Capillary gel electrophoresis and its use with complex biomolecules

Abstract

The invention described herein relates to an improved method for analytical capillary gel electrophoresis (CGE) of complex biomolecules. The improved method for CGE includes the steps of partially reducing an analyte biomolecule for use as a calibration standard, and using such partially reduced calibration samples to obtain an improved calibration curve for CGE for use with the analyte biomolecule.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The invention described herein relates to analytical capillary gel electrophoresis (CGE). [Background technology]

[0002] Background technology Capillary gel electrophoresis (CGE) is a highly sensitive and versatile analytical technique that is at the forefront of analytical technology. Its applicability is enhanced by short separation times, simple and rapid development methods, and the very small amounts of analytes required. Capillary gel electrophoresis relies on electrokinetic separation methods performed in submillimeter-diameter capillaries and micro- and nanofluidic channels. In CGE, analytes migrate through an electrolyte solution under the influence of an electric field. Analytes can be separated according to the molecular weight of the molecules present in the analytes.

[0003] CGE is often used to determine the identity and relative abundance of molecules in a test sample. Such measurements depend on the migration times of different components in the test sample. Given the inter-experiment (i.e., experiment-to-experiment) variability of such migration times, it is necessary to establish an appropriate calibration curve for each experiment. Such calibration curves are customarily obtained using a sample solution containing known amounts of a number of different molecules. These molecules are selected to cover a range of molecular weights, preferably one that encompasses the expected molecular weight of the analyte of interest.

[0004] Standard calibration molecules are routinely used in such samples / calibration solutions. In standard CGE methods, the migration time of any molecule is expected to be directly correlated to its molecular weight. Therefore, the only relevant property of the molecules in the sample / calibration solution is their molecular weight.

[0005] However, it should be 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 (these proteins can be glycosylated), may have migration times that differ from those expected based on their molecular weight. Therefore, the standard molecules provided in the calibration sample do not provide a calibration curve that can be used to identify various complex biomolecules. Misinterpretation of peaks in a CGE chromatogram can result in incorrect identification of various molecules and their relative amounts in the test sample.

[0006] Therefore, there is a need in the art for improved CGE methods that address these problems observed when performing analytical CGE on complex biomolecules. Summary of the Invention

[0007] Summary of the Invention The present invention provides a solution to the above-described problems. An improved method for analytical CGE of complex biomolecules is provided. In particular, such an improved method involves obtaining a calibration curve using a partially reduced calibration solution of the analyte biomolecule of interest. In one embodiment, a method for measuring the molecular weight of components of a test sample containing a biomolecule using capillary gel electrophoresis (CGE) is provided, the method comprising the following steps: a.) obtaining a calibration curve for the biomolecule in CGE, including partially reducing a calibration sample containing a known amount of the biomolecule, subjecting the partially reduced calibration sample to CGE, and measuring the migration time of the partially reduced biomolecule components in the partially reduced calibration sample to calculate a calibration curve for the biomolecule in CGE; b.) subjecting a test sample containing the biomolecule to CGE; c.) measuring the migration time of the components in the test sample; and d.) identifying the molecular weight of the components in the test sample by comparing the migration time with the calibration curve obtained in step a). Such a method is particularly useful for analyzing samples containing complex biomolecules, such as antibodies or fusion proteins.

[0008] In another embodiment, a method is described for identifying the composition of a test sample containing biomolecules, comprising the following steps: a.) obtaining a calibration curve for biomolecules in CGE by partially reducing calibration samples containing known amounts of biomolecules, subjecting the partially reduced calibration samples to CGE, measuring the migration times of the components of the partially reduced biomolecules in the partially reduced calibration samples, and calculating a calibration curve for biomolecules in CGE; subjecting a test sample containing biomolecules to CGE, measuring the migration times of the components in the test sample, and identifying the molecular weights of the components in the test sample by comparing the migration times with the calibration curve; and b.) identifying the components of the sample based on their molecular weights and determining the relative amounts 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 a biomolecule is described, comprising the steps of: a.) using a method described in the previous embodiment to identify the composition of the sample according to the method, and b.) measuring the relative amount of a biomolecule of interest present in the sample. [Brief explanation of the drawings]

