DESC:FIELD OF THE INVENTION
The invention relates to a method of deciphering the molecular heterogeneity of glycoproteins using mass spectrometry. In particular, the invention relates to a method of determining the intact mass of a glycosylated fusion protein in its native state using high-resolution mass spectrometry.
BACKGROUND OF THE INVENTION
Fc-fusion proteins are bioengineered polypeptides that join the crystallizable fragment (Fc) domain of an antibody with another biologically active protein domain to generate a molecule with unique structure–function properties and significant therapeutic potential. The gamma immunoglobulin (IgG) isotype is often used as the basis for generating Fc-fusion proteins because of favorable characteristics such as recruitment of effector function and increased plasma half-life. Given the range of proteins that can be used as fusion partners, Fc-fusion proteins have numerous biological and pharmaceutical applications, which has launched Fc-fusion proteins into the forefront of drug development.
Therapeutic Fc-fusion proteins are usually produced in high-yield expression systems using stably transfected cell lines such as Chinese Hamster Ovary (CHO) cells. The resulting therapeutic proteins are complex glycoproteins with complicated post-translational modifications. The proteins thus produced possess heterogeneity, which can arise from the manufacturing process or from the product itself.
Mass spectrometry (MS) is one of the most widely used techniques for the mass analysis of biopharmaceuticals such as monoclonal antibodies and Fc-fusion proteins. MS can be used to accurately determine - the mass of an intact protein, mass of the fragments of the proteins (for example light chain mass, heavy chain mass, etc.), and the mass of the peptides generated after digestion with an endoprotease such as trypsin. However, MS-based analysis of the molecular mass of an intact protein along with its glyco-variants, including monoclonal antibodies and Fc-fusion proteins, is challenging due to the complex structure of the associated glycan moieties. Glycan moieties are hydrophilic in nature which results in low ion yields in both positive and negative modes during the electrospray ionization (ESI) process. Most of the monoclonal antibodies of the IgG1 class have only one N-glycosylation site in their Fc region which translates to a low level of associated glycan moieties. Fc-fusion proteins such as, but not limited to, etanercept, abatacept, belatacept, aflibercept, luspatercept and rilonacept are heavily glycosylated as compared to monoclonal antibodies (mAbs) because such Fc-fusion proteins generally contain more than one glycosylation site (both N- and O-linked). Owing to this high level of heterogeneity, conventional analytical techniques such as matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) are used to support the molecular mass analysis for Fc-fusion proteins. The MALDI-MS technique enhances the ionization efficiency of the glyco-proteins. Although MALDI-MS is widely used for the characterization of intact glycosylated molecules, the obtained data is proven to be inadequate for the detailed molecular structural analysis. The further detailed characterization is achieved using various other approaches including chemical derivatization and enzyme dissection. Chemical derivatization involves modifying the structure of an analyte either to a form that is more easily introduced into the mass spectrometer, or into a chemical form that provides an enhanced response in terms of either improved selectivity or sensitivity. Enzyme dissection refers to the process of removal of glycan moieties associated with the protein backbone by use of enzymes such as proteases or glycosidases. The intact mass analyzed in denaturing conditions using ESI as an ionization technique results in higher charge states of the molecules thereby affecting its resolution and the ability to accurately determine the molecular mass of the glyco-variants of the protein.
There is a need for a quick and accurate method for the determination of intact mass of a protein along with its glyco-variants, without the involvement of chemical derivatization or enzyme dissection.
SUMMARY OF THE INVENTION
The present invention discloses a method for the accurate determination of molecular mass of an intact glycoprotein, preferably an Fc-fusion protein, along with its glyco-variants using high-resolution mass spectrometry. The method involves the determination of mass of the intact protein in its native state, which preserves the quaternary and tertiary structure of the protein and at the same time also leads to decreased spectral complexity and increased resolution. The method may be used to decipher the molecular heterogeneity associated with heavily glycosylated Fc-fusion proteins in a quick and efficient manner and may be quite useful in speeding up the therapeutic Fc-fusion protein development process.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 represents the mass spectrum obtained for +19, +20 and +21 charge states of the sample Fc-fusion protein as described in example 1.
