DESC:Field of invention
The present invention relates to an LCMS (Liquid Chromatography – Mass Spectrometry) based method for determination of polymer conjugation sites and characterization of different species formed after treatment of said polymer conjugated protein with a protease enzyme and a chemical agent.
Background of the invention
Therapeutic proteins at times are covalently conjugated with non-protein polymers in order to improve their solubility, stability, serum half-life and bio-distribution by altering their physicochemical and biochemical properties. However, the site of conjugation plays an important role in determining the efficacy of the therapeutic protein. Conjugation at undesirable sites can hinder the interaction of a protein with its receptor or impair the catalytic site of an enzyme making the therapeutic preparation ineffective.
Granulocyte colony stimulating factor (G-CSF) is a cytokine hormone released by body cells and involved in the proliferation of neutrophils. Therapeutic GCSF is a recombinant human protein methionylated at N-terminus and finds application in treatment of neutropenia associated with cancer chemotherapy and other related disorders. Further, polymer conjugation of G-CSF with polyethylene glycol at N-terminus to form PEG-GCSF improves its serum half-life significantly and enhances its stability. However, presence of multiple pegylation sites on G-CSF leads to structural heterogeneity as several mono and multi pegylated isoforms of PEG-GCSF with compromised therapeutic efficacies are generated. While it is relatively easier to distinguish between mono and multi pegylated isoforms, it is difficult to distinguish between different mono pegylated isoforms that are pegylated at different amino acids. Assessment of these isoforms requires a robust analytical method that distinctly separates each isoform and identifies the key sites that have been pegylated. Therefore there is a need in the art to provide a method to determine the pegylation site in a polymer conjugated protein studies.
The main objective of the present invention is to identify the site of conjugation on a polymer conjugated protein. The invention provides a robust and effective LCMS (Liquid Chromatography-Mass Spectrometry) based method for determination of site of Pegylation more particularly in PEG-GCSF.
Summary of invention
The present invention discloses an LCMS based method to identity the conjugation site on a polymer conjugated protein. The present invention further characterizes and quantifies different species formed after treating said polymer conjugated protein with an enzyme followed by treatment with a pseudohalogen chemical compound.
Brief description of the drawings:
Figure 1a: Depicts trypsin and CNBr cleavage sites on GCSF and PEG-GCSF
Figure 1b: Depicts GluC and CNBr cleavage sites on GCSF and PEG-GCSF
Figure 2: Depicts the flow chart for processing and treatment of GCSF and PEG-GCSF samples
Figure 3: Depicts UV chromatographic overlay of reduced, IAA alkylated and trypsin digested GCSF and PEG-GCSF
Figure 4: Depicts UV chromatographic overlay of GCSF and PEGGCSF after reduction and IAA alkylation followed by Trypsin and CNBr digestion
Figure 5: Depicts Extracted ion chromatogram of a) GCSF P2 b) GCSF P5 C) PEG-GCSF P2 d) PEG-GCSF P5

