Capillary gel electrophoresis and its use in complex biomolecules

Abstract

The invention described herein relates to an improved method for analytical capillary gel electrophoresis (CGE) of complex biomolecules. The method for improved CGE comprises the following steps: partially reducing a bioanalyte molecule used as a calibration standard; and using this partially reduced calibration sample to obtain an improved calibration curve for CGE of the bioanalyte molecule.

Description

Technical Field

[0001] The invention described herein relates to analytical capillary gel electrophoresis (CGE). Background Art

[0002] Capillary gel electrophoresis (CGE) is a sensitive and versatile technique that has become the forefront of analytical methodology. Its applicability is enhanced by short separation times, easy and rapid method development, and the fact that only very small amounts of analytes are required. Capillary gel electrophoresis relies on electrokinetic separation methods performed in sub-millimeter diameter capillaries as well as micro- and nanofluidic channels. In CGE, analytes migrate through an electrolyte solution under the influence of an electric field. Analytes can be separated based on the molecular weight of the molecules present in the analyte.

[0003] CGE is usually used to determine the identity of molecules in a test sample and their relative amounts. Such determinations depend on the migration times of the different components in such test samples. Taking into account the inter-experimental (i.e., experiment-to-experiment) variation of such migration times, it is necessary to establish an appropriate calibration curve for each experiment. Such calibration curves are usually obtained using sample solutions containing a variety of different molecules of known amounts. These molecules are selected to cover a molecular weight range, preferably a molecular weight range that includes the expected molecular weight of the target analyte.

[0004] In such sample / calibration solutions, standard calibration molecules are usually used. In standard CGE methods, the migration time of any molecule is expected to be directly related to its molecular weight. Therefore, the only characteristic associated with a molecule in the sample / calibration solution is its molecular weight.

[0005] However, it has been noted that this direct correlation between migration time and molecular weight is not consistently observed for complex biomolecules. Some complex biomolecules, particularly fusion proteins and antibodies (which may be glycosylated), may have migration times that differ from the expected migration time based on their molecular weight. Therefore, the standard molecules provided in the calibration samples do not provide a calibration curve that can be used to identify various complex biomolecules. Misinterpretation of the peaks identified in the CGE chromatogram may lead to misidentification of the various molecules and their relative amounts in the test sample.

[0006] Therefore, there is a need in the art for an improved CGE method that will overcome the above-mentioned problems observed when performing CGE analysis using complex biomolecules. Summary of the invention

[0007] The present invention provides a solution to the above-mentioned problem. An improved method for analyzing complex biomolecules CGE is provided. In particular, the improved method includes obtaining a calibration curve using a partially reduced calibration solution of the target analyte biomolecule. In one embodiment, a method for determining the molecular weight of a component of a test sample containing biomolecules using capillary gel electrophoresis (CGE) is provided, the method comprising the following steps: a.) obtaining a calibration curve for the biomolecule in CGE, comprising the following steps: partially reducing a calibration sample containing a known amount of biomolecules, submitting the partially reduced calibration sample to CGE, determining the migration time of the components of the partially reduced biomolecules in the partially reduced calibration sample, and calculating the calibration curve for the biomolecule in CGE; b.) submitting a test sample containing biomolecules to CGE; c.) determining the migration time of the components in the test sample; and d.) determining the molecular weight of the components in the test sample by comparing the migration time with the calibration curve obtained in step a). This method is particularly suitable for analyzing samples containing complex biomolecules such as antibodies or fusion proteins.

[0008] In another embodiment, a method for determining the composition of a test sample containing biomolecules is described, comprising: a.) determining the molecular weight of components of the sample according to the method described in the previous embodiment, comprising the following steps: obtaining a calibration curve of biomolecules in CGE by partially reducing a calibration sample containing a known amount of biomolecules, submitting the partially reduced calibration sample to CGE, determining the migration times of components of the partially reduced biomolecules in the partially reduced calibration sample, and calculating the calibration curve of biomolecules in CGE, submitting the test sample containing biomolecules to CGE, determining the migration times of components in the test sample, and determining the molecular weight of components in the test sample by comparing the migration times with the calibration curve; b.) determining the components of the sample based on their molecular weights, and determining the relative amount of each component based on the CGE performed in step a).