Figure 2 is a representation of the deconvolution of the mass spectrum obtained in Figure 1 in a mass range of 74 kilo Daltons (kDa) to 122 kDa, showing that the maximum peak height is in the mass range of 90 kDa to 96 kDa.
Figure 3(a) shows the deconvolution of the mass spectrum as shown in Figure 2 in the mass range from 90 kDa to 96 kDa and Figure 3(b) is a representation of the peaks in Figure 3(a) in centroid form.
Figure 4 shows the plot of drift time (Y axis) vs m/z range (x axis) as obtained from ion mobility spectrometry of the protein sample as described in example 1. The three bubbles corresponding to a drift time of 16 - 20 ms and m/z range of 4000 – 5000 represent the charge states of a set of conformational species of the Fc-fusion protein used in example 1. Conformational isomers (for example, higher order conformational isomers; not shown), if present, would be visible in the similar m/z range, corresponding to a different drift time range. Hence, the method can also be used to identify the conformational isomers (for example, higher order conformational isomers) of the Fc-fusion protein, in addition to the determination of intact mass.
DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
The term “glycoprotein” as used herein refers to protein or polypeptide having at least one glycan moiety attached to it. Thus, any polypeptide attached to a saccharide moiety is termed as a glycoprotein.
"Fc-fusion protein" as used herein is a protein that contains the Fc region of an immunoglobulin fused or linked to a heterologous polypeptide. The heterologous polypeptide fused to the Fc region may be a polypeptide from a protein other than an immunoglobulin protein. For instance, the heterologous polypeptide may be a ligand polypeptide, a receptor polypeptide, a hormone, cytokine, growth factor, an enzyme, or other polypeptide that is not a component of an immunoglobulin. Such Fc fusion proteins may comprise an Fc region fused to a receptor or fragment thereof or a ligand from a receptor including, but not limited to, any one of the following receptors: both forms of TNFR (referred to as p55 and p75), Interleukin-1 receptors types I and II (as described in EP Patent No. 0460846, US Patent No. 4,968,607, and US Patent No. 5,767,064, which are incorporated by reference herein in their entirety), Interleukin-2 receptor, Interleukin-4 receptor (as described in EP Patent No. 0 367 566 and US Patent No. 5,856,296, which are incorporated by reference herein in their entirety), Interleukin-15 receptor, Interleukin-17 receptor, Interleukin-18 receptor, granulocyte- macrophage colony stimulating factor receptor, granulocyte colony stimulating factor receptor, receptors for oncostatin-M and leukemia inhibitory factor, receptor activator of NF-kappa B (RANK, as described in US Patent No. 6,271,349, which is incorporated by reference herein in its entirety), CTLA-4 receptor, VEGF receptors, EGF receptor, FGF receptors, receptors for TRAIL (including TRAIL receptors 1,2,3, and 4), and receptors that comprise death domains, such as Fas or Apoptosis-Inducing Receptor (AIR). Fc fusion proteins also include peptibodies, such as those described in WO 2000/24782, which is hereby incorporated by reference in its entirety.
“N-linked glycosylation” as used herein refers to the attachment of an oligosachharide to a nitrogen atom (the amide nitrogen) of an asparagine (N) residue of a protein. Oligosachharides added to the asparagine group are also called as N-glycans.
Oligosachharide refers to a sachharide polymer containing a small number of monosachharides. An oligosachharide generally contains three to ten monosachharide units.
“O-linked glycosylation” as used herein refers to the attachment of a sugar molecule to the oxygen atom of a serine (S) or threonine (T) residues in a protein. Sugars added to the serine or threonine groups are also called as O-glycans.
The term “glyco-variant” as used herein refers to an isoform of the protein which differs only with respect to the number of type of attached glycan moiety.
“Non-denaturing conditions”, as described herein refer to the buffer conditions which preserve the tertiary and quaternary structure of the glyco-protein. Examples of buffer solutions known in the art which preserve the native structure of the protein and are also compatible for use as mobile phase with mass spectrometry are ammonium acetate and ammonium formate.
