DESC:The invention relates to the field of biotechnology. More specifically the invention discloses a method for selective reduction of disulfide bonds in a therapeutic protein composition. In particular, the invention relates to a method for selective reduction of disulfide bonds in a composition comprising immunoglobulins.
BACKGROUND OF THE INVENTION
The recombinant monoclonal antibodies (mAbs) of the immunoglobulin (IgG) class comprise a rapidly growing group of protein therapeutics. The introduction of mAbs for the treatment of chronic diseases such as cancer and autoimmune diseases has changed the scenario of the healthcare industry. mAbs are effective targeted therapeutic agents. The high specificity of such antibodies makes them ideal to reach their intended target and hence is useful to treat a wide variety of diseases.
Monoclonal antibodies are highly complex glycoproteins with a large molecular weight of approximately 150 kilo Daltons (~150 kDa). They consist of two heavy chains and two light chains of polypeptides linked together by inter-chain disulfide bonds and non-covalent interactions. The tertiary structure of mAbs is required to be present in a certain conformation in order to be biologically active. The inter- and intra-chain disulfide bonds in a mAb play a key role in maintaining its conformational stability. Consequently, mAbs include several disulfide bonds, and the patterns of inter-chain disulfide bonds characterize different IgG subclasses. The disulfide bond structure of IgG is highly conserved through evolution and was once considered a uniform and homogeneous structural feature. However, detailed characterization of a large number of IgG molecules has revealed several new structural features in both recombinant and natural human IgG antibodies. Disulfide bond structures of the four subclasses of IgG, namely IgG1, IgG2, IgG3, and IgG4, were established in the 1960s. It is already well established that there are many similarities and some differences with regard to the disulfide bond structures in the four subclasses of IgG antibodies. Each IgG contains a total of 12 intra-chain disulfide bonds; each disulfide bond is associated with an individual IgG domain. The two heavy chains are connected in the hinge region by a variable number of disulfide bonds: 2 for IgG1 and IgG4, 4 for IgG2 and 11 for IgG3. The light chain of the IgG1 is connected to the heavy chain by a disulfide bond between the last cysteine residue of the light chain and the fifth cysteine residue of the heavy chain. However, for IgG2, IgG3 and IgG4, the light chain is linked to the heavy chain by a disulfide bond between the last cysteine residue of the light chain and the third cysteine residue of the heavy chain. The level of solvent exposure is different between intra-chain and inter-chain disulfide bonds. Generally, inter-chain disulfide bonds are highly solvent exposed whereas the intra-chain disulfide bonds are buried inside and hence not solvent exposed.
Antibody-drug conjugates (ADCs) are a class of targeted cancer therapeutics that benefit from the specificity of antibodies to deliver potent cytotoxic drug molecules directly to cancer cells. ADCs that are conjugated via partial reduction of the inter-chain disulfides form a heterogeneous distribution of conjugated molecules with varying numbers of drug-linkers per mAb. The number of conjugated drug-linkers can range from 0 – 8 for an IgG1 ADC, or 0 – 12 for an IgG2 ADC.
In several industrial applications, including various types of analytical assays, the inter- and/or intra-chain disulfide bonds need to be reduced for various purposes, such as for preparing the antibody composition for peptide mass fingerprinting, for analyzing the mass of light chains and heavy chains of an immunoglobulin, for manufacturing ADCs, etc. The reduction process is most commonly carried out using denaturing and/or reducing agents such as guanidine hydrochloride, urea, TCEP (tris(2-carboxyethyl)phosphine), DTT (dithiothreitol), DTE (dithioerythritol), ß-mercaptoethanol, etc. There are two major disadvantages of using such chemicals for the reduction of disulfide bonds – (i) lack of selectivity, and (ii) incompatibility with most chromatography and mass spectrometry based methods. So, an additional sample purification step has to be included in the process to remove these chemicals from the antibody composition before subjecting to further analyses. Additionally, few chemicals such as DTT are quite expensive, which drives up the cost of the analytical assay. Moreover, the lack of selectivity is especially a problem in the case of manufacture of ADCs that are conjugated via partial reduction of the inter-chain disulfides.
