DESC:FIELD OF THE INVENTION
The present invention relates to an analytical method for identification and characterization of a novel protein sequence variant in biotherapeutic preparations. Particularly, the invention uses a characterization workflow combining CEX chromatography, partial proteolytic digestion, peptide mapping and LC-MS for precise, error-free detection and characterization of a relatively less abundant sequence variant, for better clone selection and product development thereby contributing to increase in product quality.
BACKGROUND OF THE INVENTION
Monoclonal antibodies are a rapidly growing class of biotherapeutic drugs with therapeutic relevance in oncology, auto-immune and chronic inflammatory diseases, among others. Production of these monoclonal antibodies from a single clone is critical and technically challenging. Protein expression in cells is governed through the biological processes including DNA replication, RNA transcription and protein translation where each of these biosynthesis steps 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 results 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 polymorphism in triplet codons.
Any unintentional amino acid substitution, omission, or insertion in the protein sequence, generated during protein biosynthesis can result in often less-abundant ‘sequence variants’ (SVs) in a population of the otherwise intended/desired biotherapeutic protein molecule. Many of these modifications induce charge heterogeneity. Understandably, these sequence variants (SV) contribute to an undesired heterogeneity in the biotherapeutic protein preparation. For this reason, establishing a sequence variant profile of a biotherapeutic-producing cell line is essential to prove its structure, manufacturing consistency, stability and most importantly, quality. Identification and characterization of such charge variants is also of utmost importance for regulatory submissions. Hence, investigations of these heterogenic variants take priority in the early stages of product and process development. Sequence variants can also be charged variants, as one amino acid change in protein sequence can lead to change in charge of the protein. Obviously, such variants seriously compromise the quality of therapeutic protein in terms of folding, aggregation, ultimately raising concerns over safety and efficacy of the drug product. Hence critical examination of such substitution mutations or sequence variants in the therapeutic preparation is necessary.
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. Importance of integrated and effective analytical method development are discussed in characterization of sequence variants from Nivolumab. (Li Y et al., Characterization of alanine to valine sequence variants in the Fc region of nivolumab biosimilar produced in Chinese hamster ovary cells. InMAbs 2016 Jul 3 (Vol. 8, No. 5, pp. 951-960). Taylor & Francis).
There is hence a need to develop an improved method to detect and characterize sequence variants even in the presence of limited sample availability and relatively lower abundance of variant forms. Present invention discloses a sensitive method for rapid characterization and detection of a novel sequence variant in an anti-TNF a monoclonal antibody preparation with high specificity. Particularly, the method discloses a method to specifically characterize a glycine to arginine transition mutation identified in the heavy chain of the anti TNF-a monoclonal antibody.
SUMMARY OF THE INVENTION
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 TNF-a monoclonal antibody preparation. The novel sequence variant is identifiable as a ‘shoulder peak’ towards the right side of the main peak on a CEX chromatogram, having a distinct mass peak with additional molecular weight of ~99 Da in comparison to the main peak (Figure 1). Preparative chromatography cannot be employed to purify the said ‘shoulder peak’ due to several limitations including reduced abundance during process development. Present invention provides a novel method with a characterization workflow combining CEX chromatography, partial proteolytic digestion, peptide mapping and LC-MS for precise, error-free detection and characterization to specifically characterize a glycine to arginine transition mutation in the heavy chain of the anti TNF-a monoclonal antibody. Thus claimed method utilizes characterization techniques to identify and characterize unanticipated variants and monitor for better clone selection and superior product development.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1. Elution profile of biosimilar anti-TNFa monoclonal antibody and its RMP by cation exchange chromatography, demonstrating the main peak and lysine variants with a distinct shoulder peak in biosimilar sample across the variants.
Figure 2. Ion exchange chromatogram: A – Purity profiles of main peak (K0) and its shoulder peak (K0+), shown in comparison with control sample treated with and without CpB, respectively. B – Purity profiles of main peak (K0) and its shoulder peak (K0+) with and without CpB treatment.