Mass spectrometry is an analytical technique that is used to identify unknown compounds, quantify known materials, and elucidate the structural and physical properties of ions. Mass Spectrometry can be used in conjunction with chromatography techniques, such as LC-MS and GC-MS. Examples of mass spectrometry tools for use as detection agents include, but are not limited to, electron ionisation (EI), chemical ionisation (CI), fast atom bombardment (FAB)/liquid secondary ionisation (LSIMS), matrix assisted laser desorption ionisation (MALDI), and electrospray ionisation (ESI). See, for example, Gary Siuzdak, Mass Spectrometry for Biotechnology, Academic Press, San Diego, 1996.
DETAILED DESCRIPTION OF THE EMBODIMENTS
The present invention discloses a mass spectrometry-based method for the determination of intact mass of a glycoprotein along with its glyco-variants.
In an embodiment, the invention discloses the use of native mass spectrometry for the determination of intact mass of a CTLA4-Ig fusion protein along with its glyco-variants using electrospray ionization platform based mass spectrometry.
In an embodiment, the invention discloses a method for the determination of intact mass of a protein including the protein backbone and the glyco-variants in the native state, i.e., under non-denaturing conditions, using 75 mM ammonium acetate as sample diluent and mobile phase in electrospray ionization based mass spectrometry.
In another embodiment, the method is used to determine the mass of an intact Fc-fusion protein along with its glyco-variants in the native state using electrospray ionization mass spectrometry.
In an embodiment, the method is used to identify the conformational isoforms of the intact protein and its glyco-variants.
In any of the above embodiments, the protein is glycosylated on at least one asparagine (N) residue.
In any of the above embodiments, the protein has O-linked glycosylation on at least one serine (S) residue.
In any of the above embodiments, the mass of the intact protein along with its glyco-variants is determined accurately without the use of enzymatic dissection.
In any of the above embodiments, the level of glycosylation including N-linked glycosylation and O-linked glycosylation is not less than 10%.
In any of the above embodiments, the protein is diluted with ammonium acetate or ammonium formate before subjecting the protein to ESI-MS.
In any of the above embodiments, ESI-MS is coupled with liquid chromatography.
In any of the above embodiments, liquid chromatography coupled with ESI-MS is size exclusion chromatography.
In any of the above embodiments, the protein is an Fc-fusion protein.
In any of the above embodiments, the Fc-fusion protein may be selected from a group of CTLA4-Ig fusion proteins, TNFR:Fc fusion proteins, VEGFR:Fc fusion proteins, ActRIIb-Ig fusion protein and IL-1R1:Fc fusion proteins.
The method disclosed in any of the above embodiments may be used to quickly and accurately determine the molecular mass of an Fc-fusion protein glycosylated at multiple asparagine (N) residues and serine (S) residues without the need to release the associated glycan moieties by use of enzyme dissection.
Specific embodiments of the invention are more fully defined by reference to the following examples. These examples should not, however, be construed as limiting the scope of the invention.
EXAMPLES
Example 1: Intact mass analysis under native conditions
Sample Fc-fusion protein produced in-house was diluted to 1 mg/mL with 75 mM ammonium acetate and loaded onto an Acquity UPLC Protein BEH Column 200A, 1.7 µm. 75 mM ammonium acetate was used as the mobile phase for LC-MS. Details of the LC-MS parameters used are shown in Table 1.
Parameter Specification
LC Method used for separation SEC
Mobile Phase 75mM Ammonium Acetate
Sample Diluent 75mM Ammonium Acetate
pH of the mobile phase 6.63
Injection volume 10 µL
Flow Rate (mL/min) 0.2
Run Time (mins) 12
Column Specification Acquity UPLC Protein BEH Column 200A, 1.7 µm Part: 186005225; S.No:02913817317124
On column load (µg) 10
Column Temperature (°C) 25
Cone Voltage (V) 120
Capillary voltage (kV) 3
Source Offset 40
Source temperature °C 40 – 150
Desolvation temperature °C 350
Cone gas (L/h) 50
Desolvation gas (L/h) 800
Nebulizer gas (Bar) 6.5
Table 1: LC and MS parameters for intact mass analysis using Native mass spectrometry
Results
The acquired data was processed using UNIFI® software. The mass spectrum was obtained in the range of 4000 - 6000 m/z. The spectral complexity is reduced in this technique as compared to intact mass analysis under denaturing conditions and the obtained spectra was hence successfully deconvoluted by the software. The mass of the protein along with one glyco-variant species is listed in Table 2.
