WO2011042024A1

WO2011042024A1 - Pharmacokinetic assessment of recombinant proteins in complex biological samples by pk-chromatography

Info

Publication number: WO2011042024A1
Application number: PCT/DK2010/050254
Authority: WO
Inventors: Henrik Naested; Jette Wagtberg Sen; Nina Terp Moenster; Pernille Foged Jensen
Original assignee: Symphogen A/S
Priority date: 2009-10-09
Filing date: 2010-10-06
Publication date: 2011-04-14

Abstract

Abstract The present invention relates to a method for pharmacokinetic assessment of at least two recombinant proteins, such as therapeutic mono and polyclonal antibodies in a complex biological sample such as human, monkey or mouse serum.

Description

Pharmacokinetic assessment of recombinant proteins in complex biological samples by PK-Chromatography

All patent and non-patent references cited in the application are also hereby incorporated by reference in their entirety.

Field of invention The present invention relates to a method for pharmacokinetic (PK) assessment of at least two recombinant proteins in a complex biological sample. The method involves an affinity chromatography step resulting in up-concentration/enrichment of at least two recombinant proteins followed by another chromatography step such as weak cation exchange High Performance Liquid Chromatography (CIEX-HPLC) for detection and quantification of at least two recombinant proteins in the complex biological sample of interest. In particular, the present invention relates to a method for pharmacokinetic assessment of individual therapeutic antibodies constituting a recombinant polyclonal antibody composition.

Background of invention

There is a need for pharmacokinetic assessment of recombinant proteins in various complex biological samples such as e.g. human or animal serum. Traditionally, pharmacokinetic evaluations are often solved by radioimmunoprecipitation (RIPA), electrochemiluminescence (ECL), flow cytometry, Enzyme-Linked Immunosorbent Assay (ELISA), and immunopurification in combination with mass spectrometric quantification. Immunoassays such as ELISA-based techniques are often the assay of choice, however, when the drug lead consists of more than one antibody with identical or overlapping affinities there is a need for anti-idiotype antibodies.

The method of the present invention provides an alternative to conventional methods used for pharmacokinetic assessment of more than one recombinant protein in complex biological samples. One advantage of the method of the present invention is that the technique allows the simultaneous analysis of individual therapeutic antibodies such as a recombinant polyclonal antibody composition in a complex biological sample such as serum or plasma. Another advantage of the method of the present invention is that it is independent of anti-idiotypic antibodies, which are essential for e.g. ELISA- based techniques in multiplex analysis of antibodies with overlapping epitopes.

Summary of invention

The present invention relates to a method for pharmacokinetic assessment of at least two recombinant proteins in a complex biological sample comprising the steps of:

i) providing a complex biological sample comprising said at least two recombinant proteins,

ii) applying said complex biological sample to one or more affinity chromatographic column(s),

iii) eluting from said one or more affinity chromatographic column(s) an eluate comprising said at least two recombinant proteins,

iv) applying the eluate comprising said at least two recombinant proteins obtained in step iii) to one or more additional chromatographic column(s),

v) measuring absorbance of said at least two recombinant proteins,

vi) collecting absorbance signals by chromatographic peak integration corresponding to said at least two recombinant proteins thereby obtaining a chromatogram, and vii) analysing the chromatogram and annotating recombinant proteins to detected peaks of said chromatogram thereby detecting and quantitating said at least two recombinant proteins. The method of the present invention can be used for detection, analysis and

quantification of at least two recombinant proteins, such as two or more recombinant polyclonal antibodies, in a complex biological sample. The sample can be a serum or whole blood sample or any bodily fluid wherein the two or more recombinant proteins of interest are present.

The method can be used for simultaneous analysis of the in vivo clearance of individual antibodies constituting a recombinant polyclonal antibody composition in serum for e.g. pharmacokinetic studies without the need for anti-idiotypic antibodies as in e.g. ELISA- based techniques. Definitions and abbreviations

The term "antibody" describes a functional component of serum and is often referred to either as a collection of molecules (antibodies or immunoglobulins, fragments, etc.) or as one molecule (the antibody molecule or immunoglobulin molecule). An antibody molecule is capable of binding to or reacting with a specific antigenic determinant (the antigen or the antigenic epitope), which in turn may lead to induction of immunological effector mechanisms. An individual antibody molecule is usually regarded as monospecific, and a composition of antibody molecules may be monoclonal (i.e., consisting of identical antibody molecules) or polyclonal (i.e., consisting of different antibody molecules reacting with the same or different epitopes on the same antigen or on distinct, different antigens). The distinct and different antibody molecules

constituting a polyclonal antibody may be termed "members". Each antibody molecule has a unique structure that enables it to bind specifically to its corresponding antigen, and all natural antibody molecules have the same overall basic structure of two identical light chains and two identical heavy chains.

The term "recombinant polyclonal antibody" refers to a collection of antibodies manufactured using recombinant technology. In the context of the present invention, an antibody is considered recombinant if its coding sequence is known, i.e. also if it is expressed from a hybridoma or an immortalized B-cell. In the context of the present invention the term "recombinant protein" includes a "recombinant polyclonal antibody". A recombinant polyclonal antibody describes a composition of different antibody molecules which is capable of binding to or reacting with several different specific antigenic determinants on the same or on different antigens. A polyclonal antibody can also be considered to be a "cocktail of monoclonal antibodies". The variability of a polyclonal antibody is located in the so- called variable regions of the individual antibodies constituting the polyclonal antibody, in particular in the complementarity determining regions CDR1 , CDR2 and CDR3 regions. The polyclonal antibodies that may be characterized by the method of the invention may be of any origin, e.g.

chimeric, humanized or fully human. The recombinant polyclonal antibody according to the invention preferably comprises a population of at least two different antibodies. The term "anti-idiotypic antibody" or "anti-idiotype antibody" refers to a full-length antibody or fragment thereof (e.g. a Fv, scFv, Fab, Fab Or F(ab)2) which specifically binds to the variant part of an individual member of a polyclonal protein, such as another antibody. The anti-idiotype antibody specificity is preferably directed against the antigen-specific part of an individual member of a polyclonal antibody or a polyclonal T cell receptor.

The term 'bind' includes any physical attachment or close association, which may be permanent or temporary. Generally, reversible binding includes aspects of charge interactions, hydrogen bonding, hydrophobic forces, van der Waals forces, etc. , that facilitate physical attachment between the molecule of interest and the analyte being measured.

The term "protein" refers to any chain of amino acids, regardless of length or post- translational modification. Proteins can exist as monomers or multimers, comprising two or more assembled polypeptide chains, fragments of proteins, polypeptides, oligopeptides, or peptides.