Hence, there is a requirement of an improved method for the selective reduction of disulfide bonds in antibody compositions that overcomes the above mentioned shortcomings.
SUMMARY OF THE INVENTION
The inventors of the present invention have surprisingly found that the inter-chain disulfide bond between the light chain and the heavy chain of an immunoglobulin can be selectively reduced, leaving intact the intra-chain disulfide bonds of the immunoglobulin. And that the selective reduction is achieved without the use of commonly used reducing agents such as TCEP, DTT, DTE, ß-mercaptoethanol, etc. Accordingly, the present invention discloses a method for the selective reduction of inter-chain disulfide bond between the heavy chain and light chain of an immunoglobulin, without the use of chemicals such as DTT, DTE, ß-mercaptoethanol, etc. The method involves the following steps:
(a) obtaining a sample comprising an immunoglobulin composition and fragments thereof,
(b) adding to the sample an aqueous solution comprising citrate buffer at a specific concentration and adjusting the pH to a value within a specific range,
(c) incubating the composition of step (b) at a specific temperature for a specific period to obtain a composition comprising the immunoglobulin and free light chains of the immunoglobulin.
The method is used to selectively reduce the inter-chain disulfide bond between the light chain and the heavy chain of an immunoglobulin, while keeping the intra-chain disulfide bonds intact.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Overlay of chromatograms from SEC-HPLC for samples incubated for 4 weeks showing two post-shoulder peaks in F13 (bottom panel), which are significantly higher than in F10 (top panel).
Figure 2: Chromatogram obtained from mass spectrometer confirming peak 2 identified as the pyro-Q form of free light chain.
Figure 3: Electropherogram obtained from CE-SDS showing further distribution of the LMW content for 7 formulation samples, namely F01, F07, F10, F13, F18, F25 and F26, after 4 weeks of incubation at 40°C. CE-SDS was performed as an orthogonal technique to confirm the impurity levels as obtained from SEC-HPLC analysis. Details of monomer content and other impurities as obtained from CE-SDS for the abovementioned 7 samples is tabulated in Table 5.
DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
The term “composition” as used herein comprises the protein of interest and one or more contaminants, including but not limited to heavy chains, light chains, low molecular weight fragments, high-molecular weight aggregates etc. The composition may be "partially purified" (i.e., having been subjected to one or more purification steps) or may be obtained directly from a host cell or organism producing the antibody (e.g., the composition may comprise harvested cell culture fluid).
A "contaminant" or an “impurity” as used interchangeably herein is a material that is different from the desired polypeptide product. The contaminant may be a low molecular fragment or a high molecul aggregates of the desired polypeptide, a variant of the desired polypeptide (e.g. a deamidated variant or an aminoaspartate variant of the desired polypeptide) or another non-product related polypeptide, for e.g., host cell protein, host cell nucleic acid, endotoxin, etc. A contaminant can also be process related, for 25 example - Protein-A-leachates.
“High molecular weight aggregates” as referred herein encompasses association of at least two molecules of a product of interest, e.g., antibody or any antigen- binding fragment thereof. The association of at least two molecules of a product of interest may arise by any means including, but not limited to, non-covalent interactions such as, e.g., charge-charge, hydrophobic and van der Waals interactions; and covalent interactions such as, e.g., disulfide interaction or non-reducible crosslinking. An aggregate can be a dimer, trimer, tetramer, or a multimer greater than a tetramer, etc.
The term “immunoglobulin” referred herein is interchangeable with “antibody”, which comprises two identical heavy chains and two identical light chains.
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.
The term “heavy chain” is one of the polypeptides of immunoglobulin which comprises variable region, hinge region and constant region. Approximate molecular weight of heavy chain is 50 kDa. Further, it comprises at least four intra chain disulphide bonds.
The term “light chain” is also one of the polypeptides of immunoglobulin which is shorter than heavy chain and has variable region and constant region and it contains two intra chain disulphide bonds. Approximate molecular weight of light chain is 25 kDa. Further, heavy chain and light chain of immunoglobulin is connected by two inter chain disulphide bonds.
The term “free light chain” or “free light chains” as used herein refers to light chain(s) of immunoglobulin that are not connected to heavy chain by disulphide bonds, and thus free light chain(s) expose and generate “free thiol groups”. The free thiol groups are then available to react or bond with any compound /chemical species or can enter into a chemical reaction further.