Sample Theoretical Mass (Da) Observed Mass (Da)
FC1 (Protein + 1 hexose + 1 acetyl hexosamine) 92665 92654
Table 2: Theoretical and Observed mass for sample Fc-fusion protein

,CLAIMS:CLAIMS
We claim:
1. A method for determining the mass of an intact Fc-fusion protein in a sample comprising the Fc-fusion protein and one or more glycovariants thereof, the method comprising steps of:
(a) providing a sample comprising the Fc-fusion protein and one or more glycovariants thereof,
(b) diluting the sample provided in step (a) to 1 mg/mL with 75 mM ammonium acetate,
(c) passing the sample diluted in step (b) through a chromatography column comprising ethylene bridged hybrid particles using 75 mM ammonium acetate as mobile phase to obtain an eluate,
(d) introducing the eluate obtained in step (c) to an electrospray ion based mass spectrometer and determining the intact mass of the Fc-fusion protein,
wherein the method is devoid of enzymatic dissection, and
wherein the parameters used for the determination of the intact mass of the Fc-fusion protein are listed in Table (a);
Parameter Value
LC Method used for separation SEC
Column Temperature (°C) 20 – 30
Cone Voltage (V) 100 – 140
Capillary voltage (kV) 2.5 – 3.5
Source Offset 30 – 50
Source temperature °C 40 – 150
Desolvation temperature °C 300 – 400
Cone gas (L/h) 25 – 75
Desolvation gas (L/h) 700 – 900
Nebulizer gas (Bar) 5.5 – 7.5
Table (a)
2. A method for determining the mass of an intact CTLA4-Ig fusion protein in a sample comprising the CTLA4-Ig fusion protein and one or more glycovariants thereof, the method comprising steps of:
(a) providing a sample comprising the CTLA4-Ig fusion protein and one or more glycovariants thereof,
(b) diluting the sample provided in step (a) to 1 mg/mL with 75 mM ammonium acetate,
(c) passing the sample diluted in step (b) through a chromatography column comprising ethylene bridged hybrid particles using 75 mM ammonium acetate as mobile phase to obtain an eluate,
(d) introducing the eluate obtained in step (c) to an electrospray ion based mass spectrometer and determining the intact mass of the CTLA4-Ig fusion protein,
wherein the method is devoid of enzymatic dissection, and
wherein the parameters used for the determination of the intact mass of the CTLA4-Ig fusion protein are listed in Table (a).
2. The method as claimed in claim 1, wherein the method is used to determine the mass of the intact CTLA4-Ig fusion protein in its native state, i.e., under non-denaturing conditions.
3. The method as claimed in claim 1 or claim 2, wherein the Fc-fusion protein is glycosylated on at least one asparagine (N) residue.
4. The method as claimed in claim 1 or claim 2, wherein the Fc-fusion protein has O-linked glycosylation on at least one serine (S) residue.
5. The method as claimed in claim 1 or claim 2, wherein the level of glycosylation of the Fc-fusion protein, including N-linked glycosylation and O-linked glycosylation, is not less than 10%.
6. The method as claimed in claim 1, wherein the the Fc-fusion protein may be selected from a group comprising of CTLA4-Ig fusion proteins, TNFR:Fc fusion proteins, VEGFR:Fc fusion proteins, ActRIIb-Ig fusion protein and IL-1R1:Fc fusion proteins.
7. The method as claimed in claim 1 or claim 2, wherein the method is used to quickly and accurately determine the molecular mass of the intact Fc-fusion protein glycosylated at multiple asparagine (N) residues and serine (S) residues without the need to release the associated glycan moieties by use of enzyme dissection.