The term "complex biological sample" refers to a sample containing a multitude of naturally occurring proteins similar to the recombinant proteins of interest, such as naturally occurring antibodies in serum.

The term "immunoglobulin" is commonly used as a collective designation of the mixture of antibodies found in blood or serum. Hence a serum-derived polyclonal antibody is often termed immunoglobulin. However, immunoglobulin may also be used to designate a mixture of antibodies derived from other sources, e.g. recombinant immunoglobulin. All immunoglobulins independent of their specificity have a common structure with four polypeptide chains: two identical heavy chains, each potentially carrying covalently attached oligosaccharide groups depending on the expression conditions; and two identical non-glycosylated light chains. A disulphide bond joins a heavy chain and a light chain together. The heavy chains are also joined to each other by disulphide bonds. All four polypeptide chains contain constant and variable regions found at the carboxyl and amino terminal, respectively. Immunoglobulins are divided into five major classes according to their heavy chain components: IgG, IgA, IgM, IgD, and IgE. There are two types of light chain, K (kappa) and λ (lambda). Individual molecules may contain kappa or lambda, but never both. IgG and IgA are further divided into subclasses that result from minor differences in the amino acid sequence within each class. In humans four IgG subclasses, lgG1 , lgG2, lgG3, and lgG4 are found. In mouse four IgG subclasses are also found : lgG1 , lgG2a, lgG2b, and lgG3. In humans, there are three IgA subclasses, lgA1 , lgA2, and lgA3.

The term "eluate" refers to the material emerging from a chromatographic column.

PK-CIEX is an abbreviation for "pharmacokinetic evaluation by CIEX chromatography"

A B-cell receptor is a transmembrane receptor protein located on the outer surface of B-cells. The receptor's binding moiety is composed of a membrane-bound antibody that, like all antibodies, has a unique and randomly-determined antigen-binding site. A T cell receptor or TCR is a molecule found on the surface of T lymphocytes (or T cells) that is, in general, responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. It is a heterodimer consisting of an alpha and beta chain in 95% of T cells, whereas 5% of T cells have TCRs consisting of gamma and delta chains.

Description of Drawings

Figure 1 : A drug lead consisting of two antibodies; A992 and A1024 was injected in to a Balb/c Nu/Nu mouse and the PK-curve of the individual antibodies was measured by PK-chromatography. The figure shows the serum concentration of each antibody as a function of time after injection. The total drug lead shown is the sum of the two antibodies.

Figure 2: Representative chromatogram showing the two peaks corresponding to A992 and A1024 (marked by arrows) on a background of Cynomolgus Ig co-purified with the two antibodies by protein A. The sample was drawn from the monkey 1 hour after injection of the drug. The two peaks were integrated for quantitation.

Figure 3: The plasma concentration-time curves for A992 and A1024 in a Cynomolgus monkey administered with one dose of drug lead (18mg) as measured by PK- chromatography. Each time point was measured in triplicate and the mean and the standard deviation is shown.

Figure 4: Standard curves generated by spiking A992 and A1024 at different concentrations into control serum or plasma, and the area of the individual peak was correlated to the concentration of antibody in the sample. Comparison of standard curves for two antibodies spiked in to cynomolgus monkey plasma and serum is shown. The samples were analyzed in triplicate and each data point is shown. Dotted lines indicate the 95% confidence band. A: The two curves corresponding to plasma and serum for A992, and B: The curves corresponding to plasma and serum for A1024. Figure 5: Comparison of chromatograms showing eluates of pooled human serum samples spiked with the drug lead after a purification on a protein A plate with citrate phosphate wash buffers with different pH values. The samples have been washed with 1 . citrate phosphate pH 5.5, 2. citrate phosphate pH 5.25, 3. citrate phosphate pH 5, 4. citrate phosphate pH 4.7 and 5. citrate phosphate pH 4.6. The table shows the sample number together with pH value of citrate phosphate wash buffer and the total peak area of A992 and A1024.

Figure 6: Raw data chromatograms of Cynomolgus monkey serum samples in a dose range finding study. All three time point originates from the same animal, sampled at day 1 (after 1 injection of drug), day 8 (after 1 injection of drug) and day 22 (after 3 injections of drug). In the table the relative concentration of A1024 is shown. The calculation is based on the integrated areas of A992 and A1024, mean relative peak for A1024 is the relative area of A1024 as compared to total A1024+A992 calculated from duplicate runs.

Figure 7: Chromatograms of Cynomolgus monkey serum samples from a toxicity study analysed by the PK-Chromatography method. The eight chromatograms are all from a single animal receiving 8 weekly injections of the drug lead. The samples are taken at day 1 : 4, 24 and 72 hours after 1 injection, day 22: 4, 24 and 72 hours after 4 injections and day 50: 4, and 24 hours after 8 injections. In the table the relative peak area of A1024 is shown in percent for the different sampling points. The relative peak area of A1024 is calculated from the percentage of the peak area of A1024 compared to the total peak area of the two peaks A992 and A1024. Detailed description of the invention

The present invention relates to a method for pharmacokinetic assessment of at least two recombinant proteins in a complex biological sample, such as two or more recombinant antibodies in serum. The method involves an affinity chromatography step resulting in up-concentration/enrichment of at least two recombinant proteins followed by another chromatography step leading to detection of the at least two recombinant proteins in the complex biological sample of interest. The present invention relates to a method for pharmacokinetic assessment of at least two recombinant proteins in a complex biological sample comprising the steps of: i) providing a complex biological sample comprising said at least two recombinant proteins,

v) measuring absorbance of said at least two recombinant proteins,

vi) collecting absorbance signals by chromatographic peak integration corresponding to said at least two recombinant proteins thereby obtaining a chromatogram, and vii) analysing the chromatogram and annotating recombinant proteins to detected peaks of said chromatogram thereby detecting and quantitating said at least two recombinant proteins.

The method of the present invention can be used for the simultaneous detection, analysis and quantification of at least two recombinant proteins, such as two or more recombinant antibodies, in a complex biological sample. The sample can be a serum or whole blood sample or any bodily fluid wherein the two or more recombinant proteins are present. The quantification can be a relative quantification or an absolute quantification.

Affinity chromatography The affinity chromatographic step described herein above can comprise any method described in the art for up-concentration/enrichment of at least two recombinant proteins. In one embodiment the up-concentration/enrichment captures intact proteins such as intact recombinant proteins or such as intact recombinant polyclonal antibodies.

