DESC:FIELD OF THE INVENTION
The present invention relates to an analytical method for identification and characterization of a protein sequence variant in a biotherapeutic preparation. Particularly, the invention discloses detection and characterization of a relatively less abundant protein sequence variant by liquid chromatography-mass spectrometry (LC-MS).
BACKGROUND OF THE INVENTION
Protein-based drugs are often produced from a single cell clone, and such production is technically challenging. Protein expression in cells is governed through biological processes such as DNA replication, RNA transcription and protein translation, each of which has a finite fidelity, with error rates ranging from 10-9 per base pair for DNA replication to 10-4 to 10-5 per codon for protein translation (O. Borisov et al., Sequence Variants and Sequence Variant Analysis in Biotherapeutic Proteins, ACS Symposium Series, Vol. 1201). This can result in molecular heterogeneity of the resultant product in a solution. The underlying cause of such heterogeneity can be correlated to cell culture conditions such as amino acid starvation, accelerated rate of protein expression, inefficient codon optimization and single nucleotide polymorphisms in triplet codons.
Any resultant unintentional amino acid substitution, omission, or insertion in the protein sequence can result in the often less-abundant ‘sequence variants’ (SVs) in a population of otherwise intended/desired biotherapeutic protein molecule. Some of these modifications could hinder product activity and/or quality of the final drug product. For this reason, establishing a sequence variant profile of a biotherapeutic is essential to ensure sequence fidelity, manufacturing consistency and stability. Identification and characterization of such variants is also of utmost importance for regulatory submissions. Therefore, these investigations take priority in the early stages of product and process development.
Major challenges faced in the characterization of sequence variants are: availability of limited amount of sample during cell line development, lower abundance of variant forms and inefficient in silico and software tools, which often generate misleading false positives. Integrated and effective analytical methods help in characterizing these sequence variants.
There is hence a need to develop an improved method to detect and characterize the sequence variants even in the presence of limited sample availability and relative lower abundance of variant forms. Present invention discloses a sensitive method for rapid characterization and detection of a low abundant sequence variant in an anti-RANKL antibody preparation with high specificity. Particularly, the method helps in characterizing a tyrosine to asparagine transversion mutation identified in the CDR region of heavy chain of the antibody.
SUMMARY OF THE INVENTION
Accordingly, it is an objective of the present invention to provide an analytical method to identify and characterize a novel sequence variant present in an anti-RANKL antibody preparation. The novel sequence variant is identifiable as an additional distinct peak with a lower molecular weight of approximately -49 Da when in comparison with the corresponding expected mass spectra (Figure 1). Preparative chromatography cannot be employed to separate the said peak primarily due to reduced abundance. Present invention provides a novel method with a characterization workflow combining partial proteolytic digestion, peptide mapping and LC-MS for precise, error-free detection and characterization to specifically characterize a tyrosine to asparagine transversion mutation in the heavy chain of the anti RANKL antibody. Thus, claimed method utilizes characterization techniques to detect an unanticipated variant and monitor the same for better clone selection and superior product development.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1. Deconvoluted mass spectra of intact protein of DRL mAb from 3 clones. The arrows indicate an additional peak with about 49 Da lower mass in Clone 2 and Clone 3 when compared to the expected mass spectra of Clone 1.
Figure 2. Deconvoluted mass spectra of subunit proteins (light chain vs. heavy chain of immunoglobulin) of DRL mAb from 3 clones. The arrows indicate an additional peak with about 49 Da lower mass only on the heavy chain in Clone 2 and Clone 3, when compared to the expected mass spectra of Clone 1. The additional peak is not detected in the mass spectra of light chain, indicating transversion mutation in the heavy chain.
Figure 3. Peptide mapping base peak ion (BPI) chromatogram comparing reference medicinal product (RMP) with that of clone 2 and clone 3. Peptide mapping identified the site of substitution mutation as, tyrosine Y59 to asparagine N59.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person having ordinary skill in the art to which the invention pertains.
The term "biotherapeutic" herein is used in the broadest sense and it covers proteins that are genetically engineered through recombinant DNA technology, which are of therapeutic significance in the treatment of ailments. Biotherapeutics include monoclonal antibodies, fusion proteins, polyclonal antibodies, multispecific antibodies and antibody fragments so long as they exhibit the desired biological activity.