Figure 3. Deconvoluted mass spectra of intact mass analysis of biosimilar anti-TNFa monoclonal antibody (K0 and K0+ fraction) and RMP demonstrating the masses of glycovariants with exceptional peak of + 99 Da in K0+ fraction
Figure 4. Deconvoluted mass spectra of subunit mass analysis of biosimilar anti-TNFa monoclonal antibody (K0 and K0+ fraction) and RMP demonstrating the masses of light chain and heavy chain across the samples with additional +99 Da mass in K0+ fraction
Figure 5. Deconvoluted mass spectra of Ides treated samples of biosimilar anti-TNFa monoclonal antibody (K0 and K0+ fraction) and RMP showing the masses of Fc`/2 and F(ab`)2 with ~123 kDa mass seen only in K0+ fraction.
Figure 6. Deconvoluted mass spectra – Comparative mass spectra of control and Ides treated samples of K0 and K0+ demonstrating the heavy chain masses in control and absence of the same in K0 fraction with exceptional peak of HC + 99 Da in K0+ fraction, in latter conditions.
Figure 7. Peptide mapping profile of tryptic digests of K0+, K0 and RMP samples demonstrating the presence of HC18 peptide across the samples with relatively significant amount of HC18+99 Da peak in K0+ fraction.
DETAILED DESCRIPTION OF THE INVENTION
Analysis of charge or sequence variants of a therapeutic protein form a part of critical quality attribute testing as presence of these variants may 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.
In an attempt to overcome aforementioned challenges, present invention discloses a sensitive method for rapid characterization and detection of a novel sequence variant in an anti-TNF a monoclonal antibody preparation with high specificity. The charge variant is formed due to a glycine?arginine transition in the hinge region of the antibody. Various embodiments of the disclosed invention are carried out to detect and characterize the charged variants. The novel sequence variant is identifiable as a ‘shoulder peak’ towards the right side of the main peak on a CEX chromatogram, having a distinct mass peak in comparison to the main peak. Carboxypeptidase B (CpB) 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 reveals increase in the mass of the variant species by ~99 Da from native peak of desired anti-TNFa monoclonal antibody. The sequence variant is resistant to IdeS digestion. Characterization of mutation is performed by peptide mapping which shows substitution mutation at G241 (G241R) of the heavy chain (227THTCPPCPAPELLGR241). 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 glycine to arginine (G241R) in an anti-TNF-a antibody using a combination of CEX elution, mass analysis of intact and subunit protein, peptide mapping and also by resistance of variant to IdeS digestion.
In an embodiment the claimed invention describes an analytical method for identifying and characterizing a novel sequence variant in a monoclonal antibody composition comprising:
a. loading the composition on an ion exchange chromatography (IEX) column and eluting wherein the eluted fractions correspond to ‘main peak’ and ‘variant peak’ on the IEX chromatogram; wherein the loading and eluting are performed in sequence for a plurality of times;
b. separately pooling the fractions corresponding to ‘main peak’ and ‘variant peak’ at the end of step a)
c. enriching each pooled fraction of step b) using a molecular weight cut-off filter to obtain an enriched fraction;
d. partially digesting the enriched fraction;
e. detecting the novel sequence variant in digested fraction of step d) by liquid chromatography – mass spectrometry (LC – MS)
wherein the sequence variant has an amino acid substitution of glycine to arginine (G241R) on the heavy chain;
wherein the sequence variant is observed as an additional shoulder peak of ~ 99 Da of basic nature along the main peak and;
wherein the relative abundance of the variant is as low as 1%.
