glycosylated and is called analyte-protein herein below;

optionally removal of unbound protein;

the conjugate is detected by a nucleotide hybridization depending technique which is employed for detecting the conjugate.

2. The method of claim 1 wherein the nucleic acid is DNA and the modified DNA is a DNA having a backbone more resistant to hydrolytic attacks than the native DNA, such as PNA.

3. The method of claim 1 wherein the analyte-protein is of pathophysiological relevance.

4. The method of claim 2 wherein the analyte-protein of patho-physiological relevance is selected from the group consisting of a blood clotting factor, transport proteins, protease inhibitors, immunoglobulins cell related plasma proteins, apolipoproteins, growth factors, antiangionetic proteins, highly glycosylated proteins, other human blood proteins or human blood plasma proteins, blood factors.

5. The method of claim 4 wherein the blood clotting factors are selected from the group consisting of fibrinogen, fibrin monomer, prothrombin, thrombin, FV, FVa, FX, FXa, FIX, FIXa, FVII, FVIIa, FVIII, FXI, FXIa, FXII, FXIIa, FXIII, FXIIIa, von Willebrand factor, ADAMTS13 and derivatives and muteins thereof.

6. The method of claim 4 wherein the transport proteins are selected from the group consisting of such as albumin, transferrin, ceruloplasmin, haptoglobin, hemoglobin, hemopexin, etc.

7. The method of claim 4 wherein the protease inhibitors are selected from the group consisting of such as β-antithrombin, oc-antithrombin, oc2- macroglobulin, Cl-inhibitor, tissue factor pathway inhibitor (TFPI), heparin cofactor II, protein C inhibitor (PAI-3), Protein C, Protein S, Protein Z, etc.

8. The method of claim 4 wherein the immunoglobulins are selected from the group consisting of such as polyclonal antibodies (IgG), monoclonal antibodies, IgGl, IgG2, IgG3, IgG4, IgA, IgAl, IgA2, IgM, IgE, IgD, Bence Jones protein etc.

9. The method of claim 4 wherein the cell related plasma proteins are selected from the group consisting of such as fibronectin, thromboglobulin, platelet factor4, etc.,

10. The method of claim 4 wherein the apolipoproteins are selected from the group consisting of such as apo A-I, apo A-II, apo E, complement factors such as Factor B, Factor D, Factor H, Factor I, C3b-Inactivator, properdin,

C4-binding protein etc.

11. The method of claim 4 wherein the growth factors are selected from the group consisting of like Platelet derived growth factor (PDGF), Epidermal growth factor (EGF), Transforming growth factor alfa (TGF-oc), Transforming growth factor beta (TGF-β), Fibroblast growth factor (FGF) and Hepatocyte growth factor.

12. The method of claim 4 wherein the antiangionetic proteins are selected from the group consisting of latent-antithrombin and prelatent- antithrombin, etc.

13. The method of claim 4 wherein the highly glycosylated proteins are selected from the group consisting of alfa-l-acid glycoprotein, antichymotrypsin, inter-oc-trypsin inhibitor, α-2-HS glycoprotein, C- reactive protein.

14. The method of claim 4 wherein the other human blood proteins or human blood plasma proteins are selected from the group consisting of histidine- rich glycoprotein, mannan binding lectin, GC-globulin, plasminogen, oc-1 microglobulin.

15. The method of claim 4 wherein the blood factors are selected from the group consisting of such as erythropoeitin, interferon, tumor factors, tPA, gCSF and derivatives and muteins thereof.

16. The method of at least one of the foregoing claims wherein the nucleic acid or modified nucleic acid in said conjugate consists of a nucleic acid of 10-500 nucleotides, in particular 50 to 180 nucleotides

17. The method of at least one of the foregoing claims wherein the compound having an affinity to a carbohydrate moiety on glycosylated protein is selected from the group consisting of lectins having affinity to GIcNAc, Glc/Man, Mannose, Terminal GkNAc, Alpha Gal, Beta Gal, and GaVGalNAc.

18. The method of at least one of the foregoing claims wherein the nucleic acid or modified nucleic acid of the conjugate is conjugated to a lectin, by conventional protein DNA conjugation e.g. coupling of protein molecules on the nucleic acid through the 5 ^' -phosphate group, which binds specifically to moieties in glycosylation units of the selected protein.

19. The method of any of the foregoing claims wherein nucleic acid sequence of the nucleic acid of the conjugate is Seq ID No. 1.

20. The method of one of the foregoing claims wherein the hybridization dependent technique comprises a primer pair for nucleic acid amplification methods.

21. The method of one of the foregoing claims wherein the hybridization dependent technique comprises a hybridization probe or oligo nucleotide, labeled or unlabeled with markers.

22. A conjugate of a sialic-moiety binding lectin protein with a nucleic acid or modified nucleic acid.

23. The conjugate of claim 22 consisting wherein the sialic-moiety binding lectin is conjugated with the nucleic acid or modified nucleic acid through coupling of protein molecules on the nucleic acid through the 5 ^'- phosphate group.

24. The conjugate of claim 22, wherein a linker group is selected from the group consisting of 10-500 nucleotides, in particular the nucleotide sequence

5' -TCTCCTCATCTCACCTACCAAGCCTGCGCCTTGCTCCTTTGGTTCGAGTTGGTGATTGGTGAGGAG-3' .

25. Use of the conjugate of any one of the claims 22 to 24 in a method of any one of the claims 1 to 21.

26. A kit for performing the method of any one of the claims 1 to 21 comprising the conjugate of any one of claims 22 to 24, primers hybridising with the nucleic acid of the conjugate for performing a nucleic acid amplification reaction, optionally nucleotides, polymerases and buffers.