The term “about” as used herein, means an acceptable error range for the particular value as determined by one of ordinary skill in the art. For example, “about” can mean a range of up to 20%.
DETAILED DESCRIPTION OF THE EMBODIMENTS
The present invention discloses a method for the selective reduction of the inter-chain disulfide bond between the light and heavy chain of an immunoglobulin.
In an embodiment, the invention discloses a method for the selective reduction of the inter-chain disulfide bond between the light and heavy chain of an immunoglobulin, the method comprising the addition of citrate buffer to the immunoglobulin composition, at a specific pH, followed by incubating the composition at a specific temperature.
In an embodiment, the invention discloses a method for the selective reduction of the inter-chain disulfide bond between the light and heavy chain of an immunoglobulin, the method comprising the steps of addition of citrate buffer to the immunoglobulin composition, at a specific pH, followed by incubating the composition at a specific temperature.
In an embodiment, the invention discloses a method for the selective reduction of the inter-chain disulfide bond between the light and heavy chain of an immunoglobulin, the method comprising the steps of addition of citrate buffer at a specific concentration to the immunoglobulin composition, at a specific pH, followed by incubating the composition at a specific temperature.
In an embodiment, the invention discloses a method for the selective reduction of the inter-chain disulfide bond between the light and heavy chain of an immunoglobulin, the method comprising the steps of addition of citrate buffer at a specific concentration to the immunoglobulin composition, at a specific pH, followed by incubating the composition at a specific temperature for a specific period.
In an embodiment, the invention discloses a method for the selective reduction of the inter-chain disulfide bond between the light and heavy chain of an immunoglobulin, the method comprising the steps of
(a) obtaining a sample comprising an immunoglobulin composition and fragments thereof,
(b) adding to the sample an aqueous solution comprising citrate buffer at a specific concentration and adjusting the pH to a value within a specific range,
(c) incubating the composition of step (b) at a specific temperature for a specific period to obtain a composition comprising the immunoglobulin and free light chains of the immunoglobulin.
In any of the abovementioned embodiments, the immunoglobulin belongs to IgG1, or IgG2, or IgG3, or IgG4 subclass.
In any of the abovementioned embodiments, the immunoglobulin belongs to IgG1 subclass.
In any of the abovementioned embodiments, the immunoglobulin is a monoclonal antibody.
In any of the above-mentioned embodiments, the immunoglobulin is a fusion protein, wherein the immunoglobulin or fragment thereof, is fused to a protein.
In any of the above-mentioned embodiments, the immunoglobulin is a conjugate, wherein the immunoglobulin moiety is fused to a chemical compound or a drug. Examples are antibody-drug conjugates.
In any of the above embodiments, the disclosed method selectively reduces the inter-chain disulfide bond between the light and heavy chain of an immunoglobulin, wherein the method results in formation of free light chains containing free thiol group(s) and these thiol group(s) are available for conjugation with a drug or compound to prepare antibody drug conjugate.
In yet another aspect, the invention discloses a method of preparing an antibody drug conjugate, comprising:
(a) obtaining a sample comprising an antibody composition,
(b) adding to the sample an aqueous solution comprising citrate buffer at a pH value of specific range,
(c) incubating the composition of step (b) at a specific temperature to obtain a composition comprising the antibody and free light chains of the antibody;
wherein the free light chains of the antibody generate free thiol groups,
and the free thiol groups obtained are available to react or conjugate with a drug or compound to obtain an antibody drug conjugate.
In the above mentioned embodiment, the drug in the preparation of antibody drug conjugate is a cytotoxic drug or a chemical compound. Examples of cytotoxic drug include, but not limited to emtansine, vedotine, deruxtecan, ozogamicin or auristatin.
In any of the above embodiments, the immunoglobulin or antibody belongs to IgG1, or IgG2, or IgG3, or IgG4 subclass.
In any of the abovementioned embodiments, the immunoglobulin or antibody belongs to IgG1 subclass.