[0010] [Figure 1] Figure 1 shows the migration times of samples containing calibration molecules of conventional molecular weights spiked with a complex biomolecule (cetuximab (Erbitux®)). The discrepancy in migration times of the standard sized molecule (Std) and the cetuximab molecular subunits / species (considering their theoretical weights) (Erbitux LC and Erbitux HC) is clearly shown. [Figure 2] FIG. 2 shows the predicted molar ratio of light chain (LC) to heavy chain (HC) contained in each different molecular subunit / species of partially reduced antibodies. [Figure 3] FIG. 3 shows the differences in migration time and calculated molecular weight of molecular subunits / species of a partially reduced human IgG2 aglycosylated antibody (anti-TIM3). [Figure 4]FIG. 4 shows the different migration times of molecular subunits / species of a partially reduced human IgG2 aglycosylated antibody (anti-TM3) and the calibration curve obtained based on the observed migration times. [Figure 5] Figure 5 compares the migration times of the molecular subunits / species of the two partially reduced antibodies with those of commercially available size standard species, showing the relative calibration curves and the large discrepancies observed in the tree curve comparison. [Figure 6] Figure 6 shows the differences in migration times of molecular subunits / species of a partially reduced human IgG2 aglycosylated antibody (anti-TM3), as well as a calibration curve obtained based on the observed migration times and a comparison of the measured and theoretical molar relative proportions. [Figure 7] FIG. 7 shows the differences in migration times of molecular subunits / species of partially reduced IgG1 antibodies, a calibration curve based on the observed migration times, and a comparison of the relative percentage molar ratios between the observed and theoretical values. [Figure 8] FIG. 8 shows the differences in migration times of molecular subunits / species of a partially reduced IgG1 antibody with a κ light chain, a calibration curve based on the observed migration times, and a comparison of the relative percentage molar ratios between the observed and theoretical values. [Figure 9] FIG. 9 shows the migration times of different molecular subunits / species of partially reduced IgG1.4 moieties conjugated to Dolaflexin with a maleimide-based Cys linker glycosylated antibody, the resulting calibration curve based on the observed migration times, and a comparison of the relative percentage molar ratios between the observed and theoretical values. [Figure 10] FIG. 10 shows the migration times of molecular subunits / species of a partially reduced chimeric mouse / human IgG1 aglycosylated antibody, a calibration curve based on the observed migration times, and a comparison of the relative percentage molar ratios between the observed and theoretical values. [Figure 11] Figure 11 shows the difference in migration time between the molecular subunits / species of Sirp-α domain-linked partially reduced fusion IgG1 antibody and LC-glycosylated antibody, a calibration curve based on the observed migration times, and a comparison of the relative percentage molar ratios between the observed and theoretical values. DETAILED DESCRIPTION OF THE INVENTION

[0011] Detailed Description Capillary gel electrophoresis (CGE) is an analytical method frequently used to determine the composition of an analyte sample. This method can be used qualitatively or semi-quantitatively to determine the composition of an analyte sample, such as those produced in the manufacture of active pharmaceutical ingredients. As the first step in such an analytical method, a calibration curve is generated using molecules of known molecular weight. The identity of a molecule present in a sample relative to its molecular weight is based on its migration time compared to that of a molecule of known molecular weight in a calibration curve generated with the same molecule under test. The method relies on a direct correlation between migration time and the molecular weight of the molecule in the test sample. However, the migration time of complex biomolecules in CGE may not always directly correlate to molecular weight when compared to the migration time of conventional standard molecules in a calibration sample.

[0012] Figure 1 shows an example of a mixture of a sample containing standard molecular weight standards and a fixed amount of the antibody cetuximab (known as Erbitux®). It is clear that the 23 kD and 53 kD cetuximab subunits / species migrate at different migration times compared to conventional standards of approximately the same molecular weight. The results confirmed that the 23 kD cetuximab subunit / species migrates at a migration time that appears to be approximately 30 kD compared to the 25 kD standard. Similarly, the 53 kD cetuximab subunit / species has a migration time that appears to be closer to that of a molecule of approximately 75 kD compared to the 50 kD (and 100 kD) standards. Such discrepancies may be due to the breakdown of complex molecules into multiple components upon exposure to reducing agents, or because the molecules are of such size or contain certain exposed charges and glycoconjugates that they migrate under the electric field and interaction with the dextran-based gel matrix (commonly used) in CGE and behave differently from the largely unglycosylated conventional standard.

