DESC:A BIOTHERAPEUTIC PREPARATION WITH REDUCED HETEROGENEITY AND METHODS THEREOF

FIELD OF INVENTION
The present invention relates to analytical attributes of compositions and methods relating to biologic preparations. The invention particularly provides post-translational modification profile of the biologic protein targeting IL-6R wherein the profile includes glycosylation profile, charged species profile and methods thereof.
BACKGROUND OF THE INVENTION
Biopharmaceuticals or biotherapeutics are engineered biological molecules of therapeutic significance. They have revolutionized the treatment of many diseases where existing therapeutic measures haven’t been successful. Therapeutic monoclonal antibodies (mAbs), which are engineered recombinant proteins, form a large class of biotherapeutics and are the fastest growing class in the treatment of cancer, autoimmune, and inflammatory disorders among others.
N-linked glycosylation is a post-translational modification (PTM) which plays an important role in the physiological function of therapeutic mAbs. The pharmacokinetics, pharmacodistribution, solubility, stability, receptor binding and enhancement of receptor function is highly influenced by the glycosylation profile of the mAb. For example, antibody-dependent cell cytotoxicity (ADCC), a functional effect of some mAbs, is influenced by the specific N-glycosylation at the Fc domain. Synthesis and processing of N¬-linked oligosaccharides is a multi-enzyme regulated process that takes place in several subcellular compartments. Clearly, a highly regulated manufacturing process with sensitive analytical monitoring is required to reduce changes in the oligosaccharide structures in the therapeutic preparation. Resultant heterogeneity can be largely mitigated by glycoengineering, the aim of which is to generate correct or optimized glycosylation profile, mainly by altering the process conditions.
Charged species also influence stability and biological activity, differences in the relative proportions of which can pose a challenge in demonstrating consistency. Deamidation, sialylation and C-terminal lysine cleavage can result in an increase in the net negative charged, thus generating acidic species. Basic species are formed with C-terminal lysine or glycine amidation, succinimide formation, amino acid oxidation or removal of sialic acid, which introduce additional positive charge. As mentioned earlier, manufacturing process and conditions contribute to a large extent, to glycan and charge-related heterogeneity in biotherapeutic preparations. Hence, developing a biotherapeutic preparation that exhibits reduced heterogeneity remains a challenge.
The present invention provides tocilizumab preparations with reduced heterogeneity in glycan and charged species profile and methods thereof.
SUMMARY OF THE INVENTION
The PTM profile of a therapeutic monoclonal antibody (mAb) has a bearing on its stability, safety and efficacy. PTM profile mainly includes glycan profile and charged species profile among others. The present invention provides biotherapeutic preparations with reduced heterogeneity by analysing glycosylation and charged species profile of therapeutic preparation and methods thereof. Reduced heterogeneity refers to that PTM profile of analysed parameters, which translates to improvement in the desired functionality of the therapeutic preparation, thereby resulting in a high quality preparation. The present invention provides a test biotherapeutic preparation with reduced heterogeneity wherein, the preparation is produced by a method that comprises: a) analytical testing of the test biotherapeutic preparation for glycosylation and charged species, b) acquiring the values for the plurality of parameters as listed in Table 1 and Table 2., c) assessing whether the values for the plurality of parameters fall within the corresponding acceptable range as listed in Table 1 and Table 2. d) altering manufacturing processes such that the values for the plurality of parameters fall within the corresponding acceptance range as list e) thereby producing a test biotherapeutic preparation with reduced heterogeneity.
The invention is of particular importance as it can form the part of methods that demonstrate batch-to-batch consistency and predict shelf-life of complex protein molecules.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1. Amino acid sequence in the complementarity determining regions in the variable region of tocilizumab
Figure 2. A representative UPLC glycan chromatographic profile of Tocilizumab sample after integration of all the glycan species in the time frame of 10 to 35 minutes
Figure 3. IEX profile of Tocilizumab depicting the integration of total acidic and basic isoforms/species along with the main peak

DESCRIPTION OF THE INVENTION
The present invention discloses compositions and methods relating to biotherapeutic preparation targeted against anti-IL-6R. The invention provides analytical profile of the biotherapeutic. The invention further provides a method of reducing heterogeneity in glycosylation and charged species profile and the resultant biotherapeutic preparation thereof.