In any of the abovementioned embodiments, the immunoglobulin or antibody is rituximab, infliximab, abciximab, adalimumab, aducanumab, alemtuzumab, alemtuzumab, alirocumab, amivantamab, anifrolumab, ansuvimab, atezolizumab, atoltivimab maftivimab and odesivimab, avelumab, basiliximab, belimumab, benralizumab, bevacizumab, bezlotoxumab, blinatumomab, brodalumab, brolucizumab, burosumab, canakinumab, caplacizumab, capromab pendetide, cemiplimab, cetuximab, crizanlizumab, daratumumab, denosumab, dinutuximab, dinutuximab beta, dostarlimab, dupilumab, durvalumab, eculizumab, elotuzumab, emapalumab, emicizumab, erenumab, evinacumab, evolocumab,,faricimab, fremanezumab, galcanezumab, golimumab, guselkumab, ibalizumab, idarucizumab, infliximab, ipilimumab, isatuximab, ixekizumab, lanadelumab, margetuximab,mepolizumab, mogamulizumab, natalizumab, naxitamab, necitumumab, nivolumab, obiltoxaximab, obinutuzumab, ocrelizumab, ofatumumab, omalizumab, palivizumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, ranibizumab, ravulizumab, raxibacumab, reslizumab, risankizumab, rituximab, romosozumab, sarilumab, satralizumab, secukinumab, siltuximab, tafasitamab, teprotumumab, tildrakizumab, tocilizumab, trastuzumab, ustekinumab, vedolizumab, bimekizumab, eptinezumab, inebilizumab, lecanemab, mosunetuzumab, nivolumab and relatlimab, retifanlimab, spesolimab, sutimlimab, teclistamab, teplizumab, tezepelumab, tralokinumab, tremelimumab, ublituximab, or fusions or conjugates thereof.
In any of the above mentioned embodiments, the immunoglobulin or antibody composition comprises one or more pharmaceutically acceptable excipients, wherein the pharmaceutically acceptable excipients are sugars, polyols, salts and surfactants.
In any of the abovementioned embodiments, the concentration of citrate buffer is about 20 mM to about 50 mM.
In any of the abovementioned embodiments, the concentration of citrate buffer is about 30 mM to about 50 mM.
In any of the abovementioned embodiments, the pH of the buffer solution is about 6 to about 7.
In any of the abovementioned embodiments, the pH of the buffer solution is about to 5 to about 6.
In any of the abovementioned embodiments, the pH of the buffer solution is about to 6 to about 6.5.
In any of the abovementioned embodiments, the specific temperature is about 60 °C.
In any of the abovementioned embodiments, the specific temperature is about 50 °C.
In any of the abovementioned embodiments, the specific temperature is about 40 °C.
In any of the abovementioned embodiments, the specific temperature is about 30°C.
In any of the abovementioned embodiments, the specific temperature is about 25 °C.
In any of the abovementioned embodiments, the immunoglobulin or antibody composition is incubated at a specific temperature for a period of 4 weeks or less.
In the abovementioned embodiment, the immunoglobulin or antibody composition is incubated at a specific temperature for a period ranging from 1 hour to 1 week, or 1 week to 2 weeks, or 2 weeks to 3 weeks, or 3 weeks to 4 weeks.
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: Study of drug product of an immunoglobulin from IgG1 subclass for selective reduction
The drug substance of a sample monoclonal antibody (Mab) belonging to IgG1 subclass, manufactured in-house, was obtained and formulated into the drug product using excipients in various concentrations, which included sodium chloride (NaCl), polysorbate 80 (PS 80), and citrate buffer. The different formulations of the drug product in citrate buffer, were incubated at 40°C for one week and up to four weeks, and before and after incubation, were subjected to size exclusion chromatography for the determination of monomer, high-molecular weight (HMW) and low-molecular weight (LMW) content. Details of various formulations of the drug product are shown in Table 1.