The term “biotherapeutic sample” or “biotherapeutic preparation” used interchangeably, refers to a population of biotherapeutic molecules or fragments thereof that is produced by mammalian cell culture. The population of biotherapeutic molecules may have sequence variants, one or several post translational modifications (PTM), imparting the antibody molecules a different molecular weight, charge, solubility or combinations thereof.
The term “sequence variant” or “SV” refers to unintentional amino acid substitution, omission, transversion or insertion in the protein sequence, generated during protein biosynthesis, leading to heterogeneity in a population of the otherwise intended biotherapeutic protein molecule.
The term “variant peak” refers to the peak on a chromatogram or mass spectra corresponding to the unintended sequence variant form of the polypeptide.
The term “reference medicinal product” or “RMP” refers to a currently or previously marketed recombinant protein, also described as the "originator product" or "branded product" serving as a comparator in the studies.
The term “RANKL” refers to Receptor activator of nuclear factor kappa-? ligand.
The term “peptide mapping” refers to an analytical method of identifying confirmation of a protein therapeutic and to monitor any degradative change in protein structure such as oxidation or deamidation.
Identification of sequence variants of the therapeutic protein forms a part of critical quality attribute testing as presence of these variants can impact protein activity and pose a risk of immunogenicity. Major challenges faced in the characterization of such sequence variants are: availability of limited amount of sample during cell line development, lower abundance of variant forms in the presence of the more abundant intended form of biotherapeutic and inefficient in silico and software tools which often generate misleading false positives.
To overcome the aforementioned challenges, present invention discloses a sensitive method for rapid characterization and detection of a sequence variant in an anti-RANKL antibody preparation with high specificity. The sequence variant of present disclosure is formed due to a tyrosine?aspargine transversion mutation (Y59N) in the CDR region of the heavy chain of the antibody at position. Various embodiments of the disclosed invention are carried out to detect and characterize the variant species. The sequence variant is identifiable as an additional distinct peak with a lower molecular weight of approximately -49 Da when in comparison with the corresponding expected mass in the mass spectra.
Carboxypeptidase B (CP-B) based CEX is performed to confirm peak purity and presence of basic charge variant. Following this, ultra-performance liquid chromatography-mass spectrometry (LC-MS) analysis is performed. Characterization of mutation is performed by peptide mapping which shows transversion mutation at Y59 (Y59N) of the heavy chain. Characterization is confirmed by peptide mapping of tryptic digests.
Claimed invention discloses a sensitive and specific method to identify and characterize amino acid substitution of tyrosine to aspargine (Y59N) in an anti-RANKL antibody using a combination of intact, subunit and middle-up analysis, combined by peptide mapping.
In an embodiment the claimed invention describes a method for detecting a sequence variant in a protein sample comprising:
a) optionally performing sample preparation;
b) loading the sample onto a gel filtration column;
c) eluting the sample from the gel filtration column;
d) digesting the fraction with trypsin to generate peptide fractions;
e) separating the peptide fractions in the sample by loading the digested fraction of step d) onto a liquid chromatography column;
f) separately loading a preparation containing synthetic peptide bearing the known sequence variation onto a second liquid chromatography column;
g) detecting the presence of sequence variant in the samples of step e) and step f) by mass spectrometry (LC – MS); and
h) detecting and quantitating the sequence variant by comparing the mass spectra of step g).
In an embodiment, the protein sample comprises intact protein, subunit protein or middle up protein.
In another embodiment, the liquid chromatography column has a pore size of about 300 Å;
In yet another embodiment, the sample preparation comprises one or more of denaturation, reduction, alkylation, Carboxypeptidase-B treatment, IdeS treatment, PNGase treatment and N-glycanase treatment.
In a further embodiment, the sequence variant has an amino acid transversion of tyrosine to asparagine.
In yet another embodiment, the relative abundance of the variant is as low as about 1.4%.
Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of this invention.