[0013] In the present invention, instead of using a conventional calibration curve, a calibration curve is obtained using a sample of the analyte biomolecule of interest. Furthermore, the calibration curve is obtained using a novel method for partially reducing a complex analyte biomolecule of interest. The method for partially reducing a complex analyte biomolecule of interest allows for the generation of a calibration curve using various components (or combinations thereof) of the analyte biomolecule of interest. For example, the analyte biomolecule of interest is an antibody protein. Such an antibody protein is a complex molecule composed of multiple components (subunits or species) that form the antibody protein. Two light chain and two heavy chain proteins constitute a complete antibody. As shown in Figure 2, partial reduction of an antibody sample results in multiple components (subunits / species) of different molecular weights in a single specific molar ratio. Figure 2 shows the possible subunits / species of antibodies in such partial reduction, including individual light chains (LC), individual heavy chains (HC), a single combination of light and heavy chains (1H1L), a combination of two heavy chains (HH), a combination of two heavy chains and one light chain (2H1L), and the intact antibody molecule (2H2L). For the human IgG2 aglycosylated model antibody (anti-TIM3), the molecular weight of each molecular subunit / species is known, as shown in Figure 3.

[0014] Analyte biomolecules of interest for use in the methods of the present invention include antibodies, single-chain antibodies, fusion proteins, chimeric proteins, and any other proteins having one or more subunits or one or more reduction sites (such as disulfide bonds). Preferably, for such analyte biomolecules of interest, the molar ratio of molecular subunits / species is known. While particularly useful for analyte biomolecules, the methods of the present invention are not limited to use with complex biomolecules. Any analyte molecule containing a reducible subunit can be used in the methods of the present invention.

[0015] The partial reduction of an analyte biomolecule involves at least two major steps. In the first step, a reducing agent is used to partially reduce the complex biomolecule. In the second step, an alkylating agent is used to quench the mixture of the complex biomolecule and the reducing agent, preventing further reduction of the complex biomolecule. Thus, the partial reduction of a calibration sample of an analyte biomolecule of interest involves: 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 period of time such that the biomolecule is not completely reduced. Within this period of time, the method may further include: 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.

[0016] In the above method for partially reducing complex biomolecules, the first step (step a.) is preferably carried out at room temperature for a period of about 30 minutes or less. The step of incubating the partially reduced calibration sample with an alkylating agent (step d.) is preferably carried out at a temperature ranging from room temperature to about 75°C, preferably from about 50°C to about 75°C, more preferably about 70°C. The incubation with the alkylating agent in the partial reduction calibration step is for a period of up to about 15 minutes, preferably from about 5 minutes to about 15 minutes, more preferably about 10 minutes. As used herein, the term "about" is defined as a value plus or minus 10 percent of the stated value. Thus, about 50°C refers to 50°C ± 5°C, and about 30 minutes refers to 30 minutes ± 3 minutes.

[0017] The concentrations of the reducing agent and alkylating agent used in the partial reduction method of the present invention are 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 alkylating agent are preferably used at a reducing agent:alkylating agent concentration ratio of about 0.16 to 0.80. Thus, the reducing agent:alkylating agent concentration ratio is in the range of 0.15:3, preferably 1:6.5 to 3:1. More preferably, the reducing agent:alkylating agent ratio is 1:1. Preferably, the reducing agent and alkylating agent concentrations are 250 mM.

[0018] The reducing agent can be any conventional reducing agent. Preferably, the reducing agent is 2-mercaptoethanol, 2-mercaptoethylamine, dithiothreitol (DTT), tris(2-carboxyethly)phosphine (TCEP), or dithiobutylamine (DTBA). More preferably, the reducing agent is TCEP.