Formulation ID pH NaCl (mg/mL) PS 80 (mg/mL) Citrate (mM) Protein (mg/mL)
F01 6.5 9 0.7 35 10
F02 6.5 7 0.7 25 10
F03 6.7 8 0.8 30 10
F04 6.5 9 0.7 25 10
F05 6.3 10 0.8 20 10
F06 6.3 8 0.8 20 10
F07 6.3 8 0.8 30 10
F08 6.3 8 0.6 20 10
F09 6.5 11 0.7 25 10
F10 6.5 9 0.7 25 10
F11 6.7 10 0.8 30 10
F12 6.5 9 0.5 25 10
F13 6.3 8 0.6 30 10
F14 6.7 8 0.8 20 10
F15 6.7 8 0.6 30 10
F16 6.7 8 0.6 20 10
F17 6.5 9 0.7 25 10
F18 6.1 9 0.7 25 10
F19 6.9 9 0.7 25 10
F20 6.3 10 0.6 20 10
F21 6.7 10 0.6 20 10
F22 6.5 9 0.9 25 10
F23 6.7 10 0.6 30 10
F24 6.7 10 0.8 20 10
F25 6.3 10 0.6 30 10
F26 6.3 10 0.8 30 10
F27 6.5 9 0.7 15 10
Table 1: Details of excipients used for various formulations
After every week, the samples from each formulation of Table 1 were subjected to size exclusion chromatography for determination of monomer, LMW and HMW content.
The details of the monomer, HMW and LMW content at the initial time point (after addition of citrate buffer and pH adjustment, before incubation) and up to four weeks of incubation are tabulated in Tables 2, 3 and 4, respectively.
Formulation ID T0 T1W T2W T4W
RI_CCD_DP_F01 99.2 98.9 97.1 98.7
RI_CCD_DP_F02 99.2 98.9 97.3 98.7
RI_CCD_DP_F03 99.1 98.8 97.2 98.6
RI_CCD_DP_F04 99.2 98.9 96.8 98.6
RI_CCD_DP_F05 99.2 98.9 96.9 98.5
RI_CCD_DP_F06 99.2 98.9 96.9 98.4
RI_CCD_DP_F07 99.2 96.6 94.8 92.4
RI_CCD_DP_F08 99.2 98.9 96.8 99.0
RI_CCD_DP_F09 99.2 98.9 97.3 98.6
RI_CCD_DP_F10 99.2 98.9 97.0 98.7
RI_CCD_DP_F11 99.1 98.9 97.1 96.4
RI_CCD_DP_F12 99.2 98.9 96.6 96.3
RI_CCD_DP_F13 99.2 96.7 94.5 91.9
RI_CCD_DP_F14 99.1 98.8 97.0 96.3
RI_CCD_DP_F15 99.1 98.7 96.7 96.5
RI_CCD_DP_F16 99.2 98.8 96.4 96.5
RI_CCD_DP_F17 99.2 98.9 97.0 96.9
RI_CCD_DP_F18 99.3 99.0 97.0 96.5
RI_CCD_DP_F19 99.1 98.8 96.8 96.5
RI_CCD_DP_F20 99.2 98.9 96.7 96.2
RI_CCD_DP_F21 99.1 98.8 96.7 96.1
RI_CCD_DP_F22 99.2 98.9 96.9 96.5
RI_CCD_DP_F23 99.1 98.7 96.7 96.3
RI_CCD_DP_F24 99.1 98.8 96.8 96.2
RI_CCD_DP_F25 99.2 96.7 94.7 92.1
RI_CCD_DP_F26 99.2 97.0 95.0 92.1
RI_CCD_DP_F27 99.2 98.9 97.2 96.6
Table 2: Monomer content at initial time point (T0), after 1 week (T1W), 2 weeks (T2W) and 4 weeks (T4W) of incubation at 40°C
Formulation ID T0 T1W T2W T4W
RI_CCD_DP_F01 0.8 0.9 0.9 0.9
RI_CCD_DP_F02 0.8 0.9 0.8 0.9
RI_CCD_DP_F03 0.9 1.0 1.0 1.0