The separation by affinity chromatography is based on differences in affinity towards a specific detector molecule, ligand and/or protein. The detector molecule, ligand and/or protein, or a plurality of these (these different options are just termed ligand in the following), is immobilized on a chromatographic medium such as a resin and the sample containing the recombinant proteins is applied to the affinity column under conditions that favor interaction between the individual members and the immobilized ligand. Proteins showing no affinity towards the immobilized ligand are collected in the column flow-through and wash, and proteins showing affinity towards the immobilized ligand are subsequently eluted from the column under conditions that counteract the binding (e.g. low pH, high salt concentration or high ligand concentration).

The affinity chromatography can be performed by Protein A. Protein A can be immobilized onto a support and used for purification of total IgG from a crude protein mixture such as serum. Protein A binds with high affinity to human lgG1 ,lgG2 and lgG4 as well as mouse lgG2a and lgG2b. Protein A binds with moderate affinity to human IgM, IgA and IgE as well as to mouse lgG3 and lgG1 . Protein A can also be used for purification of antibodies from other animals, including monkeys. The human IgG subclasses lgG1 , lgG2 and lgG4 can be separated on Protein A by variation of the pH of the elution buffer. One recombinant form of Protein A is called MabSelect. In one embodiment affinity chromatography on a matrix consisting of Staphylococcal protein A immobilized to agarose beads is used. Alternatives include Protein A-SEPHAROSE, protein A immobilized to agarose, Protein A coupled to Activated Arginine-agarose, and Protein A coupled to magnetic, latex and agarose beads.

In addition to Protein A, other immunoglobulin-binding proteins such as

immunoglobulin-binding bacterial proteins like e.g. Protein G, Protein A/G and Protein L can be used in the affinity chromatography step. Each of these immunoglobulin-binding proteins has a different antibody binding profile in terms of the portion of the antibody that is recognized and the species and type of antibodies it will bind. The invention also relates to use of other immunoglobulin-binding proteins, such as Streptococcal protein G, anti-mouse IgG immunoglobulins such as e.g. rabbit anti-mouse IgG

immunoglobulins, and anti-human IgG immunoglobulins and anti-monkey IgG immunoglobulins generated in suitable species. Such anti-species antibodies are commercially available.

The present invention also relates to use of other affinity based chromatography methods. These include Fc receptors, Con A (Concanavalin A e.g. from Canavalia ensiformis (Jack bean); recognizes glycoproteins), other types of lectin affinity chromatography, antibodies against the Fc part of an antibody or antibodies against the constant part of an antibody.

The affinity determining ligand, such as Protein A, can be immobilized on any column packing material suitable for performing affinity chromatography known to a person skilled in the art such as silica gel, alumina, agarose, cellulose, dextran, crushed glass, and beads such as acrylamide, styrene, ceramic, magnetic or latex beads.

In one embodiment, only a single affinity chromatographic column is used.

In one embodiment, more than one affinity chromatographic column is used.

In one embodiment, the eluate is collected in a series of fractions, wherein said fractions comprise different amounts of the at least two recombinant proteins. One or more of such fractions can be used in the further analysis.

In one embodiment, the affinity chromatography step comprising the use of one or more affinity chromatography columns, such as enrichment by protein A, can be performed in 12, 24, 48, 96, or 384-well formats.

Chromatographic techniques for separation and detection of individual recombinant proteins

The chromatographic step described above in step iv) for separation and detection of the at least two recombinant proteins by applying the eluate comprising the at least two recombinant proteins to one or more additional chromatographic column(s) can comprise any chromatographic method described in the art suitable for separation of at least two recombinant proteins. In one embodiment of the present invention the second chromatography step does not comprise affinity chromatography.

In one embodiment the chromatography step comprises liquid chromatography and leads to detection of two or more recombinant proteins such as at least two

recombinant antibodies constituting a recombinant polyclonal antibody composition. The liquid chromatography in the present invention can be either high performance liquid chromatography (HPLC) or fast protein liquid chromatography (FPLC).

Liquid chromatographic separation of the at least two recombinant proteins, such as the individual members of a recombinant polyclonal antibody composition may be based on differences in physico-chemical properties such as i) net charge (exemplified by ion- exchange chromatography (IEX)), or ii) hydrophobicity (exemplified by reverse- phase chromatography (RP-HPLC), hydrophobic interaction chromatography based on salt concentration (HIC), or hydrophobic charge induction chromatography (HCIC)). A third well-known chromatographic technique is based on the physico-chemical property of size. Size exclusion chromatography (SEC) is a chromatographic method in which molecules in solution are separated based on their size, or in more technical terms, their hydrodynamic volume. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel filtration chromatography, versus the name gel permeation chromatography which is used when an organic solvent is used as a mobile phase. SEC is not a particularly suitable technique for separation of individual members of a recombinant polyclonal antibody composition, since all the recombinant antibodies constituting a recombinant polyclonal antibody composition are essentially the same size.

Methods for achieving sufficient separation in the chromatographic separation technique lie within the capabilities of the person skilled in the art, who can adjust the buffer used, gradient, temperature, flow rate, pressure, column material, etc. Ion-exchange chromatography

In one embodiment of the present invention, ion-exchange (IEX) chromatography is used to separate individual recombinant proteins, such as members of a recombinant polyclonal antibody or a sub-population of individual members of a polyclonal protein. The separation by ion-exchange chromatography is based on the net charge of the individual proteins in the composition to be separated. Depending on the pl-values and conformation of the proteins, and the pH values and salt concentrations of the chosen column buffer, the individual proteins can be separated, at least to some extent, using either anion or cation-exchange chromatography. For example, all the individual proteins in the sample applied to the chromatographic column will normally bind to a negatively charged cation-exchange media as long as the pH is well below the lowest pl-value of the individual proteins in the sample. The individual members of the bound proteins can subsequently be eluted from the column depending on the net charge of the individual proteins, typically using an increasing gradient of a salt (e.g. sodium chloride) or an increasing pH value.

The general principles of cation and anion-exchange are well known in the art, and columns for ion-exchange chromatography are commercially available. Any type of ion- exchange chromatography column suitable for separating the at least two proteins can be used in the present invention. Types of ion exchangers include, but are not limited to polystyrene resins, cellulose and dextran ion exchangers (gels) and controlled-pore glass or porous silica. In a preferred embodiment of the present invention the liquid chromatographic column used for the detection of the individual recombinant proteins is a weak cation exchange high performance liquid chromatography (CIEX-HPLC) column.

In another embodiment the IEX chromatographic column can be selected from the group consisting of: strong cation exchange, weak anion exchange, and strong anion exchange chromatography columns.