[0009] In another embodiment, a method for determining the purity of a sample containing biomolecules is described, comprising: a.) determining the composition of the sample according to the method described in the previous embodiment, and b.) determining the relative amount of the target biomolecule present in the sample.

[0010] BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 ; shows a calibration molecule containing a conventional molecular weight spiked with a complex biomolecule (Cetuximab The difference between the migration times of standard size molecules (Std) and of Cetuximab molecular subunits / substances (considering their theoretical weight) (Erbitux LC and Erbitux HC) is clearly shown.

[0012] Figure 2 ; Shows the expected molar ratio of both the light chain (LC) and the heavy chain (HC) included in the subunits / substances of each different molecule of the partially reduced antibody.

[0013] Figure 3 ; Shows the different migration times and calculated molecular weights of the molecular subunits / substances of partially reduced human IgG2 aglycosylated antibody (anti-TIM3).

[0014] Figure 4 ; Shows the different migration times of molecular subunits / substances of partially reduced human IgG2 aglycosylated antibody (anti-TM3) and a calibration curve based on the observed migration times.

[0015] Figure 5 ; Shows the different migration times of the molecular subunits / species of the two partially reduced antibodies compared to the migration of a commercial size standard; Relative calibration curves as well as large differences were found by comparing the dendrograms.

[0016] Figure 6 ; Shows the different migration times of molecular subunits / substances of partially reduced human IgG2 aglycosylated antibody (anti-TM3), a calibration curve based on the observed migration times, and a comparison between the relative percentages of experimental and theoretical molar ratios.

[0017] Figure 7 ; Shows the different migration times of molecular subunits / substances of partially reduced human IgG1 glycosylated antibodies, a calibration curve based on the observed migration times, and a comparison between the relative percentages of experimental and theoretical molar ratios.

[0018] Figure 8 ; shows the different migration times of molecular subunits / species of partially reduced IgG1 with a kappa light chain glycosylated antibody, a calibration curve based on the observed migration times, and a comparison between the relative percentages of experimental and theoretical molar ratios.

[0019] Fig. 9 ; Shows the different migration times of the molecular subunits / species of the glycosylated antibody of the partially reduced IgG1.4 portion conjugated to Dolaflexin via a maleimide-based Cys linker, the calibration curve obtained based on the observed migration times, and the comparison between the relative percentages of the experimental and theoretical molar ratios.

[0020] Fig.10 ; Shows the different migration times of the molecular subunits / substances of a partially reduced chimeric mouse / human IgG1 aglycosylated antibody, a calibration curve based on the observed migration times, and a comparison between the relative percentages of the experimental and theoretical molar ratios.

[0021] Fig.11 ; Shows the different migration times of the molecular subunits / species of partially reduced fused IgG1 with the Sirp-α domain attached to the LC glycosylated antibody, the calibration curve based on the observed migration times, and the comparison between the relative percentages of the experimental and theoretical molar ratios. Specific implementation plan

[0022] Capillary gel electrophoresis (CGE) is a common analytical method for determining the composition of analyte samples. The method can be used qualitatively and semi-quantitatively to determine the composition of analyte samples, such as analyte samples generated, for example, in the production of active pharmaceutical ingredients. As an initial step of this analytical method, calibration of the method is performed with the aid of a calibration curve of molecules with known molecular weights. The identity of the molecules present in the sample is based on the comparison of their migration times with the migration times of molecules of known molecular weight compared to their molecular weight, as in a calibration curve made with the same molecules under investigation. The method relies on a direct correlation between the migration time and the molecular weight of the molecules in the test sample. However, the migration time of complex biomolecules in CGE is not always directly related to their molecular weight compared to the migration time of conventional standard molecules in the calibration sample.

[0023] exist Figure 1 In this paper, an example is provided in which a sample containing a standard molecular weight marker is mixed with a certain amount of the antibody cetuximab (called ) mixed. Obviously, the subunits / substances of cetuximab with molecular weights of 23kD and 53kD have different migration times compared to conventional standards with approximately the same molecular weight. Therefore, compared with the 25kD standard, the migration time of the 23kD cetuximab subunit / substance migration appears to be around 30kD. Similarly, compared with the 50kD (and 100kD) standard, the 53kD cetuximab subunit / substance has a migration time that looks closer to a molecule of about 75kD. Any such differences may be due to the fact that these complex molecules break down into multiple components when exposed to reducing agents, or that the molecules have such a size or contain certain exposed charges and complex sugars that will cause them to migrate under the electric field and interact with the dextran-based gel matrix (commonly used) in the CGE, thereby exhibiting different behavior from most non-glycosylated conventional standards.