Abbreviations
BPI: base peak ion
CDR: Complementarity determining region
CEX: Cation exchange chromatography
CP-B: Carboxypeptidase-B
DNA: Deoxyribonucleic acid
DTT: Dithiothreitol
IAM: Iodoacetamide
LC-MS: liquid chromatography-mass spectrometry
PNGase F: Peptide:N-glycosidase F
PTM: post translational modifications
RANKL: Receptor activator of nuclear factor kappa-? ligand
RMP: reference medicinal product
RNA: Ribonucleic acid
SV: sequence variants
EXAMPLES
The invention will now be described in greater detail by reference to the following examples which further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
Example 1: Sample preparation for mass analysis of intact, subunit and middle-up protein
For intact protein mass analysis, one of the following was used: untreated sample, CP-B treated, deglycosylated, CP-B treated and deglycosylated. For untreated sample, equivalent volume of biotherapeutic sample was dilute with water to get a final concentration of 1 mg/mL. For CP-B treated sample, untreated sample (1 mg/mL) was mixed with CP-B (1 mg/mL) in protein: enzyme ratio 17.5:1 and incubated at 37 °C for 2 h. For deglycosylation, untreated sample (1 mg/mL) was mixed with 20 µL of 5X Tris buffer, 2 µl of N-glycanase enzyme in the protein:enzyme ratio of 100 µg : 2 µL, mixed and incubated at 37 °C for at least 17h. For obtaining CP-B treated and deglycosylated sample, the deglycosylated sample was further treated with CP-B as described above.
For subunit protein mass analysis, one of the following was used: reduced sample, CP-B treated, deglycosylated, CP-B treated and deglycosylated. For obtaining reduced sample, equivalent volume of biotherapeutic sample diluted with water (final concentration of 1 mg/mL) was reduced by addition of 1 µL of 0.5 M DTT. Sample was mixed gently and incubated at 37 °C for 30 minutes. For CP-B treatment followed by reduction, 100 µg of sample was mixed with CP-B (1 mg/ml), volume made up to ~1 mg\mL and incubated at 37 °C for 2 h, followed by reduction with 0.5 M DTT as described above. For deglycosylation followed by reduction, 100 µg of sample mixed with 5X reaction buffer, 2 µL of PNGase F was added and volume made up with water to get final concentration of ~1 mg\mL, mixed and incubated at 37 °C for 17 h, followed by reduction with 0.5 M DTT as described above. For deglycosylation and CP-B treatment followed by reduction 100 µg of sample mixed with 5X reaction buffer and 2 µL of PNGase F was incubated at 37 °C for 17 h. 5 mg/mL CP-B was mixed with 100 µg of the deglycosylated sample and volume made up to get final concentration of ~1 mg\mL. This was followed by reduction with 0.5 M DTT as described above.
For middle-up protein mass analysis, one of the following was used: non-reduced sample and reduced sample. For middle-up non-reduced sample, equivalent volume of sample was diluted with water to get a final concentration of 1 mg/mL. Sample was mixed with Ides/Fabricator using 1:1 protein to enzyme ratio and incubated at 37°C for 1 h. For middle-up reduced sample, equivalent volume of sample diluted with water (final concentration of 1 mg/mL) was mixed with IdeS/Fabricator using 1:1 protein to enzyme ratio and incubated at 37°C for 1 h. The same sample may be reduced to break the inter chain disulfide linkages using DTT (final concentration of 10 mM) and incubating for 30 minutes at 37 °C.
Example 2: Sample preparation for peptide mapping analysis
For sample denaturation, 1 mg of sample was mixed with denaturation (8.2 M GdnHCl, 1 mM EDTA and 0.1 M Tris, pH =7.5±0.2)buffer to give a final protein concentration of 1 mg/mL. After mixing, protein was incubated at room temperature for a few minutes. For reduction, 10 µL of 0.5 M DTT was added to the above solution (final concentration ~5 mM-10mM), mixed gently and incubated at 37 °C for 30 minutes. After reduction, the solution was incubated at room temperature for a few minutes. For alkylation, (to prevent the reduced free sulfhydryl groups from again forming disulfide bond), 20 µL of 0.5 M iodoacetamide (IAM) was added to the above solution (final concentration ~ 10 mM to 20mM) and incubated at room temperature for 40 minutes in dark.