Description of the invention
The present invention discloses an LCMS based method to identify the site of conjugation on a polymer conjugated protein, wherein the method involves
i. treating said polymer conjugated protein with a protease enzyme that cleave either at C-terminal of lysine (K) and Arginine (R) (using Trypsin) or glutamic acid (E) and Aspartic acid (D) (using Glu-C).
ii. treating the fragments obtained in step (i) with a chemical compound that cleaves C-terminal to methionine;
iii. separating the fragments obtained after step (i) and (ii) by a chromatographic method; and
iv. determining the mass and intensities of the separated fragments using mass spectrometry.
The polymer conjugated protein as disclosed in the present invention is any protein or therapeutic drug, conjugated with a polymer to improve the therapeutic property of the drug. The said polymer is a PEG (Polyethylene Glycol) and said polymer conjugated protein of the present invention is PEG-GCSF.
Therapeutic GCSF (r-metHuG-CSF) is a 175 amino acid methionylated human recombinant protein obtained from E. coli. Pegylation of G-CSF (PEG-GCSF) involves covalently attaching hydrophilic polyethylene glycol polymer to the N-terminal methionine residues of G-CSF. The process of pegylation of GCSF is commonly known in the art that ensure abundant pegylation at the N-terminal methionine of GCSF. However, presence of several lysine residues with free amino group in the side chain increases the possibility of pegylation at multiple sites generating different isoforms of PEG-GCSF with varied therapeutic efficacy.
The said polymer conjugated protein is treated with protease enzymes, such as trypsin, Glu-C or the like. Trypsin cleaves peptide bond at C-terminus of a lysine (K) and arginine (R) residue in the PEG-GCSF, provided the side chain epsilon amino group is free and not covalently linked to any chemical group such as a polymer. Any covalent modification of lysine at the side chain amino group strictly resists the hydrolysis of the C-terminal peptide bond by trypsin. Similarly, Glu-C cleaves the peptide bond at C-terminus of glutamic acid (E) or aspartic acid (D).
The PEG-GCSF conjugate after treatment with trypsin, is treated with a pseudohalogen compound such as cyanogen bromide (CNBr). CNBr cleaves the peptide bond at C-terminus to methionine residue in any protein particularly when methionine has a free, unoxidized, -SH group and when it is not followed by the amino acids serine and threonine.
Participation of the SH group into disulfide bond or oxidation of methionine (Mo) renders PEG-GCSF uncleavable. Furthermore, when methionine is followed by serine or threonine it undergoes homoserine (HS) formation in presence of CNBr and the cleavage is further prevented.
In one embodiment, the present invention discloses an LCMS based method to identify the site of conjugation on a polymer conjugated protein, wherein the method involves
i. treating separately said polymer conjugated protein and non-conjugated protein with an enzyme that cleaves C-terminal to lysine (K) or glutamic acid (E);
ii. treating independently the fragment obtained in step i. for polymer conjugated protein and non-conjugated protein with a chemical compound that cleaves C-terminal to methionine;
iii. separating independently the fragments obtained in step i and ii from polymer conjugated protein and non-conjugated protein by a chromatographic method; and
iv. determining the mass and intensities of the separated fragments for polymer conjugated protein and non-conjugated protein using mass spectrometric system.
In one another embodiment, the present invention discloses an LCMS based method to identify the site of conjugation on a polymer conjugated protein, wherein the method involves identification of different peptide fragments formed and characterization of various species such as Homoserinised (HS) and Oxidized methionine (Mo) or the like.
G-CSF molecule comprises nine trypsin and thirteen GluC cleavage site and four CNBr cleavage sites as provided in Figure 1a & Figure 1b respectively. Ten different fragments would be obtained while GCSF sample with reduced disulfide bonds is subjected to trypsin; and fourteen different fragments with GluC treatment; and five fragments upon CNBr treatment. Theoretically double digestion of G-CSF with trypsin followed by CNBr would yield fourteen peptide fragments of different chain lengths; whereas double digestion with GluC followed by CNBr would cleave at seventeen sites yielding eighteen peptide fragments of different chain length and molecular mass. The key fragments of GCSF with amino acid sequence and theoretical masses formed upon trypsin digestion include P1, P2, P3, P4 and P5 as referred in Table 1a.
The Peptide P1 with 17 amino acid comprises two possible sites for pegylation, namely 1st amino acid (Methionine residue) and 17th amino acid (Lysine Residue). Pegylation of GCSF at N-terminal methionine or the intermittent lysine residues alter the fragmentation of the protein and the mass profile of the individual fragments based on the site and extent of pegylation.

Table 1a: Relevant peptides with theoretical mass obtained from tryptic digest of GCSF
Peptide No. Amino acid Range Theoretical mass
(M+H)+ Sequence
P1 1- 17 1786.98 MTPLGPASSLPQSFLLK
P2 18- 23 804.38 CLEQVR
P3 24- 24 147.106 K
P4 25- 35 1129.578 IQGDGAALQEK
P5 36-41 755.37 LCATYK

In case of pegylation at the 1st amino acid Methionine residue, the PEG GCSF when treated with trypsin is cleaved at the indicated sites and Peg-P1, P2, P3, P4 and P5 is formed prominently. In subsequent cleavage with CNBr, the N terminal Methionine is separated and PEG-M fragment and the remaining P1a (2-17) (as referred in Table 1b) amino acid fragment are obtained. In case of Methionine Oxidation (Mo) or Homoserine Formation (HS), the methionine is not cleaved by CNBr and the entire P1 peptide is eluted out in the pegylated form at the end because of enormous (~18-20 kDa) size of PEG moiety. Pegylation at K17 would prevent the trypsin cleavage at this site and hinder the formation of P1 and P2. Similarly pegylation of the GCSF at other lysine residues (K24, K35 and K41) would impact the fragmentation and alter the formation of peptides P3, P4 and P5.
In one embodiment, during LCMS and MS/MS analysis the presence of peptide with mass corresponding to the P1a, P2, P3, P4 and P5 in the digestion confirms the attachment of PEG moiety to N-terminal methionine residue. In one another embodiment the absence of mass corresponding to P2 peptide indicates pegylation at K17.