[0024] In the present invention, a sample of the target analyte biomolecule is used to obtain a calibration curve instead of using a conventional calibration standard. In addition, a new method is used to obtain a calibration curve for a partially reduced target complex analyte biomolecule. The method of partially reducing the target complex analyte biomolecule enables the acquisition of a calibration curve with various components (or combinations thereof) of the target analyte biomolecule. For example, the target analyte biomolecule is an antibody protein. This antibody protein is a complex molecule composed of multiple elements (subunits or substances) to form an antibody protein. Two light chains and two heavy chain proteins make up a complete antibody. Figure 2 As shown, partial reduction of an antibody sample results in multiple distinct elements (subunits / species) with different molecular weights and a single specific molar ratio. Figure 2 This possible subunit / substance of the antibody after partial reduction is shown, and the antibody includes a single light chain (LC), a single heavy chain (HC), a single combination of light chain and heavy chain (1H1L), a combination of two heavy chains (HH), a combination of two heavy chains and one light chain (2H1L) and a complete antibody molecule (2H2L). For human IgG2 non-glycosylated exemplary antibodies (anti-TIM3), the molecular weight of the subunit / substance of each molecule is known, such as Figure 3 shown.

[0025] Target analyte biomolecules for use in the methods of the present invention include antibodies, single chain antibodies, fusion proteins, chimeric proteins, and any other protein having one or more subunits or one or more reduction sites (e.g., disulfide bonds). Preferably, for such target analyte biomolecules, the molar ratio of molecular subunits / substance is known. Although particularly useful for analyte biomolecules, the methods of the present invention are not limited to use with complex biomolecules. The methods of the present invention can be used for any analyte molecule that includes reducible subunits.

[0026] Partial reduction of the analyte biomolecule comprises at least two main steps. In the first step, a reducing agent is used to partially reduce the complex biomolecule. In the other step, an alkylating agent is used, which quenches the mixture of the complex biomolecule and the reducing agent and prevents further reduction of the complex biomolecule. Thus, partial reduction of a calibration sample of the target analyte biomolecule comprises: a.) adding a reducing agent to a buffer solution containing a known amount of the biomolecule, and b.) incubating the calibration sample of step a) for a certain period of time, wherein the length of the period of time is such that the biomolecule is not completely reduced. During such a period of time in which the biomolecule is not completely reduced, the method may further comprise: c.) adding an alkylating agent to the partially reduced calibration sample of step b), and d.) incubating the partially reduced calibration sample of step c) for a period of time.

[0027] In the above method for partially reducing a complex biomolecule, the first step (step a.) is preferably carried out at room temperature for a time of about 30 minutes or less. The step of incubating the partially reduced calibration sample with the alkylating agent (step d.) is preferably carried out at a temperature in the range of room temperature to about 75°C, preferably about 50°C to about 75°C, more preferably about 70°C. The incubation time of the partially reduced calibration step with the alkylating agent is up to about 15 minutes, preferably about 5 minutes to about 15 minutes, more preferably a time period of about 10 minutes. The term "about" herein is defined as an amount of plus or minus 10% of the stated value. Thus, about 50°C means 50°C ± 5°C, and about 30 minutes means 30 minutes ± 3 minutes.

[0028] The concentration of the reducing agent and the alkylating agent used in the partial reduction method of the present invention is about 15 to 500 mM, preferably about 30 to about 470 mM, more preferably about 125 mM to about 375 mM, and even more preferably about 200 mM to about 300 mM. The reducing agent and the alkylating agent are preferably used in a reducing agent / alkylating agent concentration ratio of about 0.16 to 0.80. Therefore, the reducing agent / alkylating agent concentration ratio is in the range of 0.15:3, suitably 1:6.5 to 3:1. More preferably, the reducing agent / alkylating agent ratio is 1:1. Preferably, the reducing agent and the alkylating agent concentration is 250 mM.