Hydrophobic interaction chromatography In further embodiments of the present invention, hydrophobic interaction

chromatography is used to separate individual recombinant proteins, such as the members of a recombinant polyclonal antibody or a sub-population of individual members of a recombinant polyclonal antibody. The separation by hydrophobic interaction chromatography is based on differences in hydrophobicity of the individual proteins in the composition to be separated. The recombinant proteins are bound to a chromatography media modified with a hydrophobic ligand in a buffer that favours hydrophobic interactions. This is typically achieved in a buffer containing a low percentage of organic solvent (RP-HPLC) or in a buffer containing a fairly high concentration of a chosen salt (HIC). The individual recombinant proteins of interest are subsequently eluted from the column depending on the hydrophobicity of the individual proteins, typically using an increasing gradient of organic solvent (RP-HPLC) or decreasing gradient of a chosen salt (HIC).

The general principles of hydrophobic interaction chromatography are well known in the art, and columns for RP-HPLC as well as HIC are commercially available.

In further embodiments of the present invention, hydrophobic charge induction interaction chromatography (HCIC) is used to separate individual recombinant proteins of interest, such as the members of a recombinant polyclonal antibody or a sub- population of individual members of a recombinant polyclonal antibody. The separation by HCIC is based on differences in hydrophobicity of the individual proteins in the composition to be separated. Adsorption is based on mild hydrophobic interaction and is performed without the addition of salts. Desorption is based on charge repulsion achieved by altering the mobile phase pH. Optimal separation of the individual recombinant proteins of interest, following adsorption to the HCIC resin, may be achieved by gradient optimization, e.g. by changing the pH and buffer salt in the mobile phase.

The general principles of hydrophobic charge induction chromatography are well known in the art, and columns for HCIC are commercially available. An example of a commercially available HCIC resin is MEP HyperCel(TM) (PALL, East Hills, NY, USA). The MEP HyperCel(TM) sorbent is a high capacity, highly selective chromatography material specially designed for the capture and purification of monoclonal and polyclonal antibodies.

In one embodiment of the present invention, the eluate obtained from the one or more chromatographic column(s) is collected in a series of fractions, wherein said fractions comprise different amounts of the at least two recombinant proteins. One or more of such fractions can be further analyzed.

In one embodiment, the chromatography step comprising the use of one or more chromatography columns is performed in an automated way, such as by using automated flash chromatography systems (typically referred to as LPLC, low pressure liquid chromatography, around 50-75 psi). Automated systems will include components normally found on more expensive HPLC systems such as a gradient pump, sample injection ports, a UV detector and a fraction collector to collect the eluent. The resolution (or the ability to separate a mixture) on an LPLC system will always be lower compared to HPLC, as the packing material in an HPLC column can be much smaller, typically only 5 micrometre thus increasing stationary phase surface area, increasing surface interactions and giving better separation. However, the use of this small packing media causes the high back pressure and is why it is termed high pressure liquid chromatography. The LPLC columns are typically packed with silica of around 50 micrometres, thus reducing back pressure and resolution, but it also removes the need for expensive high pressure pumps. Medium pressure liquid chromatography (MPLC) systems which operate above 150 psi can also be used in the present invention. Detection of the recombinant proteins

Detection of the individual recombinant proteins in the sample is performed by exposing the column eluate to light of a specific wavelength and measuring the absorbance of the sample. Any wavelength of light suitable for detection of proteins in a sample can be used.

In one embodiment of the present invention the absorbance of the sample is measured after exposing the sample to ultraviolet (UV) light. The preferred wavelength of the UV light is from 200 to 220 nm, for example 200 nm, such as 201 nm, for example 202 nm, such as 203 nm, for example 204 nm, such as 205 nm, for example 206 nm, such as 207 nm, for example 208 nm, such as 209 nm, for example 210 nm, such as 21 1 nm, for example 212 nm, such as 213 nm, for example 214 nm, such as 215 nm, for example 216 nm, such as 217 nm, for example 218 nm, such as 219 nm, for example 220 nm. More preferred is detection with UV light at 215 nm.

In another embodiment, the absorbance of the sample is measured after exposing the sample to ultraviolet (UV) light at from 270 to 290nm, preferably at 280 nm.

In another embodiment, the recombinant proteins are detected by exposing the sample to UV light at more than one wavelength. In one such embodiment the sample is exposed to 200 to 220 nm, such as at 215 nm, and at 270 to 290 nm, such as at 280 nm.

The absorbance signals are collected by chromatographic peak integration thereby obtaining a chromatogram. The at least two recombinant proteins in the sample can be annotated to detected peaks of the chromatogram, thereby detecting said at least two recombinant proteins, such as individual therapeutic antibodies constituting a recombinant polyclonal antibody composition. In one embodiment, the recombinant proteins in the eluate are detected by a fluorescence detector.

Quantification of recombinant proteins Analysis of the chromatogram provides information of the quantity of the individual recombinant proteins in the sample. The quantification can be relative, absolute or evaluation of chromatogram compared to a standard.

Relative quantification of the at least two recombinant proteins in the sample can be performed by direct comparison of the collected absorbance signal i.e. by comparison of the sizes of the individual peaks of the chromatogram obtained.

In another embodiment of the present invention, the recombinant proteins can be quantified in an absolute manner by normalizing the obtained absorbance signals to the slope of a standard curve and adjusting for sample size and dilution. The standard curve is derived from a corresponding recombinant protein preparation of known concentration spiked into a complex biological sample such as serum, wherein the recombinant protein is analyzed using the same procedure as described for the recombinant proteins of interest - i.e using steps i) to vii) herein above.

In yet another embodiment of the invention the chromatogram is evaluated with reference to a known sample, and the presence and/or pattern of the individual peaks is used to evaluate the biological turnover of the protein(s) In a preferred embodiment of the present invention, the standard curve is derived from a corresponding antibody preparation of known concentration spiked into serum. Such standard curves can be used for the absolute quantification of antibodies in serum for e.g. pharmacokinetic assessment of individual therapeutic antibodies constituting a recombinant polyclonal antibody composition.

Sample to be analysed

The recombinant proteins to be analyzed can comprise any recombinant protein of interest including one or more individual antibodies of a recombinant polyclonal antibody composition, a recombinant monoclonal antibody, an immunoglobulin, and a glycoprotein.

In one embodiment the recombinant proteins to be analyzed comprise one or more recombinant B-cell receptors.

In another embodiment the recombinant proteins to be analyzed comprise one or more recombinant T-cell receptors.