Table 1b: Sequence and theoretical mass of N terminal peptide from Trypsin and CNBr digested GCSF
Peptide No. Amino acid Range Theoretical
Mono (M+H)+ Sequence
P1a 2- 17 1785.98 TPLGPASSLPQSFLLK

In case of pegylation at the 17 amino acid Lysine residue, trypsin does not cleave at the C-terminal of Lysine, hence no fragmentation occurs with the P1 peptide. However, the next trypsin cleavage site at K23 is cleaved. In subsequent cleavage with CNBr, the N terminal Methionine is separated and a new peptide P1a+P2 (2-23 amino acids) is obtained. This P1a+P2 peptide possess the PEG moiety at Lysine 17.
In another embodiment, during LCMS and MS/MS analysis the absence of peptide with mass corresponding to the P1a in the respective digestion confirms the attachment site of the PEG Moiety to lysine (K17) residue.
In yet another embodiment of the invention, the retention time and relative percentage intensity of different fragments obtained is analyzed and a quantitative comparison of P2 with P4 or P2 with P5 is performed. The said quantitative comparison determines the percentage occupancy at the site of pegylation as the pegylated fragment poses difficulty in detection in both on UV chromatogram and in mass analysis. The peptides P4 and P5 are robustly formed in both GCSF and PEG-GCSF and hence they act as an internal markers for quantitation. Complete formation of P2 peptide for all PEG-GCSF molecules indicate no pegylation at Lysine (K17) residue. Similarly, for the same amount of sample, the complete formation of P4 and P5 for all PEG-GCSF molecules indicates no pegylation at Lysine K24, K35 and K41 residues.
In yet another embodiment, the site of pegylation is determined by comparing the ratios of intensities of P2:P4 (or) P2:P5 of PEG-GCSF to GCSF. A decreased P2:P4 or P2:P5 ratio indicates increased pegylation at 17 amino acid lysine.
In one embodiment the present invention discloses a novel method to identify the site of pegylation by determining the ratios of intensities of the non-pegylated fragments (P2 and P4) generated upon trypsin and CNBr treatment.
In one embodiment, the present invention provides an improved method to determine the efficiency of pegylation process, more particularly the pegylation of PEG moiety to GCSF.
In one another embodiment, double digestion of GCSF with the enzyme GluC and CNBr leads to formation of key fragments as shown in the table 2. Pegylation at N-terminal methionine lead to formation of Pc1a and is detected on a UV chromatogram and in the mass spectra. The uncleaved, oxidized and homoserinized forms of N-terminal methionine species would also be visible at the end of the UV chromatogram.
In case of pegylation at lysine (K17) Pc1a is attached with the enormous peg moiety and is visible at the end of the UV chromatogram. Mass corresponding to Pc1a peptide (Table 2) is absent.
Table 2: Relevant peptides with theoretical mass and sequence obtained after GluC and CNBr digestion
Peptide No. Enzyme digest Amino acid Range Theoretical (M+H)+ Da. Sequence
Pc1 Glu C 1-20 2189.1 MTPLG PASSL PQSFL LKCLE
Pc1a Glu C and CNBr 2-20 2058.07 TPLG PASSL PQSFL LKCLE

Various aspect of the disclosed invention provides an LCMS based biophysical method to identify the site of conjugation on a polymer conjugated protein, wherein the method involves step
i. treating the polymer conjugated protein with an enzyme that cleaves C-treminal to amino acid lysine (K) or glutamic acid (E).
ii. treating the fragment obtained in step i. with a chemical compound that cleaves C-terminal to methionine.
iii. separating the fragments obtained in step (i) and (ii) by a chromatographic method and
iv. characterizing the separated fragments with respect to their retention time, mass of fragments and its relative intensity.