[0019] The alkylating agent may be any conventional alkylating agent. Preferably, the alkylating agent is maleimide or a derivative thereof, 9-anthracenemethanol or a derivative thereof, 1-naphtalinemethanol, 2,2-biphenyldimethanol, 2-indanol, iodoacetamide, dithiothreitol (DTT), ammonium carbonate, streptozocin, 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-diiododecane, 5-chlorovaleroyl chloride, R(-)-chloroethylnorapomorphine, 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-benzoxazine-4-propionic acid, Ellagic acid, Phenethylamine, Decarbazine, cis-1,5-Dimethylbicyclo[3.3.0]octane-3,7-dione, 2,4'-Dichloroacetophenone, 3,4-Dichlorobenzyl chloride chloride, trimethylsilyl bromoacetate, (3-bromopropoxy)-tert-butyldimethylsilane, dimethyl sulfate, 2-(2-bromoethyl)-1,3-dioxolane, 1,3-benzodithiolylium tetrafluoroborate, 4-(4-nitrobenzyl)pyridine, 5-methoxyiodo-2-carboxylic acid, phosphazine base, ethyl-p-toluenesulfonate, N-Boc-5-methoxyiodide, romgatribut, and chloroacetonitrile.As provided herein, preferred derivatives of maleimide are propynylmaleimide, N-ethylmaleimide, N-methylmaleimide, N-(2-hydroxyethyl)maleimide, N-hydroxymaleimide, N-(1-phenylethyl)-maleimide, N-(4-chlorophenyl)maleimide, 2-maleinimidoethyl-mesylate, 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. Additionally, as provided herein, preferred derivatives of 9-anthracenemethanol are α-methyl-9-anthrylmethanol, 9-fluorenemethanol, methacrylic acid-9-anthracenylmethyl ester, 9-anthracenecarbaldehyde, 9-anthracenecarboylic acid, and 9-(methylaminomethyl)-anthracene. More preferably, the alkylating agent is selected from maleimide, iodoacetamide, and 9-anthracenemethanol. Even more preferably, the alkylating agent is maleimide.

[0020] Thus, the improved method of the present invention involves obtaining a calibration curve using a partially reduced calibration solution of the analyte biomolecule of interest. In one embodiment, a method is provided for measuring the molecular weight of components of a test sample containing a biomolecule using capillary gel electrophoresis (CGE), the method comprising the following steps: a.) obtaining a calibration curve for the analyte biomolecule in CGE, comprising partially reducing a calibration sample containing a known amount of the biomolecule, subjecting the partially reduced calibration sample to CGE, and measuring the migration time of the partially reduced components of the analyte biomolecule in the partially reduced calibration sample, thereby calculating a calibration curve for the analyte biomolecule in CGE; b.) subjecting a test sample containing the analyte biomolecule to CGE; c.) measuring the migration time of the components in the test sample; and d.) identifying the molecular weight of the components in the test sample by comparing the migration time with the calibration curve obtained in step a). Such a method is particularly useful for analyzing samples containing complex biomolecules such as antibodies or fusion proteins.

[0021] A calibration curve can be obtained by the method described above for obtaining a calibration curve by partial reduction of a known sample of the analyte biomolecule. Thus, in a method of the present invention for determining the molecular weight of a component of a test sample, a calibration curve for the analyte biomolecule can be obtained by: a.) partially reducing a calibration sample containing a known amount of the biomolecule; subjecting the partially reduced calibration sample to CGE; measuring the migration time of the subunits / species of the partially reduced analyte biomolecule in the partially reduced calibration sample; and calculating a calibration curve for the analyte biomolecule in CGE. Partial reduction of the calibration sample can include adding a reducing agent to a buffer solution containing a known amount of the biomolecule and incubating the calibration sample for a time period such that the biomolecule is not completely reduced. Such partial reduction can 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 up to 15 minutes. In the next step of the method (step b.), a test sample containing the target biomolecule is subjected to CGE, and in step c.), the migration time of the components in the test sample is measured. In step d.), the migration time is compared with the calibration curve obtained in step a) to identify the molecular weight of the components in the test sample.

[0022] The present invention further includes methods for determining or confirming the identity of an analyte biomolecule in a test sample. For example, such methods include using partial reduction of the analyte biomolecule as a calibration sample to obtain a calibration curve with known analyte biomolecules as described above. The identity of the biomolecule in the test sample is then determined or confirmed using partial reduction of the test sample and the CGE method of the present invention. Thus, such a method may comprise the following steps: a) obtaining a calibration curve for a biomolecule in CGE, comprising partially reducing a calibration sample containing a known amount of the biomolecule, subjecting the partially reduced calibration sample to CGE, and measuring the migration times of the subunits / species of the partially reduced biomolecule in the partially reduced calibration sample, and calculating a calibration curve for the biomolecule in CGE; b) subjecting a test sample containing the biomolecule to CGE; c) measuring the migration times of the components in the test sample, and d) identifying the molecular weights of the components in the test sample by comparing the migration times with the calibration curve obtained in step a), and determining or confirming the identity of the analyte biomolecule in the test sample based on the molecular weight subunits / species of the analyte biomolecule in the test sample and the identities of the known biomolecules in the calibration curve sample.