In order to separate and detect the at least two individual recombinant proteins in the sample, the recombinant proteins of interest, such as individual antibodies of a recombinant polyclonal composition/product, are required to exhibit net charge differences that facilitate specific and distinctive retention times for separation by ion- exchange chromatography. Likewise, the recombinant proteins of interest are required to exhibit differences in hydrophobicity when hydrophobic interaction chromatography is used to detect the recombinant proteins. The method of the present invention can be used to separate, detect and quantitate between 2 and 20 recombinant proteins, for example 2 recombinant proteins, such as 3 recombinant proteins, for example 4 recombinant proteins, such as 5 recombinant proteins, for example 6 recombinant proteins, such as 7 recombinant proteins, for example 8 recombinant proteins, such as 9 recombinant proteins, for example 10 recombinant proteins, such as 1 1 recombinant proteins, for example 12 recombinant proteins, such as 13 recombinant proteins, for example 14 recombinant proteins, such as 15 recombinant proteins, for example 16 recombinant proteins, such as 17 recombinant proteins, for example 18 recombinant proteins, such as 19 recombinant proteins, for example 20 recombinant proteins.

The sample to be analysed by the method according to the present invention can be any complex biological sample constituting a multitude of proteins and the recombinant proteins of interest. In one embodiment the sample is serum, plasma or whole blood- such as human serum, plasma or whole blood - comprising two or more recombinant proteins such as two or more recombinant polyclonal antibodies. The invention can be used both for analysis of samples from a single individual source or, for purposes of evaluating the level of a particular recombinant protein in a population, can be used to analyze pooled samples from the target population.

In one embodiment, the sample is a bodily fluid selected from the group consisting of whole blood, serum, plasma, ascites fluid, cerebrospinal fluid, amniotic fluid, aqueous humour, cerumen also known as earwax, chyme, interstitial fluid, lymph, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, sweat, tears, urine, vaginal secretion, vomit.

The method according to the present invention can also be implemented for simultaneous analysis of the in vivo clearance of individual antibodies constituting a recombinant polyclonal antibody composition/product in serum from an individual such as a human being for e.g. pharmacokinetic studies. In one embodiment, at least two recombinant proteins such as at least two recombinant antibodies constituting a recombinant polyclonal antibody composition have been administered to said individual. The method of the present invention facilitates the simultaneous analysis of individual antibodies of a polyclonal antibody composition in serum for e.g. pharmacokinetic studies without the need of anti-idiotype antibodies as in e.g. ELISA based techniques. Accordingly, the abundance and degradation rate of individual recombinant polyclonal antibodies comprising a therapeutic recombinant polyclonal antibody composition can be determined and/or monitored in an individual in need thereof.

The sample to be analysed by the method disclosed in the present invention can be a sample such as a serum, plasma or whole blood derived from an individual. Said individual can be a human being or an animal including laboratory/test animals. The animal can be a rabbit, hamster, mouse, rat, monkey, cow, pig, horse, donkey, fish or any other animal used for experimental testing.

The sample to be analysed can be obtained from one or more of the individuals selected from the group consisting of a human being, a man, a woman, a postmenopausal women, a pregnant woman, a lactating woman, an infant, a child, or an adult. The individual such as a human being can be of any age such as from newborn to 120 years old, for example from 0 to 6 months, such as from 6 to 12 months, for example from 1 to 5 years, such as from 5 to 10 years, for example from 10 to 15 years, such as from 15 to 20 years, for example from 20 to 25 years, such as from 25 to 30 years, for example from 30 to 35 years, such as from 35 to 40 years, for example from 40 to 45 years, such as from 45 to 50 years, for example from 50 to 60 years, such as from 60 to 70 years, for example from 70 to 80 years, such as from 80 to 90 years, for example from 90 to 100 years, such as from 100 to 1 10 years, for example from 1 10 to 120 years.

The individual can be of any race such as of Caucasoid race, Capoid race, Mongoloid race, Negroid race or Australoid race. Said human being or animal can be healthy or have one or more diseases. In one embodiment said disease is cancer. In another embodiment said disease is an infectious disease.

Said human being or animal can be diagnosed and/or treated for one or more diseases. In one embodiment said individual is genetically disposed for one or more diseases.

In another embodiment, said individual is immunosuppressed, said

immunosuppression being caused by either disease or immunosuppressive treatment of another disease.

EXAMPLES

The following examples are based on several experiments performed on a drug lead consisting of 2 distinct recombinant antibodies represented in a 1 :1 ratio. The antibodies are denoted A992 and A1024. The antibodies are very homologous, and thus represent an analytical challenge when they have to be analyzed simultaneously. Using the techniques described in the examples, they have been quantified in serum and plasma from Cynomolgus Monkey and in mouse serum either as actual pharmacokinetic (PK) samples or as samples spiked in serum or plasma at a known distribution and concentration. Example 1 : PK measurements in Balb/c Nu/Nu mice.

A PK profile was measured by PK-chromatography after administration of one dose (1 mg) of the drug lead, i.e. 0.5mg A1024 and 0.5mg A992 to 10 mice as a single dose injection. Samples were drawn from the mice over a period of 96 hours, the

concentration of each antibody was calculated using a standard curve of spiked samples.

Materials and methods Protein A purification:

Five μΙ serum from each of 10 mice was pooled to a 50 μΙ pool which was used for analysis at each time point. The recombinant antibodies were purified from the mouse serum using protein A affinity chromatography. A polyPep column (Biorad) for each sample was packed with 50 μΙ Protein A slurry and the columns were equilibrated using 1 ml PBS. Fifty μΙ of sample was loaded to each column, which was subsequently washed in 2 x 250 μΙ PBS. Elution was done by 100 μΙ 0.1 M glycine pH 2.7. Following elution the samples were neutralized by 1 M Tris-base and stored at -20 °C until analysis by ion exchange chromatography (IEX).

Weak cation exchange:

The specific antibody identification and quantification was based on antibody net charge giving specific retention times and UV-detection at 215nm. The UV signal(s) was collected by chromatographic peak integration corresponding to the two individual antibodies constituting the drug product. The UV signal was subsequently normalized to the slope of a standard curve, and adjusted for sample size and dilution for subsequent antibody quantification. A standard curve was derived from a

corresponding antibody preparation of known concentration spiked into to mouse serum. The antibodies were purified from the spiked serum samples and analyzed using the same procedure as for the serum samples representing samples for pharmacokinetic evaluation.

The column was run at 1 ml/min with a 0-100% gradient B over 72.25 min. The solvents were: A: 25mM Sodium Acetate, pH 5 and B: 25mM Sodium Acetate, pH 5, 0.5M NaCI.

HPLC Degasser Agilent G 1379 A

HPLC Column Oven Agilent G1316A

PolyCat ATM column PolyLC.Inc 104CT0315

(100 x 4,6mm,

Sodium acetate Merck 1 .06268

(Germany)

Acetic Acid (glacial) 100% Merck (VWR) 1 .00063

extra pure

Sodium Chloride GR for Sigma-Aldrich 31434

analysis (GER)

Results

The clearance of individual antibodies constituting the drug lead in a single Balb/c Nu/Nu mouse is illustrated in Figure 1 Error! Reference source not found.. It can be observed that the individual antibodies i.e. A1024 and A992 have almost identical PK curves. The total PK curve is also shown, illustrating the sum of the two antibodies. It is evident that the method is capable of measuring the PK curve of two very homologues antibodies simultaneously.