[0029] As the reducing agent, any conventional reducing agent can be used. Preferably, the reducing agent is 2-mercaptoethanol, 2-mercaptoethylamine, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (phophine) (TCEP) or dithiobutylamine (DTBA). More preferably, the reducing agent is TCEP.

[0030] As the alkylating agent, any conventional alkylating agent can be used. Preferably, the alkylating agent is selected from maleimide or its derivatives, 9-anthracene methanol or its derivatives, 1-naphthalene methanol, 2,2-biphenyl dimethanol, 2-indanol, iodoacetamide, dithiothreitol (DTT), ammonium bicarbonate, streptozotocin, N-nitro-N-ethylurea, procarbazine, temozolomide, busulfan, 2-chloro-2-methylpropane, carmustine, cyclophosphamide, 2-bromo-3'-methoxyacetophenone, 4-chloro-1-butanol, 3-chloropropionamide , bromoacetylcholine bromide, 1,10-diiodododecane, 5-chlorovaleryl chloride, R(-)-chloroethylnormorphine, ethyl methanesulfonate, methyl trifluoromethanesulfonate, tetrapentylammonium bromide, N,N-dimethylisopropylamine, 1-chloro-2,4-dinitrobenzene, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate, ethyl 7-bromoheptanoate, 2-fluorobenzyl bromide, 2,3-dihydro-3-oxo-4H-1,4-benzo oxazine-4-propionic acid, ellagic acid, phenethylamine, dacarbazine, cis-1,5-dimethylbicyclo[3.0]octane-3,7-dione, 2,4'-dichloroacetophenone, 3,4-dichlorobenzyl chloride, trimethylsilyl bromoacetate, (3-bromopropoxy)-tert-butyldimethylsilane, diethyl sulfate, 2-(2-bromoethyl)-1,3-dioxolane, 1,3-benzodithiopyrrole tetrafluoride boron salt, 4-(4-nitrobenzyl)pyridine, 5-methoxyindole-2-carboxylic acid, phosphazine base, ethyl p-toluenesulfonate, N-Boc-5-methoxyindole, romilutrol and chloroacetonitrile. As described herein, suitable derivatives of maleimide are propylmaleimide, N-ethylmaleimide, N-methylmaleimide, N-(2-hydroxyethyl)maleimide, N-hydroxymaleimide, N-(1-phenylethyl)-maleimide, N-(4-chlorophenyl)maleimide, 2-maleimidoethyl-methanesulfonate, 1-(4-aminophenyl)-1H-pyrrole-2,5-dione, N-phenylmaleimide, 1-(2-aminoethyl)maleimide, N-(2-aminoethyl)-maleimide-trifluoroacetate, N-tert-butylmaleimide, N-benzylmaleimide, and N-ethylmaleimide. In addition, as mentioned herein, suitable derivatives of 9-anthracene methanol are α-methyl-9-anthrylmethanol, 9-fluoromethanol, 9-anthrylmethyl methacrylate, 9-anthryloctanal, 9-anthrylcarbonic acid, 9-(methylaminomethyl)-anthracene. More suitably, the alkylating agent is selected from maleimide, iodoacetamide and 9-anthracene methanol. Even more preferably, the alkylating agent is maleimide.

[0031] Thus, the improved method of the present invention includes obtaining a calibration curve using a partially reduced calibration solution of a target analyte biomolecule. In one embodiment, a method for determining the molecular weight of a component of a test sample containing biomolecules using capillary gel electrophoresis (CGE) is provided, the method comprising the following steps: a.) obtaining a calibration curve for the bioanalyte molecules in the CGE, comprising the following steps: partially reducing a calibration sample containing a known amount of the biomolecule, submitting the partially reduced calibration sample to the CGE, determining the migration time of the partially reduced bioanalyte molecule components in the partially reduced calibration sample, and calculating the calibration curve for the bioanalyte molecules in the CGE; b.) submitting a test sample containing the bioanalyte molecules to the CGE; c.) determining the migration time of the components in the test sample; and d.) determining the molecular weight of the components in the test sample by comparing the migration time to the calibration curve obtained in step a). This method is particularly suitable for analyzing samples containing complex biomolecules such as antibodies or fusion proteins.