In an embodiment the claimed invention describes an analytical method for identifying and characterizing a novel sequence variant in a monoclonal antibody composition comprising:
f. loading the composition on a cation exchange chromatography (CEX) column and eluting wherein the eluted fractions correspond to ‘main peak’ and ‘variant peak’ on the CEX chromatogram; wherein the loading and eluting are performed in sequence for a plurality of times;
g. separately pooling the fractions corresponding to ‘main peak’ and ‘variant peak’ at the end of step a)
h. enriching each pooled fraction of step b) using a molecular weight cut-off filter to obtain an enriched fraction;
i. optionally treating the enriched fraction of step c) with carboxypeptidase B (CpB);
j. optionally treating the enriched fraction of step c) with IdeS;
k. optionally reducing the enriched fractions of step c) with a reducing agent;
l. subjecting a fraction of step c) to step f) to ultra-performance liquid chromatography – mass spectrometry (LC – MS)
m. optionally analyzing sequence of antibody by peptide mapping
wherein the sequence variant has an amino acid substitution of glycine to arginine (G241R) on the heavy chain;
wherein the sequence variant is observed as an additional shoulder peak of ~ 99 Da of basic nature along the main peak and;
wherein the relative abundance of the variant is as low as 1%.
In an embodiment the elution of sample is carried out using isocratic and linear gradient elution.
In yet another embodiment the pooled fractions demonstrate respective retention times on CEX chromatogram.
Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of this 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 preparation” 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” refers to unintentional amino acid substitution, omission, 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 “main peak” refers to the peak on a CEX chromatogram corresponding to the intended polypeptide species.
The term “variant peak” refers to the peal on a CEX chromatogram corresponding to the unintended sequence variant form of the polypeptide.
The term "heterogeneity" herein refers to a phenomenon wherein the biotherapeutic preparation exhibits a sequence variant profile that is undesired when compared to the desirable biotherapeutic preparation such that the heterogeneity may compromise the biosimilarity of the molecule.
The term “reference manufactured 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 “TNF-a” refers to “tumor necrosis factor-a”, a pro-inflammatory cytokine involved in early inflammatory events. It trigger a series of inflammatory molecules, including other cytokines, chemotactic cytokines, and chemokines.
The term “hinge region” here in this invention refers to a flexible amino acid stretch in the central part of the heavy chains of the IgG and IgA immunoglobulin classes, which links these 2 chains by disulfide bonds.
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.
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: WCX based cation exchange chromatography (CEX) of biosimilar anti-TNFa monoclonal antibody and reference medicinal product
Test anti-TNFa monoclonal antibody when analyzed on cation exchange chromatography (CEX) showed an additional shoulder peak along with main peak (K0) followed by their respective lysine variants; K1 and K2, having one and two lysine residues on their C-terminal heavy chain, respectively. All these comparisons were performed along with reference medicinal product (RMP) and the additional species were unique to our biosimilar product. Significant percentage of novel species observed in Figure 1, not present in innovator, was present as shoulder in CEX chromatograms, attributable to basic charge variant/impurity.
For CEX chromatography, 100 g of biosimilar anti-TNFa monoclonal antibody and RMP were analyzed on Ultimate 3000 system using a ProPac WCX-10 4 mm × 250 mm column (ThermoScientific, Waltham, MA, USA) equilibrated at 30°C. The mobile phases consisted of Na2HPO4 (Buffer A) and of Na2HPO4 with NaCl (Buffer B) and were buffered at pH 7. After an isocratic linear gradient was applied, the column was washed with Buffer B and further equilibrated with same buffer. Elution was monitored by UV absorbance at 280 nm. Fractions were collected after many repeated injections, performed on an Agilent HPLC system connected with a temperature controlled fraction collector. Respective fractions were pooled, enriched, and finally exchanged buffer into LCMS grade water for LCMS analysis.