Example 2: PK measurements in Cynomolgus Monkey plasma

The PK profile was measured by PK-chromatography after administration of one dose of the drug lead (18 mg) i.e. 9 mg A992 and 9 mg A1024 to a single Cynomolgus monkey. Six samples were drawn over a period of 48 hours.

Materials and methods

Protein A purification:

Protein A purification was performed in 96 well format. The PK samples are prepared by adding 25μΙ of the plasma to PBS for a total volume of 100μΙ. Standard curves were generated by preparing samples spiked with known amounts of drug lead in 25μΙ control plasma and treating these standards like the samples. Initially, the plate was washed in PBS, and the sample applied, after washing in 2 times 250 μΙ PBS pr well followed by washing in 2x 250 μΙ citrate phosphate buffer pH 5.25 the plate was eluted by citrate phosphate buffer pH 3.5 (Citrate phosphate buffer stock B) in a total volume of 400 μΙ pr well. The eluates were added 400μΙ IEX buffer A (25mM Sodium Acetate pH 5). Samples were transferred to HPLC vials and analyzed using IEX analysis. In all steps the liquid in the wells was removed by evacuation using a Univac manifold (Whatman) except elution where an Eppendorf centrifuge 5804R was used. Citrate phosphate buffers were prepared using:

Citric acid 0.1 M :

19.21 g Citric Acid dissolved in water to 1000 ml, prepared in a 1 L volumetric flask. Na₂HPO_4, 200 mM :

53.61 g Na₂HP0₄-7^*H20 dissolved in water to 1000 ml, prepared in a 1 L volumetric flask.

Citrate Phosphate pH 6.5 (Stock A):

136 ml 0,1 M Citric Acid and 364 ml 0.2 M Na₂HP0_4, adds water to 900 ml. Add water to 1000 ml.

Citrate Phosphate pH 3.5 (Stock B):

359 ml 0.1 M Citric Acid +141 ml 0.2 M Na₂HP0₄ add water to 900 ml. Fill water to 1000 ml.

Citrate Phosphate pH 5,25:

567 ml Citrate Phosphate Stock A + 433 ml Citrate Phosphate Stock B.

Thermomixer Eppendorf

Blank control monkey serum Covance Ltd, Harrogate,

UK

PBS pH 7.2 GIBCO 20012

Citric Acid Sigma C0759

Sodium acetate Merck (Germany) 1 .06268

Acetic acid (glacial) 100% extra pure Merck (Germany) 1 .00056

Sodium Chloride GR for analysis Sigma-Aldrich (GER) 31434

Hydrochloric Acid, 37% Merck (VWR) 1 .00317

Acetic Acid (glacial) 100% extra pure Merck (VWR) 1 .00063

Magnesium Chloride - MgCI2; 6H20 JT Baker 0162

Poly-Prep Column BioRad 731 -1500

Weak cation exchange:

The principle of the weak cation exchange was as described in example 1 , however, with a different column and slightly different eluents. The column was a Dionex ProPac WCX-10 (4x250 mm) column, Buffer A: 25mM sodium acetate pH 5, Buffer B: 25mM Sodium acetate, 1 M NaCI pH 5, Buffer C: 10mM sodium phosphate, 1 M NaCI pH 7.6. (wash buffer), flow: 1 ml/min. The sample was loaded in 25% buffer B. After 3 column volumes (CV) the mobile phase is increased to 35% buffer B and a gradient was run to 60% over 8 min. The column was washed with 100% buffer B for 3.2 CV and 100% buffer C for 3.2 CV. Finally, the column was re-equilibrated at 25% buffer B. After the two wash cycles the needle of the autosampler was washed in the needle wash buffer: 60% isopropanol, 30% acetonitrile, 10% water and subsequently in buffer A. The antibodies were detected at a wavelength of 215nm. Materials/instrumentation Catalog number

Dionex HPLC Summit System

equipped with :

Pump Dionex Softron (GER) 5030.0025

UVD170U detector Dionex Softron (GER)

Standard Flowcell 9mm - volume Dionex Softron (GER) 5065.1820

10μΙ

Lamp Dionex Softron (GER) 5053.1204

ASI-100T Automated sample Dionex Softron (GER) 5810.0015

injector

Carrier with 3 segments Dionex Softron (GER) 5810.9101 A

PBS pH 7.2 GIBCO 20012

Na₂HP0₄ -7Ή20 (268.03 g/mol) Merck 1 .06575.1000

NaH₂P0₄ -2Ή20 (156.01 g/mol) Merck 1 .06345.1000

Isopropanol Merck 1 .09634.1000

Acetonitrile J.T. Baker 9012

Sodium acetate Merck (Germany) 1 .06268

Acetic acid (glacial) 100% extra Merck (Germany) 1 .00056

pure

Sodium Chloride GR for analysis Sigma-Aldrich (GER) 31434

Results

The PK profile was monitored by the PK-chromatography method after administration of 18 mg of drug lead and samples were drawn at 6 time points over a period of 48 hours. An example of the chromatographic raw data is shown in Figure 2. Cynomolgus Monkey Ig is co-purified by protein A by this method and is therefore observed as a large background peak. However, the two peaks corresponding to the administered antibodies are detectable as riders on the background peak and can be integrated. The peaks corresponding to A992 and A1024 as observed in Figure 2 were integrated and the area was used to determine the concentration of the individual antibodies in the original sample using a standard curve of control serum spiked with known amounts of the individual antibodies. The principle of the standard curves is

demonstrated in example 3. The PK curve is shown in Figure 3. Samples were drawn at 6 time points. It can be observed that the pharmacokinetic profiles of the two antibodies in Cynomolgus monkey are comparable.

Example 3: Comparison of standard samples spiked in serum and plasma.

To evaluate whether it was possible to perform the analysis in either plasma or serum, spiked standard curves were made in both matrices from Cynomolgus monkey, and compared. The experiment was performed as described in example 2, with the exception that plasma exchanged for serum in one part of the experiment.

Results

The results showed that it was possible to obtain standard curves in both plasma and serum as shown in Figure 4. For A1024 the standard curves are almost identical whereas for A992 they differ. Thus it is necessary to use the same matrix as the actual sample when generating standard curves for calculating the antibody concentration in actual PK samples.