[0032] The calibration curve can be obtained according to the method for obtaining a calibration curve by partially reducing a known sample of an analyte biomolecule as described above. Therefore, in the method for determining the molecular weight of a component of a test sample of the present invention, it includes: a.) obtaining a calibration curve of bioanalyte molecules in CGE, including the following steps: partially reducing a calibration sample containing a known amount of biomolecules, submitting the partially reduced calibration sample to CGE, determining the migration time of subunits / substances of the partially reduced bioanalyte molecules in the partially reduced calibration sample, and calculating the calibration curve of the bioanalyte molecules in CGE. The partial reduction of the calibration sample includes the following steps: adding a reducing agent to a buffer solution containing a known amount of biomolecules, and incubating the calibration sample for a certain period of time so that the biomolecules are not completely reduced. This partial reduction may further include subsequently adding an alkylating agent to the partially reduced calibration sample, and incubating the partially reduced calibration sample at a temperature of about 0°C to about 75°C for a period of up to 15 minutes. In the next step of the method (step b.), a test sample containing the bioanalyte molecules is submitted to the CGE, and in step c.), the migration times of the components in the test sample are measured, and in step d.), the molecular weights of the components in the test sample are determined by comparing the migration times with the calibration curve obtained in step a).

[0033] The present invention also includes a method for determining or confirming the identity of a bioanalyte molecule in a test sample. For example, such a method includes using a partially reduced bioanalyte molecule as a calibration sample and obtaining a calibration curve using the above-mentioned known bioanalyte molecules. Subsequently, the partially reduced test sample and the CGE method of the present invention are used to determine or confirm the identity of the biomolecule in the test sample. Therefore, such a method may include the following steps: a) obtaining a calibration curve for biomolecules in CGE, including the following steps: partially reducing a calibration sample containing a known amount of biomolecules, submitting the partially reduced calibration sample to CGE, determining the migration time of subunits / substances of the partially reduced biomolecules in the partially reduced calibration sample, and calculating a calibration curve for biomolecules in CGE; b) submitting a test sample containing biomolecules to CGE; c) determining the migration time of components in the test sample; and d) determining the molecular weight of the components in the test sample by comparing the migration time with the calibration curve obtained in step a), and determining or confirming the identity of the bioanalyte molecules in the test sample based on the identity of the molecular weight subunits / substances of the bioanalyte molecules in the test sample with the known biomolecules in the calibration sample.

[0034] Likewise, the present invention provides a method for determining the composition of a sample containing a target biomolecule, comprising determining the molecular weight of a substance (component and subunit) as by any of the above method steps, determining the substance (component or subunit) based on its molecular weight, and determining the relative amount of each substance (component or subunit), as obtained from capillary gel electrophoresis (CGE). This method of the present invention can be further applied to determine the purity of a sample containing a target bioanalyte molecule, determine the relative amount of each substance (component or subunit), to determine the relative amount of the entire target bioanalyte molecule compared to other molecules with different molecular weights in the test sample.

[0035] Example

[0036] The following examples are for illustrative purposes and are not intended to limit the scope of the present invention. Figure 5 Different migration times of molecular subunits / substances of two partially reduced antibodies compared to commercial scale standards are reported in. Large differences were found in the comparison of the dendrograms, ranging from 40 to 80 kDa, indicating and justifying the need for a new standard calibration curve that correlates precisely with the protocol presented herein.