In an aspect of the invention, the methods provide processes of producing biotherapeutic preparation with reduced heterogeneity. In particular, the invention provides a method of producing biotherapeutic preparation with reduced heterogeneity in the post-translational modification profile, thereby resulting in a high quality preparation.
The glycosylation profile and charged species profile of a therapeutic monoclonal antibody preparation have a bearing on its stability, safety and efficacy. Certain glycosylation profile of the therapeutic mAb are desirable based on its mechanism of action. In an embodiment, the method of the present invention may be used to produce therapeutic mAbs composition with relatively less heterogeneity.
An aim in the development of the method of present invention is to produce a high quality biotherapeutic preparation with reduced heterogeneity. Reduced heterogeneity translates to improvement in the desired functionality of the therapeutic preparation.
A person of ordinary skill in the art would be able to determine the glycosylation profile and charged species profile of the antibody composition described herein using the state of the art techniques. The methods described in the present invention are also in recognition of the fact that various methods may be used to obtain antibody composition of desired glycosylation profile and charged species profile.
Embodiments
In an embodiment the invention discloses a biotherapeutic preparation comprising IL-6R targeting protein wherein, the preparation is characterized in that it comprises one or more of the attributes comprising: about 1.2 % high mannose species; about 3.8% afucosylated glycans; about 5% total afucosylated glycoform species; about 45.6% total galactosylated glycans; about 49.8% G0F glycans; about 22% acidic species and about 4% basic species
In another embodiment, the invention discloses a method of producing a biotherapeutic preparation comprising IL-6R targeting protein wherein, the method comprises:
a) analytical testing of glycosylation profile at Asn297 and charged species profile;
b) acquiring values for one or more of the parameters listed in Table 1,
c) assessing whether the values of step b) fall within the corresponding acceptable range as listed in Table 3 and
d) tailoring the manufacturing processes such that the values of step b) fall within the corresponding acceptance range as listed in Table 3.
thereby producing the biotherapeutic preparation.
In yet another embodiment, biotherapeutic composition comprising IL-6R targeting protein comprises the CDR1, CDR2 and CDR3 sequences in the variable region of the light chain and has an amino acid sequence identity of at least 95%, 98%, 99% or 100% to SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 respectively, and wherein, the CDR1, CDR2 and CDR3 sequences in the variable region of the heavy chain has an amino acid sequence identity of at least 95%, 98%, 99% or 100% to SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 respectively.
In an embodiment, the present invention provides a method for reducing heterogeneity in the test biotherapeutic preparation wherein the test biotherapeutic preparation is tocilizumab.
In an embodiment, the invention provides the method of reducing heterogeneity wherein the test biotherapeutic preparation is one sample or belongs to a given batch.
In some embodiments, the manufacturing process comprises upstream manufacturing process. In a further embodiment, the upstream manufacturing process comprises altering the cell culture conditions that comprises culture medium, temperature, pH and cell culture process.
In some embodiments, the manufacturing process comprises downstream manufacturing process for isolation and purification of the biotherapeutic.
In some embodiments, the manufacturing process comprises formulating the test biotherapeutic preparation, processing the biotherapeutic into a drug preparation, adding excipients to the biotherapeutic, dispensing the biotherapeutic into smaller or larger aliquots and transferring the biotherapeutic to a different location.
In an embodiment, analytical testing for glycosylation comprises enzymatic or chemical digestion of the biotherapeutic, separation of digested fragments and detection of glycan species. In a further embodiment, the method of separation of digested fragments employs electromagnetic separation or chromatographic separation.
In another embodiment, the charged species are analyzed by charge-based separation techniques which includes isoelectric focusing (IEF) gel electrophoresis, capillary isoelectric focusing (cIEF) gel electrophoresis, cation exchange chromatography (CEX) and anion exchange chromatography (AEX).
Definitions
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 one or several post translational modifications (PTM), imparting the antibody molecules a different molecular weight, charge, solubility or combinations thereof.
The term "heterogeneity" herein refers to a phenomenon wherein the biotherapeutic preparation exhibits a glycosylation and charged species profile that is undesired such that the heterogeneity may compromise the safety, purity and efficacy of the drug product.
The term "reducing heterogeneity" as used herein refers to process of conversion of heterogeneous biotherapeutic preparation to substantially pure, homogenous form.