Example 4: Reducing complexity of serum samples by fractionation of IgG subclasses on Protein A

It is investigated if variation of the pH of the wash buffer in the protein A purification step can reduce the complexity of the biological sample by removing the lgG2 subclass of the total pool of endogenous antibodies. The antibodies of the lgG2 subclass elute at a higher pH compared to lgG1 subclass when using citrate phosphate buffers. The subclass lgG4 is not separated from the lgG1 and lgG2 subtypes while lgG3 antibodies are removed as they do not bind to protein A.

Materials and methods The drug lead is spiked into a pool of human serum at a concentration of 160C^g/ml and 10ΟμΙ sample is prepared per Protein A well. The Protein A procedure was performed according to example 2 except that following addition of the spiked serum samples the wells were washed first with 2χ250μΙ citrate phosphate pH 6.5 followed by washing with either 2χ250μΙ citrate phosphate pH 4.6 or citrate phosphate pH 4.7 or citrate phosphate pH 5 or citrate phosphate pH 5.25 or citrate phosphate pH 5.5.

Citrate Phosphate buffers were prepared from Citrate Phosphate pH 6.5 (stock A) and Citrate Phosphate pH 3.5 (stock B) described in example 2.

Citrate Phosphate pH 4.6

4ml Citrate Phosphate pH 6.5 (stock A) + 5.8ml Citrate Phosphate pH 3.5 (stock B)

Citrate Phosphate pH 4.7

4ml Citrate Phosphate pH 6.5 (stock A) + 5.6ml Citrate Phosphate pH 3.5 (stock B)

Citrate Phosphate pH 5.0

4ml Citrate Phosphate pH 6.5 (stock A) + 4.05ml Citrate Phosphate pH 3.5 (stock B) Citrate Phosphate pH 5.25

4ml Citrate Phosphate pH 6.5 (stock A) + 3.05ml Citrate Phosphate pH 3.5 (stock B)

Citrate Phosphate pH 5.5

4ml Citrate Phosphate pH 6.5 (stock A) + 2.3ml Citrate Phosphate pH 3.5 (stock B)

Results

As showed in Figure 5 the chromatograms show that the height of the main peak originating from endogenous antibodies decreases as the pH of the citrate phosphate wash buffer decreases. Thus, the lowest endogenous antibody background is observed when the serum sample in the protein A plate is washed with citrate phosphate pH 4,6 and the highest endogenous antibody background is observed when using citrate phosphate pH 5,5 as the wash buffer. Similarly, the total peak areas of A992 and A1024 in the table in Figure 5 show that the total area of the drug lead decreases when the pH of the citrate phosphate wash buffer is lowered. This experiment shows that it is possible to reduce sample complexity by washing with a citrate phosphate buffer in the range from 5.5- 4.6.

Whether the PK-chromatography method is used for absolute or relative quantification the relationship between reducing the amount of endogenous antibody and the recovery of the drug should be addressed. However, for absolute quantification the peak areas are normalized to a standard curve correlating drug concentration with peak area, and thus it might be beneficial to reduce the background by a lower pH in the wash, as the standard curve compensates for the recovery loss.

Example 5: Analysis of serum samples by PK-chromatography using relative quantitation In this example samples from a dose range finding study has been analyzed and PK profiles have been monitored in Cynomolgus Monkey using relative quantitation as opposed to absolute quantitation described in the previous examples. The

administered drug was the same as described in the previous examples. As the antibodies are fully resolved in the chromatogram it is possible to determine the relative peak area in percent from the total peak area of the antibody peaks.

Material and methods

The experiment was performed as described for example 2, only the study samples were analyzed in duplicates and were prepared by mixing 66 μΙ serum with 154 μΙ PBS to a total volume of 220 μΙ. For duplicate analysis 100μΙ is aliquoted to separate wells of the Protein A plate.

From the CIEX chromatograms the relative peak areas of A992 and A1024 were determined. For data simplicity the results are only reported as relative peak area of A1024 in percent. The total relative peak area for A992 and A1024 is 100%, hence the relative peak area of A992 (%) is "100% - relative peak area A1024 (%)".

Chromatograms of the Cynomolgus monkey sera containing the drug lead are shown here for one animal at day 1 (after 1 injection), day 8 (after 1 injection), day 22 (after 3 injections). The monkey was dosed with 36mg/kg in the first injection and 24 mg/kg in the following injections.

Results

As showed in Figure 6the method is able to analyze the relative distribution between two antibodies injected in to a Cynomolgus monkey. It is seen that the relative distribution is maintained over time and upon repetitive injections. This is a faster variant of the analysis which does not require a standard curve. It can be applied when information on absolute amounts of antibody is not required.

Example 6: Relative quantification of drug lead in serum samples from a 8 week toxicity study by PK-chromatography The PK-Chromatography method has been used to determine the relative distribution of the drug described in the previous examples in samples from a toxicity study in Cynomolgus monkey. The monkey described in this example received 8 weekly doses of 7mg/kg of the drug lead. Material and methods

The experiments were performed as described in example 2, except the study samples are prepared by adding 33μΙ serum to 77μΙ PBS. As described in example 5, the results are reported as the relative peak area of A1024 in percent. Results

In Figure 7 the chromatograms of the drug lead in serum following the PK- Chromatography analysis is shown for one animal at day 1 : 4, 24 and 72 hours after 1 injection, day 22: 4, 24 and 72 hours after 4 injections and day 50: 4, and 24 hours after 8 injections. The chromatograms illustrate the ability to determine the relative distribution of the antibodies A992 and A1024 in the early time points of the

pharmacokinetic profile following an injection. The results show that the relative distribution of the two antibodies in the Cynomolgus monkey serum. is comparable. Abbreviations

CIEX Cation exchange CV Column volumes IEX Ion exchange ig Immunoglobulin igG Immunoglobulin gamma

PK Pharmacokinetics

Claims

A method for pharmacokinetic assessment of at least two recombinant proteins comprising the steps of:

providing a complex biological sample comprising said at least two recombinant proteins of interest,

applying said complex biological sample to one or more affinity chromatographic column(s),

eluting from said affinity chromatographic column an eluate comprising said at least two recombinant proteins,

applying the column eluate comprising said at least two recombinant proteins obtained in step iii) to one or more additional chromatographic column(s),

measuring absorbance of said at least two recombinant proteins, collecting absorbance signals by chromatographic peak integration corresponding to said at least two recombinant proteins thereby obtaining a chromatogram, and

analysing the chromatogram and annotating recombinant proteins to detected peaks of said chromatogram thereby detecting said at least two recombinant proteins.

The method according to claim 1 , wherein the at least two recombinant proteins comprises one or more recombinant monoclonal antibodies.

The method according to claim 1 , wherein the at least two recombinant proteins comprises one or more recombinant antibodies constituting a recombinant polyclonal antibody composition.