[0023] Similarly, the present invention provides a method for identifying the composition of a sample containing a biomolecule of interest, comprising measuring the molecular weights of the species (components or subunits) of the sample via any of the method steps described above, identifying the species (components or subunits) based on their molecular weights, and determining the relative amount of each species (components or subunits) as obtained by capillary gel electrophoresis (CGE). Such methods of the present invention can be further applied to determine the purity of a sample containing an in vivo analyte molecule of interest by measuring the relative amount of each species (component or subunit), and to determine the relative amount of the total analyte biomolecule of interest compared to other molecules in a test sample of different molecular weights. [Example]

[0024] Example The following examples are illustrative and are not intended to limit the scope of the present invention. The different migration times of two molecular subunits / species of partially reduced antibodies compared to the migration of commercially available size standard species are reported in Figure 5. The large discrepancies between 40 and 80 kDa found in the tree curve comparison point out and demonstrate the need for a new type of standard calibration curve that is precisely related to the protocol presented here.

[0025] All of Examples 1-6, in which molecules with molecular characteristics were subjected to the partial reduction protocol, demonstrated a positive protocol response in terms of experimental results. In all subsequent Examples, the partial reduction protocol for biomolecules consisted of the following procedure: Biomolecules in sample buffer contained 20 μl of sample, 75 μl of sample buffer (50 mM acetate, pH 5.5 + 2% SDS), and the sample contained approximately 5 mg / ml of the biomolecule of interest. To the biomolecule sample of interest in the sample buffer, 5 μl of 250 mM TCEP was added at room temperature. This mixture was incubated at room temperature for 10 minutes, after which 5 μl of 250 mM maleimide was added. The combined mixture was then incubated at 70°C for 10 minutes. The partially reduced sample thus prepared was subjected to CGE under standard conditions (system: PA800+ capillary electrophoresis system (Beckman Coulter); detector: UV at 220 nm (DAD); capillary: bare quartz capillary (Beckman Coulter), inner diameter 50 μm, 30.2 cm, effective length from sample inlet to detection window 20.2 cm, aperture clip in cartridge 200 μm, cartridge 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: NaOH 0.1 N (20 psi, 10 min) to wash the capillary, HCl 0.1 N (20 psi, 5 min) to neutralize the capillary surface, water (20 psi, 2 min) to remove the acidic solution, filling the capillary with SDS Gel (70 psi, 10 min), MilliQ water / injection (injection pressure 7 psi, 10 seconds); Materials and consumables: IgG Purity and Heterogeneity Analysis Kit, SCIEX, Code A10663; SDS-MW Gel Buffer, SCIEX, Code A30341; Bare Fused Quartz Capillary, 50 μm inner diameter, 375 μm outer diameter, 67 cm, SCIEX, Code 33845).

[0026] Example 1 A glycosylated human IgG2 antibody (anti-TM3) was subjected to partial reduction as described above. The resulting partially reduced calibration samples were subjected to capillary gel electrophoresis (CGE) under standard conditions (described above). As shown in Figure 6, the partially reduced human IgG2 antibody (anti-TM3) was subjected to the partial reduction protocol, and the resulting calibration curve based on fitting all expected molecular antibody subunits / species and different migration times was obtained. The relative molar ratios of LC and HC of these molecular species were observed to be in perfect agreement with the theoretical values.

[0027] Example 2 An IgG1 glycosylated antibody was subjected to the partial reduction protocol described above, and the resulting partially reduced sample was subjected to CGE (as described above). As shown in Figure 7, the IgG1 glycosylated antibody produced all the expected molecular antibody subunits / species, and a calibration curve based on different migration time fitting was obtained. Note that perfect agreement was observed.