Example 2: Gel filtration and elution and trypsin digestion
Gel filtration was performed using PD-10 desalting column (GE healthcare, Catalog No.: 17085101) conditioning: After alkylation, buffer exchange the sample with digestion buffer (1 M Urea, 1 mM EDTA, 20 mM Hydroxyl ammonium chloride and 0.1 M Tris, pH 7.5±0.2 ) in order to remove guanidine hydrochloride, DTT and IAM. For cleaning and equilibrating the PD-10 column, the closure from the top was removed to allow the storage solution to drain out. After complete drainage, column was washed and equilibrated with digestion buffer. The top was closed with column closure lid until the sample is ready. Reduced and alkylated sample is desalted and buffer exchanged in respective digestion buffer by using equilibrated PD-10 column as follows:
Reduced and alkylated sample (1 mL) was loaded onto equilibrated PD-10 column. Protein was eluted by adding 10 mL of digestion buffer. 1 mL of each fraction was collected in separate eppendorf tubes. Protein content in each fraction was estimated by Bradford method. Fraction with intense blue color was selected for digestion (concentration of ~ 0.8-0.9 mg/mL).Samples were then digested with trypsin by mixing protein sample with trypsin in protein:enzyme ratio of 50:1 and incubated at 37 °C for 17 hours
Example 3: Sample preparation of synthetic peptide
The synthetic peptides in lyophilized form was reconstituted with water (1000ng/mL final stock concentration after reconstitution). Further the stock solution was diluted with trypsin digestion buffer to 25ng/µL (Working concentration). The diluted synthetic peptide was loaded onto the column (~500ng) and further analyzed.
Example 4: Liquid chromatography for peptide mass analysis
An ultra performance liquid chromatography (UPLC) method using BEH C18 column was utilized for peptide mapping analysis. 20 µL was loaded onto Waters ACQUITY UPLC, Peptide BEH C18 Column (1.7 µm particle size, 300 Å pore size, 600C column temperature). Flow rate in the column was adjusted as per gradient (Table 1). Sample was detected at 214 nm and 280 nm.
Table1: Gradient used in peptide mapping liquid chromatography run
Time (min) Flow rate (mL/min.) % Solvent A
(100% water) % Solvent B
(100% Acetonitrile) % Solvent C or D
(1.0% TFA in water)
0 0.3 87 3 10
1 0.3 87 3 10
16 0.2 78 12 10
32 0.3 70 20 10
61 0.3 50 40 10
64 0.3 10 80 10
68 0.3 10 80 10
68.2 0.3 87 3 10
75 0.3 87 3 10
Example 5: Mass spectrometry analysis of intact, subunit and peptides for detection of transversion mutant
Mass spectrometric analysis was performed on a Synapt G2-Si mass spectrometer. The corresponding parameters for analysis of intact protein, subunit protein, middle up protein and peptide analysis are given in Table 2.
Table 2: Corresponding parameters for mass spectrometry analysis
MS method parameters Intact protein Subunit protein Middle up protein Peptide analysis
Range Range Range Range
Capillary voltage (kV) 2.0-3.0 2.0-3.0 2.0-3.0 0.0-4.0
Cone voltage (V) 80-150 40-80 40-80 0-150
Desolvation gas flow (L/h) 200-1000 200-1000 200-1000 100-1200
Desolvation temperature (°C) 100-600 100-600 100-600 100-600
Mass range 500-4500 500-3995 500-3995 50-5000
Resolution = 8000 = 8000 = 8000 Minimum 15000
Source offset (V) 0-100 0-100 0-100 0-100
Source temperature (°C) 80-200 80-200 80-200 80-150

The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments and examples are therefore to be considered in all respects illustrative rather than limiting the invention described herein.
,CLAIMS:CLAIMS
We claim:
1. A method for detecting a less abundant sequence variant in a protein sample comprising:
a) optionally performing sample preparation;
b) loading the sample onto a gel filtration column;
c) eluting the sample from the gel filtration column;
d) digesting the fraction with trypsin to generate peptide fractions;
e) separating the peptide fractions in the sample by loading the digested fraction of step d) onto a liquid chromatography column;
f) separately loading a preparation containing synthetic peptide bearing the known sequence variation onto a second liquid chromatography column;
g) detecting the presence of sequence variant in the samples of step e) and step f) by mass spectrometry (LC – MS); and
h) detecting and quantitating the sequence variant by comparing the mass spectra of step g);
wherein the sequence variant has an amino acid transversion of tyrosine to asparagine at position 59 in the CDR region of the heavy chain of the antibody.
2. The method as claimed in claim 1 wherein, the protein sample comprises intact protein, subunit protein or middle up protein.
3. The method as claimed in claim 1 wherein, the liquid chromatography column has a pore size of about 300 Å.
4. The method as claimed in claim 1 wherein, the sample preparation comprises one or more of denaturation, reduction, alkylation, Carboxypeptidase-B treatment, IdeS treatment, PNGase treatment and N-glycanase treatment.
5. The method as claimed in claim 1 wherein, the relative abundance of the variant is as low as about 1.4%.