RI_CCD_DP_F04 0.8 0.9 0.9 0.9
RI_CCD_DP_F05 0.8 0.9 0.9 1.0
RI_CCD_DP_F06 0.8 0.9 0.9 1.0
RI_CCD_DP_F07 0.8 0.9 1.0 0.9
RI_CCD_DP_F08 0.8 1.0 1.0 1.1
RI_CCD_DP_F09 0.8 1.0 0.8 0.9
RI_CCD_DP_F10 0.8 1.0 0.9 0.9
RI_CCD_DP_F11 0.9 1.0 1.0 1.0
RI_CCD_DP_F12 0.8 0.9 0.9 1.0
RI_CCD_DP_F13 0.8 1.0 0.9 1.1
RI_CCD_DP_F14 0.9 1.0 1.0 1.1
RI_CCD_DP_F15 0.9 1.1 1.0 1.1
RI_CCD_DP_F16 0.8 1.0 1.0 1.1
RI_CCD_DP_F17 0.8 1.0 0.9 1.0
RI_CCD_DP_F18 0.7 0.9 0.8 0.9
RI_CCD_DP_F19 0.9 1.0 0.9 1.0
RI_CCD_DP_F20 0.9 1.0 0.9 1.1
RI_CCD_DP_F21 0.9 1.0 1.0 1.1
RI_CCD_DP_F22 0.9 0.9 0.9 0.9
RI_CCD_DP_F23 0.9 1.1 1.0 1.0
RI_CCD_DP_F24 0.9 1.0 0.9 1.1
RI_CCD_DP_F25 0.8 1.0 1.0 1.0
RI_CCD_DP_F26 0.8 0.9 1.0 1.0
RI_CCD_DP_F27 0.9 1.0 0.8 0.9
Table 3: HMW content at initial time point (T0), after 1 week (T1W), 2 weeks (T2W) and 4 weeks (T4W) of incubation at 40°C

Formulation ID T0 T1W-ST T2W-ST T4W-ST
RI_CCD_DP_F01 ND 0.2 2.1 0.5
RI_CCD_DP_F02 ND 0.2 2.0 0.4
RI_CCD_DP_F03 ND 0.1 1.8 0.5
RI_CCD_DP_F04 ND 0.2 2.3 0.5
RI_CCD_DP_F05 ND 0.2 2.3 0.6
RI_CCD_DP_F06 ND 0.2 2.2 0.5
RI_CCD_DP_F07 ND 2.5 4.2 6.7
RI_CCD_DP_F08 ND 0.2 2.2 ND
RI_CCD_DP_F09 ND 0.2 1.9 0.5
RI_CCD_DP_F10 ND 0.2 2.1 0.5
RI_CCD_DP_F11 ND 0.2 1.9 2.6
RI_CCD_DP_F12 ND 0.2 2.5 2.7
RI_CCD_DP_F13 ND 2.4 4.6 7.0
RI_CCD_DP_F14 ND 0.2 2.0 2.6
RI_CCD_DP_F15 ND 0.2 2.3 2.4
RI_CCD_DP_F16 ND 0.2 2.6 2.4
RI_CCD_DP_F17 ND 0.2 2.2 2.2
RI_CCD_DP_F18 ND 0.2 2.2 2.6
RI_CCD_DP_F19 ND 0.2 2.3 2.5
RI_CCD_DP_F20 ND 0.2 2.4 2.7
RI_CCD_DP_F21 ND 0.2 2.4 2.9
RI_CCD_DP_F22 ND 0.2 2.2 2.6
RI_CCD_DP_F23 ND 0.2 2.3 2.7
RI_CCD_DP_F24 ND 0.2 2.3 2.8
RI_CCD_DP_F25 ND 2.3 4.4 6.9
RI_CCD_DP_F26 ND 2.1 4.0 6.9
RI_CCD_DP_F27 ND 0.2 2.0 2.5
Table 4: LMW content at initial time point (T0), after 1 week (T1W), 2 weeks (T2W) and 4 weeks (T4W) of incubation at 40°C
As can be seen from Tables 2, 3, and 4, there is no significant change in the HMW content after incubation for up to 4 weeks. However, for 4 formulations, namely F07, F13, F25 and F26, there is a significant change in both monomer and LMW content after incubation for up to 4 weeks. These samples were subjected to further investigation for the identification of the LMW species by SEC-HPLC, mass spectrometry (LC-MS) and capillary electrophoresis (CE-SDS).