The method according to claim 1 , wherein the recombinant protein is a glycoprotein.

The method according to claim 1 , wherein the recombinant protein is an immunoglobulin.

The method according to claim 1 , wherein the recombinant protein is a recombinant B-cell receptor.

The method according to claim 1 , wherein the recombinant protein is a recombinant T-cell receptor.

The method according to any of the preceding claims, wherein the complex biological sample is serum.

9. The method according to any of the preceding claims, wherein the complex biological sample is human serum.

10. The method according to any of claims 1 to 7, wherein the complex biological sample is mouse serum.

1 1 . The method according to any of claims 1 to 7, wherein the complex biological sample is monkey serum.

12. The method according to any of claims 1 to 7, wherein the complex biological sample is whole blood.

13. The method according to any of claims 1 to 7, wherein the complex biological sample is blood plasma.

14. The method according to any of claims 1 to 7, wherein the complex biological sample is a bodily fluid selected from the group consisting of ascites fluid, cerebrospinal fluid, amniotic fluid, aqueous humour, cerumen also known as earwax, chyme, interstitial fluid, lymph, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, sweat, tears, urine, vaginal secretion, vomit.

15. The method according to claim 1 , wherein the affinity chromatography in step ii comprises binding to one or more immunoglobulin -binding proteins.

16. The method according to claim 1 , wherein the affinity chromatography in step ii comprises binding to one or more bacterial immunoglobulin -binding proteins.

17. The method according to claim 1 , wherein the affinity chromatography in step ii comprises binding to anti-human IgG immunoglobulins.

18. The method according to claim 1 , wherein the affinity chromatography in step ii comprises binding to anti-mouse IgG immunoglobulins.

19. The method according to claim 1 , wherein the affinity chromatography in step ii comprises binding to anti-monkey IgG immunoglobulins.

20. The method according to claim 1 , wherein the affinity chromatography in step ii comprises binding to Protein A.

21 . The method according to claim 1 , wherein the affinity chromatography in step ii comprises binding to Protein G.

22. The method according to claim 1 , wherein the affinity chromatography in step ii comprises binding to Protein A/G.

23. The method according to claim 1 , wherein the affinity chromatography in step ii comprises binding to Protein L.

24. The method according to claim 1 , wherein the affinity chromatography in step ii comprises binding to a lectin.

25. The method according to claim 24, wherein the lectin is concanavalin A.

26. The method according to claim 1 , wherein the affinity chromatography in step ii) comprises binding to a recombinant protein capable of binding to the constant region of antibodies.

27. The method according to claim 26, wherein the recombinant protein capable of binding to the constant region of antibodies comprises an Fc receptor.

28. The method according to claim 1 , wherein the chromatographic column in step iv) is a liquid chromatographic column.

29. The method according to claim 28, wherein the liquid chromatographic column is a high performance liquid chromatography (HPLC) column.

30. The method according to claim 28, wherein the liquid chromatographic column is a fast protein liquid chromatography (FPLC) column.

31 . The method according to claims 28 to 30, wherein the liquid chromatographic column is an ion exchange chromatography column.

32. The method according to claim 28 to 31 , wherein the liquid chromatographic column is a cation exchange chromatography column.

33. The method according to claim 28 to 31 , wherein the liquid chromatographic column is an anion exchange chromatography column.

34. The method according to claim 28 to 31 , wherein the liquid chromatographic column is a weak cation exchange chromatography column.

35. The method according to claim 28 to 31 , wherein the liquid chromatographic column is a strong cation exchange chromatography column.

36. The method according to claim 28 to 31 , wherein the liquid chromatographic column is a weak anion exchange chromatography column.

37. The method according to claim 28 to 31 , wherein the liquid chromatographic column is a strong anion exchange chromatography column.

38. The method according to claim 1 , wherein the chromatographic column in step iv) is a size exclusion chromatography column.

39. The method according to claim 1 , wherein the chromatographic column in step iv) is a reverse-phase chromatography column.

40. The method according to claim 1 , wherein the chromatographic column in step iv) is a hydrophobic charge induction chromatography (HCIC) column.

41 . The method according to claim 1 , wherein the chromatographic column in step iv) is a hydrophobic interaction chromatography (HIC) column.

42. The method according to claim 1 , wherein the chromatographic column in step iv) is not an affinity chromatography column.

43. The method according to claim 1 , wherein the absorbance in step v) is

measured by ultraviolet (UV) light detection.

44. The method according to claim 43, wherein the UV detection is performed at from 200 to 220 nm.

45. The method according to claim 43, wherein the UV detection is performed at 215 nm.

46. The method according to claim 43, wherein the UV detection is performed at from 270 to 290 nm.

47. The method according to claim 43, wherein the UV detection is performed at 280 nm.

48. The method according to claim 43, wherein the UV detection is performed at 215 nm and at 280 nm.

49. The method according to any of the preceding claims wherein the collected absorbance signals are used for performing relative quantification of the at least two recombinant proteins.

50. The method according to claim 49, wherein the collected absorbance signals are used for performing relative quantification of the at least two recombinant proteins, said relative quantification comprising comparison of the collected absorbance signals of the at least two recombinant proteins.

51 . The method according to any of the preceding claims, wherein the collected absorbance signals are used for performing absolute quantification of the at least two recombinant proteins by normalizing said absorbance signals to the slope of a standard curve and adjusting for sample size and dilution, wherein said standard curve is derived from a corresponding recombinant protein preparation of known concentration spiked into a complex biological sample such as serum, wherein the recombinant protein is analyzed using the same procedure as in claim 1 .

52. Use of the method according to any of claims 1 to 51 for determination of in vivo clearance of at least two individual recombinant antibodies constituting a recombinant polyclonal antibody composition in serum from an individual.

53. The method according to claim 52, wherein the individual is a human being.

54. The method according to claim 52, wherein the individual is an animal.

55. The method according to claim 54, wherein the animal is a mouse.

56. The method according to claim 54, wherein the animal is a monkey.

57. The method according to claim 1 , wherein the eluting in step iii) is performed at a pH between 7.2 and 4.5.

58. The method according to claim 57, wherein the eluting in step iii) is performed at a pH between 4.6 and 5.5.

59. The method according to claim 58, wherein the eluting in step iii) is performed at pH 4.6.

60. The method according to claim 58, wherein the eluting in step iii) is performed at pH 4.7.

61 . The method according to claim 58, wherein the eluting in step iii) is performed at pH 5.0.

62. The method according to claim 58, wherein the eluting in step iii) is performed at pH 5.25.

63. The method according to claim 58, wherein the eluting in step iii) is performed at pH 5.5.