The term “post-translational modification” or “PTM”, herein used interchangeably, refers to biochemical modification that occurs at one or more amino acids on a protein molecule after translation of the protein. PTMs are mostly chemical or enzyme-mediated, at specific target sequences in the protein and comprise inter alia, glycosylation, glycation, acetylation, amidation, deamidation, methylation, ADP-ribosylation and hydroxylation.
The term “glycosylation profile” or “glycan profile” herein refers to percentage of various glycan species attached to the proteins in the biotherapeutic preparation.
The term “charged species” refers to acidic species and basic species in the biotherpaeutic preparation.
The term “charged species profile” refers to the percentage of acidic species and basic species in the biotherapeutic preparation. When antibodies are analyzed using isoelectric focusing (IEF)-based methods, acidic species are variants with lower apparent isoelectric point (pI) and basic species are variants with higher apparent pI. In cation exchange chromatography (CEX), acidic species elute earlier than the main peak, while basic species elute later than the main.
Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of this invention. The invention will now be described in greater detail by reference to the following non-limiting examples. The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
EXAMPLES

Example I Analytical testing of glycan species by Hydrophilic Interaction Liquid Chromatography
Glycoproteins in sample was deglycosylated using PNGaseF in deglycosylation buffer containing 5% RapiGestTM (w/v). For this, solution containing 5% RapiGestTM (w/v) solution (in deglycosylation buffer) was added to test biotherapeutic sample and resultant mix was mixed well to form a homogenous solution and were incubated at 85 °C for about 3 minutes. Following this, samples were incubated at room temperature for at least 3 minutes. PNGaseF was added and samples were again incubated at 50 °C for 5 minutes The released N-Glycosylamines are labelled using GlycoWorks RapiFluor-MS reagent solution. For this, RapiFluor–MS reagent solution was added to the above deglycosylated mixture. After incubation at room temperature, samples were diluted with 100% Acetonitrile (ACN). Labelled N-Glycosylamines were captured on a GlycoWorks HILIC SPE µElutionTM plate and eluted using 200 mM Ammonium acetate (pH 7) in 5% ACN. The eluted mix was diluted in dimethylformamide (DMF)/CAN solution and mixed thoroughly. Samples were then analysed by HPLC (ACQUITY UPLC BEH Glycan Separation Technology Column). All the individual glycan species were integrated as shown in Figure 2 and peak annotation of each species were followed based on elution order for identification as per the rRT (retention time) table as indicated in Table 1. The relative proportion of glycoform species in the sample is represented according to Table 2.
Species rRT
M3NAG 0.72
M3NAGF 0.83
G0 0.88
G0F 1.00
M5A 1.10
M5B 1.12
G1A 1.16
G1B 1.20
G0F+NAG 1.23
G1AF 1.26
G1BF 1.30
M6 1.39
G2 1.43
G1F+NAG 1.45
G2F+G1FS1 1.49
G2F 1.51
G1FS1 1.54
M7 1.63
G2S1 1.63
G2F+NAG 1.66
G2FS1A 1.69
G2FS1B 1.71
M8A 1.79
M8B 1.80
M8C 1.81
G2FS2 1.87
M9 1.96
Table 1
% Glycoform Species Formula
High mannose (HM) M5+M6+M7+M8+M9
Afucosylated (AF) M3NAG+G0+G1A+G1B+G2+G2S1
TAF HM+AF
Galactose G1A+G1AF+G1B+G1BF+(G1F+NAG)+G1FS1+G2+G2F+(G2F+G1FS1)+(G2F+NAG)+G2FS1+G2FS2+G2S1
G0F G0F
Table 2
Example II Analytical testing of charged species by Ion Exchange chromatography
All the test samples were diluted to 1 mg/mL in sample diluent buffer (15 mM sodium phosphate pH 6.5 ± 0.2). HPLC column for Ion Exchange chromatography was pre-equilibrated before sample loading, with respective buffers as per gradient details. Sample diluent buffer was used as the blank. 50 µL of test sample, which is equivalent to 50 µg column load as single injection was injected into the column. Samples were analysed at 214 nm and an IEX profile was generated. The total cluster from first acidic to last acidic isoforms that elutes prior to the main peak is categorized as acidic species/isoforms. The total cluster from first basic to last basic isoforms that elutes after the main peak is categorized as basic isoform/species.