SAMPLE
NAME Size –variant (%)
LC HC HL Sp-2 2H 2H1L Sp-1 Monomer Total
Impurity
F01_T4W 1.4 0.7 0.2 ND 1.7 2.7 4.6 88.7 11.3
F07_T4W 3.1 1.2 0.4 0.2 4.4 2.8 5.8 82.1 17.9
F10_T4W 1.3 0.7 0.2 ND 1.8 2.9 5.3 87.8 12.2
F13_T4W 3.2 1.3 0.3 0.1 4.8 2.2 7.9 80.2 19.8
F18_T4W 1.3 0.6 0.2 ND 2.1 2.4 5.0 88.3 11.6
F25_T4W 3.1 1.3 0.3 0.2 5.0 2.8 5.5 81.8 18.2
F26_T4W 3.3 1.3 0.3 0.1 4.6 2.7 4.6 83.1 16.9
Table 5: Details of impurities obtained from CE-SDS for samples incubated at 40°C for 4 weeks
Further, the relative potency (by bioassay) was determined pre-incubation and 2 weeks post-incubation at 40°C for all 27 samples and melting temperatures (by differential scanning calorimetry) were determined at 4 weeks post incubation at 40°C for 7 samples, the results of which are tabulated in Tables 6 and 7.
Formulation ID Relative potency
T0 T2W
RI_CCD_DP_F01 68 91
RI_CCD_DP_F02 78 89
RI_CCD_DP_F03 111 90
RI_CCD_DP_F04 96 86
RI_CCD_DP_F05 94 78
RI_CCD_DP_F06 96 79
RI_CCD_DP_F07 94 89
RI_CCD_DP_F08 101 82
RI_CCD_DP_F09 104 86
RI_CCD_DP_F10 93 87
RI_CCD_DP_F11 92 89
RI_CCD_DP_F12 102 88
RI_CCD_DP_F13 99 90
RI_CCD_DP_F14 96 89
RI_CCD_DP_F15 104 90
RI_CCD_DP_F16 102 84
RI_CCD_DP_F17 100 94
RI_CCD_DP_F18 98 87
RI_CCD_DP_F19 96 95
RI_CCD_DP_F20 98 80
RI_CCD_DP_F21 98 94
RI_CCD_DP_F22 101 94
RI_CCD_DP_F23 102 94
RI_CCD_DP_F24 97 87
RI_CCD_DP_F25 104 94
RI_CCD_DP_F26 100 89
RI_CCD_DP_F27 106 92
Table 2: Relative potency of various formulations pre-incubation and at 2 weeks after incubation at 40°C

Formulation ID Melting Temperature (Tm) in °C
Tm1 Tm2 Tm3
RI_CCD_DP_F01 69.5 74.0 83.5
RI_CCD_DP_F07 69.9 74.1 83.5
RI_CCD_DP_F10 70.5 74.1 83.6
RI_CCD_DP_F13 70.0 74.1 83.5
RI_CCD_DP_F18 70.2 74.1 83.6
RI_CCD_DP_F25 69.9 74.0 83.5
RI_CCD_DP_F26 69.4 74.0 83.6
Table 7: Melting temperature of various formulations after 4 weeks of incubation at 40°C

The results of Tables 6 and 7 demonstrate that the formulations F07, F13, F25 and F25, despite having a higher LMW content do not lose their potency after 2 weeks post incubation, as well as maintain their Tm 4 weeks post incubation, respectively.
Alternatively, in another experiment, the monoclonal antibody (Mab) of Example-1 is formulated in various concentrations of citrate buffer, with or without excipients, the excipients being sodium chloride and polysorbate-80. These samples were incubated at 25? or at 40? for one week, with and without agitation to see impact on selective reduction of the disulfide bonds between heavy chains and light chains. Post incubation, all samples were subjected to size exclusion chromatography (SEC) for the determination of monomer, high-molecular weight (HMW) and low-molecular weight (LMW) content. Details of the formulations along with SEC data has been given in Table 8.