Example 1:
The site of polymer conjugation, here pegylation, on PEG-GCSF was determined using the above invention and GCSF as a control. Both the proteins were subjected to Trypsin and CNBr treatment after reduction and alkylation and the peptide fragments so obtained were subjected to chromatographic separation on an HPLC and their masses were profiled using mass spectrometric system. The elution pattern, retention time, molecular mass and the intensity of various fragments – P1, P1a, P1Hse, P2, P3, P4 and P5 were determined. The site of pegylation at different sites of PEG-GCSF was determined based on the appearance and intensity of each fragments and comparing the fragmentation profile with that of GCSF.
Digestion of GCSF protein with trypsin and cyanogen bromide
The GCSF and PEG-GCSF sample preparation and treatment was done as per the flow chart given in Figure 2. To get complete digestion of PEG-GCSF with trypsin, the pH of the sample was adjusted to 7.5 with tris buffer (pH-8), followed by reduction and alkylation. The sample was then trypsin digested in 1:50 (enzyme to protein ratio). The digestion was arrested by bringing down the pH to 1.5 using 1N HCl, followed by CNBr digestion for 12 hrs.

HPLC for Separation of the Peptide Fragments
The parameters used for the liquid chromatographic steps of the peptide fragments obtained after the second digestion with CNBR are as below.
Property Parameter
Mobile phase A 0.1% TFA in Milli-Q water
Mobile phase B 0.1% TFA in 99.9% acetonitrile
Column Waters, Symmetry C18 RP, 250×4.6mm, 300Å, 5µm
Column Temp 55°C
Flow Rate 0.2ml/min
Injection volume 100µl
Stop time 120.00 min
Detection Wavelength 214 nm and 280 nm

Gradient program:
Time (min) % Solvent B Flow (ml/min)
0.0 1.8 0.2
2 1.8 0.2
30 27 0.2
75 54 0.2
90 88.2 0.2
100 88.2 0.2
101 1.8 0.2
120 1.8 0.2

Mass Spectrometry of Peptide Fragments obtained after HPLC:
The mass spectrometric parameters of the peptide fragments after separation in the chromatographic are as below.

Property Parameter
Instrument Agilent’s HPLC connected with Agilent’s MSD/SL ion trap
Scan mode Standard-Normal
Tune parameters Nebulizer-50, Dry gas-10, Dry temp-355°C
Polarity Positive
Trap ICC-target-30000
Max Acc. Time -100ms,
Scan 100-2200m/z
Averages 5

Results:
The UV chromatogram and mass spectrometric data of GCSF and PEG-GCSF obtained after trypsin and CNBr digestion is as per the tables given below and the figures in the accompanying drawings.
The masses and the relative intensities of the peptide fragments and various species obtained after the trypsin and CNBr digestion and subsequent LCMS of GCSF and PEG-GCSF are given in the table below. Similar intensity ratio for PEG-GCSF and GCSF confirms absence of pegylation at any other sites than N-terminal methionine.
,CLAIMS:1. An LC-MS based method to identify the site of conjugation on a polymer conjugated protein, wherein the method involves:
i) treating said polymer conjugated protein with a protease enzyme that cleave either at C-terminal of lysine (K) and Arginine (R) or glutamic acid (E) and Aspartic acid (D).
ii) treating the fragments obtained in step (i) with a chemical compound that cleaves C-terminal to methionine;
iii) separating the fragments obtained after step (i) and (ii) by a chromatographic method; and
iv) determining the mass and intensities of the separated fragments using mass spectrometry.

2. A method according to claim 1, where the polymer is a Polyethylene glycol (PEG) molecule.
3. A method according to claim 1, where the protein is GCSF.
4. A method according to claim 1, where the protease is trypsin or Glu-C
5. A method according to claim 1, where the chemical compound is CNBr.
6. An LC-MS based method to identify the site of conjugation on PEG-GCSF, wherein the method involves:
i) treating PEG-GCSF with a protease enzyme trypsin.
ii) treating G-CSF with a protease enzyme trypsin.
iii) treating the fragments obtained in step (i) and (ii) with a chemical compound CNBr;
iii) separating the fragments obtained after step (i) and (ii) by a chromatographic method; and
iv) determining the mass and intensities of the separated fragments using mass spectrometry.
v) identifying the site of pegylation by comparing the mass intensities of peptide fragment obtained from G-CSF and PEG-GCSF
7. The method according to claim 1 and 6 wherein the fragments obtained comprises peptides P1, P1a, P2, P3 P4 and P5

8. The method according to claim 1 and 6 wherein the site of pegylation is identified by quantifying each peptide and comparing the intensities of P2, P4 and P5.

9. The method according to claim 1 and 6 wherein pegylation at N-terminal methionine is detected by presence of peptide fragments P1a, P2, P3, P4 and P5.
10. The method according to claim 1 and 6 wherein pegylation at the 17th amino acid is detected by absence of peptide fragment P2.