Example III Qualification of tocilizumab for commercial release for all analysed glycan and charged species
Table 3 details the glycan and charged species analysed and their relative abundance in different batches of selected test tocilizumab preparations. One or more of the parameters, or a subset of the same or multiple subsets of the same may be compared with a given test preparation as per the method described in invention.
A score for whether the value corresponding to the relative abundance against each glycan species matches with the values in Table 3 can be made. The test preparation that scores as positive for all types of glycan and charged species may be selected for further suitability procedures for commercial release. For example, Table 4 indicates the corresponding scores of two hypothetical test preparations X and Y. A to H indicate a given set of glycoform or charged species under consideration for qualification of the commercial batch. Test preparation X scores positive for all parameters checked, while test preparation Y does not fall within the acceptable ranges (between Min and Max values) for representative glycan species A, E and F. Thus test preparation Y may be rejected in the qualification step. Further, manufacturing processes adopted for test preparation Y may be altered to generate samples which will then be subject to subsequent qualification.
Glycoform Species Formula Min Max D_TC (% species_Average) Total (%)
High mannose (HM) M5 0.5 1 0.6 1.2
M6 0.1 0.1 0.1
M7 0.1 0.1 0.1
M8 0.3 0.4 0.4
M9 0 0 0
Afucosylated (AF) M3NAG 0 0.1 0 3.8
G0 2.2 3 2.4
G1A 0.9 0.9 0.9
G1B 0.4 0.4 0.4
G2 0.1 0.1 0.1
G2S1 0 0.1 0
TAF HM 1.2 1.2 1.2 5
AF 3.8 3.8 3.8
Galactose G1A 0.9 0.9 0.9 45.6
G1AF 23.6 28.4 26.8
G1B 0.4 0.4 0.4
G1BF 8.8 10.7 10.1
(G1F+NAG) 0.2 0.3 0.2
G1FS1 0.3 0.4 0.4
G2 0.1 0.1 0.1
G2F 4.4 6 5.5
(G2F+G1FS1) 0 0 0
(G2F+NAG) 0 0.1 0
G2FS1 0.6 0.9 0.8
G2FS2 0.3 0.5 0.4
G2S1 0 0.1 0
G0F G0F 47.1 54.4 49.8 49.8
Acidic species 20 21 20.5
Main species 71 73 72
Basic species 8 9 8.5

Table 3: Analytical parameters for test tocilizumab preparation

Score for Test Preparation X Score for Test Preparation Y
A ? X
B ? ?
C ? ?
D ? ?
E ? X
F ? X
G ? ?
H ? ?

Table 4: Qualification for commercial release: example
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. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.
,CLAIMS:We claim,
1. A biotherapeutic preparation comprising IL-6R targeting protein wherein, the preparation is characterized in that it comprises one or more of the attributes comprising: about 1.2 % high mannose species; about 3.8% afucosylated glycans; about 5% total afucosylated glycoform species; about 45.6% total galactosylation; about 49.8% G0F glycans; about 22% acidic species and about 4% basic species
2. A method of producing a biotherapeutic preparation comprising IL-6R targeting protein wherein, the method comprises:
a) analytical testing of glycosylation profile at Asn297 and charged species profile;
b) acquiring values for one or more of the parameters listed in Table 1,
c) assessing whether the values of step b) fall within the corresponding range of values as listed in Table 3 and
d) tailoring manufacturing processes such that the values of step b) fall within the corresponding range of values as listed in Table 3,
thereby producing the biotherapeutic preparation.
3. The biotherapeutic preparation as claimed in claim 1 and claim 2 wherein the IL-6R targeting protein comprises the CDR1, CDR2 and CDR3 sequences in the variable region of the light chain and has an amino acid sequence identity of at least 95%, 98%, 99% or 100% to SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 respectively, and wherein, the CDR1, CDR2 and CDR3 sequences in the variable region of the heavy chain has an amino acid sequence identity of at least 95%, 98%, 99% or 100% to SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 respectively.
4. The biotherapeutic preparation as claimed in claim 1 and claim 2 wherein the preparation belongs to a given batch of production.
5. The manufacturing process as claimed in claim 2 wherein the process comprises upstream manufacturing process or downstream manufacturing process.