Formulation ID along with composition incubation condition Monomer content at T1W HMW content at T1W LMW content at T1W
F-28-10 mg/ml Mab, 30 mM citrate buffer, at pH 6.3 At 40 ? for one week without agitation 96.9 1.6 1.5
F-29; 10 mg/ml Mab, 30 mM citrate buffer, at pH 6.3 At 25 ? for one week with agitation 97.4 1.3 1.3
F-30-10 mg/ml Mab, 30 mM citrate buffer, at pH 6.3 At 25? for 1 week, with agitation followed by incubation 40? for 1 week 96.2 2.1 1.8
F31-10 mg/ml Mab, 30 mM citrate buffer, 9 mg/ml NaCl, at pH 6.3 At 25? for 1 week, with agitation 96.9 1.7 1.3
F-32- 10 mg/ml Mab, 50 mM citrate buffer, 9 mg/ml NaCl, 0.07 mg/ml polysorabte-80 at pH 6.3 At 40? for 1 week, without agitation 97.0 1.7 1.4
F-33-10 mg/ml Mab, 50 mM citrate buffer, at pH 6.3 At 40? for 1 week, without agitation 97.0 1.7 1.3
F-34-10 mg/ml Mab, 50 mM citrate buffer, 0.07 mg/ml polysorbate-80, at pH 6.3 At 40? for 1 week, without agitation 97.0 1.7 1.4
F-35-10 mg/ml Mab, 50 mM citrate buffer, 9 mg/ml Sodium chloride, at pH 6.3 At 40? for 1 week, without agitation 97.0 1.7 1.3
Table 8: details of formulations along with SEC data ; T1W- data of formulation after one week of the respective incubation condition.
From the results/data of Table 8, it is evident that, citrate buffer is sufficient to reduce disulfide bonds between heavy chain and light chains of monoclonal antibody and there is no impact or influence of other excipients such as sodium chloride or polysorbate. Further, it has been observed that, there is no impact of agitation/mechanical stress on selective reduction of disulfide bonds between heavy chain and light chain of the antibody.
From the tables, LMW species represent the free light chains of the antibody, which result due to the selective reduction of the inter-chain disulfide bond between the light chain and the heavy chain.
Based on the data shown above, it can be concluded with confidence that the method is capable of selective reduction of the inter-chain disulfide bond between the light chain and the heavy chain of at least an immunoglobulin of the IgG1 subclass and without the loss of any potency.
,CLAIMS:We claim
1. A method for the selective reduction of the inter-chain disulfide bond between the light and heavy chain of an immunoglobulin, wherein the method comprises addition of citrate buffer to the immunoglobulin composition, at a specific pH, followed by incubating the composition at a specific temperature.
2. A method for the selective reduction of the inter-chain disulfide bond between the light and heavy chain of an immunoglobulin, the method comprising the steps of:
(a) obtaining a sample comprising an immunoglobulin composition and fragments thereof,
(b) adding to the sample an aqueous solution comprising citrate buffer at a specific concentration and adjusting the pH to a value within a specific range,
(c) incubating the composition of step (b) at a specific temperature for a specific period to obtain a composition comprising the immunoglobulin and free light chains of the immunoglobulin.
3. A method of preparing an antibody drug conjugate, comprising:
(a) obtaining a sample comprising an antibody composition,
(b) adding to the sample an aqueous solution comprising citrate buffer at a pH value of specific range,
(c) incubating the composition of step (b) at a specific temperature to obtain a composition comprising the antibody and free light chains of the antibody;
wherein the free light chains of the antibody generate free thiol groups,
and the free thiol groups obtained are available to react or conjugate with a drug or compound to obtain an antibody drug conjugate.
4. The method as claimed in claim 1 or claim 2, wherein the immunoglobulin belongs to IgG1, or IgG2, or IgG3, or IgG4 subclass.
5. The method as claimed in claim 1, claim 2 or claim 3, wherein the citrate buffer concentration is about 30 mM to about 50 mM.
6. The method as claimed in claim 1, claim 2 or claim 3, wherein the pH of the immunoglobulin or antibody solution is about 5 to about 7.
7. The method as claimed in claim 1, claim 2, or claim 3, wherein the specific pH of the immunoglobulin or antibody solution is at a pH ranging from 6 to 6.5.
8. The method as claimed in claim 1, claim 2 or claim 3, wherein the incubation temperature ranges from 25°C to 60°C.
9. The method as claimed in claim 1, claim 2 or claim 3, wherein the incubation period is 1 hour, or 1 hour to 1 week, or 2 weeks, or 3 weeks or 4 weeks.