[0037] According to the experimental results, all molecular examples 1-6 with molecular characteristics were submitted to the partial reduction scheme, showing a positive scheme response. In all subsequent embodiments, the partial reduction scheme of biomolecules includes the following steps. The biomolecules in the sample buffer contain 20 μl of sample, 75 μl of sample buffer (50 mM acetate pH 5.5 + 2% SDS), so that the sample contains about 5 mg / ml of target biomolecules. At room temperature, 5 μl of 250 mM TCEP is added to the target biomolecule sample in the sample buffer. The mixture is incubated at room temperature for 10 minutes, and then 5 μl of 250 mM maleimide is added. The combined mixture is then incubated at 70 ° C for 10 minutes. The partially reduced sample thus prepared was then submitted to CGE under standard conditions (system: PA 800 plus capillary electrophoresis system (Beckman Coulter); detector: UV at 220 nm (DAD); capillary: bare fused silica capillary (Beckman Coulter); 50 μm id 30.2 cm, effective length from sample introduction inlet to detector window 20.2 cm, aperture clamp in the box 200 μm; box temperature: 25°C; sample holder temperature: 15°C; voltage and timing: 15 kV (reverse polarity) 35-45 min, depending on the analyte; pre-injection step: 0.1 N NaOH for cleaning the capillary (20 psi, 10 min), 0.1 N HCl was used to neutralize the capillary surface (20 psi, 5 min), water was used to remove the acidic solution (20 psi, 2 min), SDS gel was used to fill the capillary (70 psi, 10 min), MilliQ water was injected each time (injection pressure 7 psi, 10 sec; Materials and Consumables: IgG Purity / Heterogeneity Analysis Kit, SCIEX, code A10663, SDS-MW Gel Buffer, SCIEX, code A30341, Bare Fused Silica Capillary - 50 μm ID, 375 μm OD, 67 cm, SCIEX, code 33845).

[0038] Example 1

[0039] As described above, human IgG2 aglycosylated antibody (anti-TM3) was partially reduced. The partially reduced calibration samples obtained were submitted to capillary gel electrophoresis (CGE) under standard conditions (as described above). Figure 6 As shown, human IgG2 aglycosylated antibody (anti-TM3) submitted to a partial reduction protocol yielded all expected molecular antibody subunits / species and calibration curves were fitted based on the different migration times. A perfect match between the experimental and theoretical relative molar ratios of LC and HC of such species was observed.

[0040] Example 2

[0041] The IgG1 glycosylated antibody was submitted to the partial reduction protocol as described above, and the partially reduced sample obtained was submitted to CGE (as described above). Figure 7 As shown, the IgG1 glycosylated antibody produced all expected molecular antibody subunits / species and a perfect match was observed based on the calibration curve fitted with the different migration times.

[0042] Example 3

[0043] IgG1 of glycosylated antibody with kappa light chain was submitted to the partial reduction protocol as described above and the obtained partially reduced sample was submitted to CGE (as described above). Figure 8 As shown, IgG1 of the glycosylated antibody with kappa light chain produced all the expected molecular antibody subunits / species, as well as calibration curves based on fitting of the different migration times. A perfect match between the experimental and theoretical relative molar ratios of LC and HC of such species was observed.

[0044] Example 4

[0045] The glycosylated antibody IgG1IgG1.4, conjugated to Dolaflexin via a maleimide-based Cys linker, was partially submitted to the partial reduction protocol as described above to obtain a partially reduced sample, which was submitted to CGE (as described above). Fig. 9 As shown, the glycosylated antibody IgG1.4 moiety conjugated to Dolaflexin via a maleimide-based Cys linker yielded all the expected molecular antibody subunits / species, as well as calibration curves based on fitting of the different migration times. A perfect match between the experimental and theoretical relative molar ratios of the LC and HC of such species was observed.

[0046] Example 5

[0047] As described above, the chimeric mouse / human IgG1 aglycosylated antibody was submitted to the partial reduction protocol to obtain a partially reduced sample, which was submitted to CGE (as described above). Fig.10 As shown, the chimeric mouse / human IgG1 aglycosylated antibody produced all the expected molecular antibody subunits / species, as well as calibration curves based on fitting of the different migration times. A perfect match between the experimental and theoretical relative molar ratios of LC and HC for such species was observed.

[0048] Example 6

[0049] As described above, the fusion IgG1 with the Sirp-α domain linked to the LC glycosylated antibody was submitted to the partial reduction protocol to obtain a partially reduced sample, which was submitted to CGE (as described above). Fig.11 As shown, the fusion IgG1 with the Sirp-α domain linked to the LC glycosylated antibody produced all the expected molecular antibody subunits / species, as well as the calibration curves derived from the fit of the different migration times. A perfect match between the experimental and theoretical relative molar ratios of LC and HC for such species was observed.