Example 2: CpB based cation exchange chromatography
Fractions of the “main peak” and “variant peak” were collected with multiple injections performed on HPLC system connected with a temperature controlled fraction collector. Respective fractions were pooled and enriched by molecular weight cut-off filters. This process resulted in significantly pure fractions, where test sample when overlaid with CpB treated, pool fraction of K0 and K0 shoulder (K0+) demonstrate the respective retention times of the species. IdeS cleaves at hinge region of HC below two inter HC-CH2 domain disulfide linkages and produces a single F(ab’)2 (~98 kDa) (two light chains and two Fd fragments of HC) and two Fc’/2 (~25 kDa) fragments in non-reducing conditions. Under reducing conditions, HC is cleaved into N-terminal Fd and C-terminal Fc’/2 fragments and LC is intact. This strategy was employed to narrow down the modification to either Fd or Fc’/2 region of the HC. Samples were treated with Ides in the non-reducing conditions and LCMS was performed. Fc’/2 and F(ab’)2 mass spectra of RMP, K0 and K0+ was similar and no peak corresponding to F(ab’)2 or Fc’/2 with additional 99 Da was observed in K0+ fraction. Data from figure 5 shows an additional mass of ~123 kDa in K0+ fraction, while the same was not observed in RMP and K0 fraction. This additional mass corresponds to mass combination of one F(ab’)2, one Fc’/2 with one water loss and additional 99 Da. Sequentially, K0 fraction under reducing condition showed LC and HC followed by LC, Fc`/2 and Fd when treated with Ides (Figure 6). In contrast, K0+ fraction showed LC and HC, HC+99 Da peaks (under reducing condition) and relatively higher abundance of HC+99 Da when treated with Ides, demonstrating its resistance to enzymatic activity.
Example 3: Mass spectral analysis of intact and subunit protein
Intact and subunit protein analysis was performed using liquid chromatography mass spectrometry (LCMS) on enriched K0 and K0+ peaks to distinguish the basic charge variant observed as sequence and/or charge variant. Intact protein mass spectra in figure 3 shows control, K0 and K0+ fractions were similar with exceptions on K0+ fraction having a distinct mass peak with additional ~99 Da in comparison to control’s G0F/G0F peak. It was observed to be relatively higher in intensity amidst background/artefact mass peaks. Subunit protein analysis, performed to localize the presence of additional mass, were partially reduced and subjected to LCMS analysis. Figure 4 discusses about the subunit protein mass spectra of RMP and K0 fraction found to be similar except for the absence of G0F+K1 mass, attributed to CpB treatment. K0+ fraction shows an additional peak with a mass ~+99 Da on heavy chain (HC) G0F peak observed in relatively higher intensity, with no difference in light chain (LC) mass across the samples.
Mass spectrometric analysis was performed on a Waters Synapt G2-Si Q-TOF MS system with a positive ion mode. The desolvation gas and source temperature was set to 350°C and 120°C. The capillary and cone voltage was set at 3,000 and 40 V. The m/z scan range was set to 500–3,000. The system was controlled by MassLynx 4.1. The deconvolution of ESI mass spectra of Intact and reduced antibody samples were analyzed by Biopharmalynx 1.3.3 using MaxEnt 1 algorithm. 100 µg of biosimilar anti-TNFa monoclonal antibody and RMP was analyzed on UPLC H class Bio system using a micro C4 Desalting catridge (Waters MassPREP®) equilibrated at 80°C and operated at a flow rate of 0.2 ml/min. The mobile phases consisted of water with 0.1 % formic acid (Buffer A) and acetonitrile (Buffer B). A linear gradient was applied from 5% to 90% of B in 2 min at 0.2 ml/min. Elution was monitored by UV absorbance at 280 nm with the above mentioned MS parameters. 100 µg of biosimilar anti-TNFa monoclonal antibody and RMP was reduced with 10 mM DTT at 37°C for 30 mins and analyzed on UPLC H class Bio system with Waters Synapt G2-Si Q-Tof MS. The mobile phases consisted of water with 0.1 % formic acid (Buffer A) and acetonitrile (Buffer B). A linear gradient was applied and elution was monitored by UV absorbance at 280 nm with above mentioned MS parameters.