[0028] Example 3 The glycosylated IgG1 antibody with kappa light chain was subjected to the partial reduction protocol described above, and the resulting partially reduced sample was subjected to CGE (as described above). As shown in Figure 8, the glycosylated IgG1 antibody with kappa light chain generated all the expected molecular antibody subunits / species, and a calibration curve based on fitting different migration times was obtained. The relative molar ratios of LC and HC of these species were confirmed to be in perfect agreement with the experimental and theoretical values.

[0029] Example 4 The IgG1.4 moiety conjugated to Dolaflexin with a maleimide-based Cys-linker glycosylated antibody was subjected to the partial reduction protocol described above to obtain a partially reduced sample that was subjected to CGE (as described above). As shown in Figure 9, the IgG1.4 moiety conjugated to Dolaflexin with a maleimide-based Cys-linker glycosylated antibody yielded a calibration curve based on fitting the migration times of all expected antibody subunits / species. The relative molar ratios of LC and HC for these species were confirmed to be in perfect agreement with the experimental and theoretical values.

[0030] Example 5 A partially reduced chimeric mouse / human IgG1 aglycosylated antibody was subjected to the partial reduction protocol as described above, and the partially reduced sample was subjected to CGE (as described above). As shown in Figure 10, the chimeric mouse / human IgG1 aglycosylated antibody exhibited all expected molecular antibody subunits / species, and a calibration curve was obtained based on the fitting of different migration times. The relative molar ratios of LC and HC of these species were confirmed to be in perfect agreement with the experimental and theoretical values.

[0031] Example 6 The fusion IgG1 containing Sirp-α domain and LC-glycosylated antibody was subjected to the partial reduction protocol as described above to obtain a partially reduced sample, which was then subjected to CGE (described above). As shown in Figure 11, the fusion IgG1 containing Sirp-α domain and LC-glycosylated antibody produced all the expected molecular antibody subunits / species, and a calibration curve was obtained based on fitting different migration times. The relative molar ratios of LC and HC in these species were confirmed to be in perfect agreement with the experimental and theoretical values.

Claims

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

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

3. The step of partially reducing the calibration sample comprises the steps of: a) adding a reducing agent to the buffer; b) incubating the calibration sample at room temperature for 30 minutes or less, and further comprising the following steps c) and d): c) adding an alkylating agent to the partially reduced calibration sample of step b; and d) incubating the partially reduced calibration samples of step c) at a temperature between 0°C and 75°C for a maximum of 15 minutes; The method of claim 2 , comprising:

4. The method described in claim 3, wherein the step of incubating the partially reduced calibration sample in step c) is carried out at 70°C and / or for 10 minutes.

5. 5. The method of claim 3 or 4, wherein the reducing agent and alkylating agent are added to the solution in a concentration ratio of reducing agent:alkylating agent of 0.15:

3.

6. 5. The method of claim 3, wherein the reducing agent and alkylating agent are added to the solution in a concentration ratio of reducing agent:alkylating agent of 1:6.5 to 3:

1.

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

1.

8. 8. The method of claim 3, 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.

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

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

11. The method of any one of claims 3 to 10, wherein the alkylating agent is maleimide or an analog thereof.

12. The method of any one of claims 3 to 11, wherein the calibration sample is incubated with the reducing agent at room temperature for up to 20 minutes.

13. 13. The method of claim 12, wherein the period is about 20 minutes.

14. The method of any of claims 1 to 13, wherein the biomolecule is a complex molecule comprising two or more subunits / species.

15. 15. The method of claim 14, wherein the biomolecule is selected from a recombinant protein, an antibody, and a fusion protein.

16. 1. A method for identifying the composition of a sample containing biomolecules, comprising the steps of: a) determining the molecular weights of components of a sample according to the method of any one of claims 1 to 15, b) identifying components of the sample based on the molecular weight of the sample; c) and determining the relative amounts of each component based on the CGE performed in step a; 1. A method for identifying components of a sample containing a biomolecule, comprising:

17. 1. A method for determining the purity of a sample containing a biomolecule, comprising the steps of: a) identifying the composition of the sample according to the method of claim 16; and b) determining the relative amounts of each subunit or fragment of the biomolecule present in the sample; A method for measuring the purity of a sample containing a biomolecule, comprising:

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