Claims

1. A method for determining the molecular weight of a component of a test sample by using capillary gel electrophoresis (CGE), the test sample containing biomolecules, the method comprising the following steps: a) obtaining a calibration curve of a biomolecule in CGE, comprising the steps of: subjecting a calibration sample comprising a known amount of the biomolecule to partial reduction, submitting the partially reduced calibration sample to CGE, determining the migration time of subunits / species of the partially reduced biomolecule in the partially reduced calibration sample, and calculating a calibration curve of the biomolecule in CGE; b) submitting a test sample containing the biomolecule to CGE; c) determining the migration time of components in the test sample; and d) determining the molecular weight of the component in the test sample by comparing the migration time with the calibration curve obtained in step a).

2. The method of claim 1, wherein the step of subjecting the calibration sample to partial reduction comprises: a) adding a reducing agent to a buffer solution containing a known amount of a biomolecule, and b) incubating the calibration sample of step a) for a period of time, wherein the length of the period of time is such that the biomolecule is not completely reduced.

3. The method of claim 2, wherein the step of subjecting the calibration sample to partial reduction comprises: a) adding the reducing agent to the buffer solution, b) incubating the calibration sample at room temperature for a period of 30 minutes or less, and further comprising the steps of c) adding an alkylating agent to the partially reduced calibration sample of step b), and d) incubating said partially reduced calibration sample of step c) at a temperature of 0°C to 75°C for a period of time of up to 15 minutes. 4 . The method according to claim 3 , wherein the reducing agent and the alkylating agent are added to the solution at a concentration ratio of reducing agent / alkylating agent of 0.15 to 3. 5 . The method according to claim 3 , wherein the reducing agent and the alkylating agent are added to the solution at a concentration ratio of reducing agent / alkylating agent of 1:6.5 to 3:

1.

6. The method according to any one of claims 3 to 5, wherein the reducing agent and the alkylating agent are added to the solution at a concentration ratio of reducing agent / alkylating agent of 1:

1.

7. The method according to any one of claims 3 to 5, wherein the reducing agent is added at a concentration of 15 to 500 mM and the alkylating agent is added at a concentration of 15 to 500 mM.

8. The method according to any one of claims 3 to 5, wherein the reducing agent is added at a concentration of 250 mM and the alkylating agent is added at a concentration of 250 mM.

9. The method according to any one of claims 2 to 5, wherein the reducing agent is tris(2-carboxyethyl)phosphine (TCEP).

10. The method according to any one of claims 3 to 5, wherein the alkylating agent is maleimide or a derivative thereof.

11. The method according to any one of claims 2 to 5, wherein the calibration sample is incubated with the reducing agent at room temperature for a period of up to 20 minutes.

12. The method of claim 11, wherein the time period is 20 minutes.

13. The method according to any one of the preceding claims 2 to 5, wherein the biomolecule is a complex molecule comprising two or more subunits / substances. The method according to claim 13 , wherein the biomolecule is at least one selected from a recombinant protein, an antibody and a fusion protein.

15. The method according to claim 3, wherein the step d) is carried out at a temperature of 70-75°C.

16. The method of claim 3, wherein in step d), the partially reduced calibration sample of step c) is incubated for 10-15 minutes.

17. The method according to claim 10, wherein the derivative of maleimide is at least one of propylmaleimide, N-methylmaleimide, N-(2-hydroxyethyl)maleimide, N-hydroxymaleimide, N-(1-phenylethyl)-maleimide, N-(4-chlorophenyl)maleimide, 2-maleimidoethyl-methanesulfonate, 1-(4-aminophenyl)-1H-pyrrole-2,5-dione, N-phenylmaleimide, 1-(2-aminoethyl)maleimide, N-(2-aminoethyl)-maleimide-trifluoroacetate, N-tert-butylmaleimide, N-benzylmaleimide and N-ethylmaleimide.

18. A method for determining the composition of a sample containing biomolecules, the method comprising: a) determining the molecular weight of the components of the sample by the method of any one of claims 1 to 17, b) determining said components of said sample based on their molecular weights, c) and, determining the relative amount of each component based on the CGE performed in step a.

19. A method for determining the purity of a sample containing biomolecules, comprising: a) determining the composition of the sample according to the method of claim 18, and b) determining the relative amount of each subunit or fragment of said biomolecule present in said sample.

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