Example 4: Peptide mapping analysis of sequence/charge variant (G241R)
Peptide mapping analysis of the samples would determine the type and site of mutation/modification attributable to the charge variant. Probable mass based assignments to additional 99 Da included chemical modification by EGC crosslinker/N-isopropylcarboxamidomethyl and amino acid substitutions. Absence of these chemicals in process development directed investigations towards sequence variants, an aftermath of amino acid substitutions/mutations. Peptide mapping data analysis indicated the presence of peptide with additional 99 Da (HC tryptic peptide HC18) along with many peptide possibilities of similar differences due to background interference. Peptide with IdeS cleavage site HC 18, was identified in all the samples of RMP, K0 fraction and K0+ fraction as shown in Figure 7. HC 18 with additional 99 Da (HC18 + 99Da, G to R mutation) was found only in K0+ fraction with trace amounts in K0 fraction in the ratio as shown in Table 1. HC 18 peptide has two Gly residues at HC 240 and 241, one of which was mutated to Arg.

Table 1
Tryptic Peptide HC 18 Theoretical mass (Da) Observed mass (Da) RT (Min) RMP % K0 (%) K0+ (%)
THTCPPCPAPELLGGPSVFLFPPKPK 2843.5 2843.5 46.1 100.0 100.0 100.0
THTCPPCPAPELLGRPSVFLFPPKPK 2942.5 2942.5 43.0 ND ~1.0 ~25.0
THTCPPCPAPELLR 1647.8 ND ND ND ND ND
THTCPPCPAPELLGR 1705.8 1705.8 27.7 ND ND 100.0

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:WE CLAIM:

1. A method for identification and characterization of a novel sequence variant in a monoclonal antibody composition comprising:
a. loading the composition on an ion exchange chromatography (IEX) column and eluting wherein the eluted fractions correspond to ‘main peak’ and ‘variant peak’ on the IEX chromatogram; wherein the loading and eluting are performed in sequence for a plurality of times;
b. separately pooling the fractions corresponding to ‘main peak’ and ‘variant peak’ at the end of step a)
c. enriching each pooled fraction of step b) using a molecular weight cut-off filter to obtain an enriched fraction;
d. partially digesting the enriched fraction;
e. detecting the novel sequence variant in digested fraction of step d) by liquid chromatography – mass spectrometry (LC – MS)
wherein the sequence variant has an amino acid substitution of glycine to arginine (G241R) on the heavy chain;
wherein the sequence variant is observed as an additional shoulder peak of ~ 99 Da of basic nature along the main peak on the IEX chromatogram;
wherein the relative abundance of the variant is as low as 1%; and
wherein the partial digestion comprises one or more of treatment with carboxypeptidase B (CpB), IdeS protease and reducing agent.
2. A method for identification and characterization of a novel sequence variant in a monoclonal antibody composition comprising:
a. loading the composition on a cation exchange chromatography (CEX) column and eluting wherein the eluted fractions correspond to ‘main peak’ and ‘variant peak’ on the CEX chromatogram; wherein the loading and eluting are performed in sequence for a plurality of times;
b. separately pooling the fractions corresponding to ‘main peak’ and ‘variant peak’ at the end of step a)
c. enriching each pooled fraction of step b) using a molecular weight cut-off filter to obtain an enriched fraction;
d. optionally treating the enriched fraction of step c) with carboxypeptidase B (CpB);
e. optionally treating the enriched fraction of step c) with IdeS protease;
f. optionally reducing the enriched fractions of step c) with a reducing agent;
g. subjecting a fraction of step c) to step f) to ultra-performance liquid chromatography – mass spectrometry (LC – MS);
h. optionally analyzing sequence of antibody by peptide mapping;
wherein the sequence variant has an amino acid substitution of glycine to arginine (G241R) on the heavy chain;
wherein the sequence variant is observed as an additional shoulder peak of ~ 99 Da of basic nature along the main peak and;
wherein the relative abundance of the variant is as low as 1%.
3. The method as claimed in claim 1 and claim 2 wherein elution is carried out using isocratic and linear gradient elution.
4. The method as claimed in claim 1 to claim 3 wherein the pooled fractions demonstrate respective retention times on CEX chromatogram.