DESC:FIELD OF THE INVENTION
The present invention relates to a method for estimation of reversible aggregate content in a protein sample wherein the protein sample is subjected to pretreatment prior to subjecting the sample to size exclusion chromatography.
BACKGROUND OF THE INVENTION 5 Protein based therapeutics, in particular, monoclonal antibodies are highly successful for treatment of various diseases including oncological disorders. Proteins/antibodies expressed by recombinant DNA methods are typically associated with impurities such as host cell proteins (HCP), host cell DNA (HCD), endotoxins, variants, viruses etc., including aggregates. 10 Formation of protein aggregates is a common phenomenon which can be encountered during various stages of a commercial therapeutic antibody manufacturing processes such as fermentation, purification, formulation and during storage of the protein drug substance or final protein product. Protein aggregation can occur through different mechanisms. These mechanisms 15 are not mutually exclusive, and more than one mechanism can influence the same product. Such mechanisms include reversible association of native protein monomers, conformationally altered monomer and nucleation controlled aggregation. The protein aggregates thus formed can be due to covalent or non-covalent self-association. Non-covalent self-association of proteins include 20 reversible aggregates such as dimers/ oligomers and sometimes these reversible aggregates lead to the formation of irreversible aggregates. The reversibility of protein aggregation is the ability of the aggregates to dissociate into monomers in equilibrium conditions induced by pH, temperature, ionic strength and concentration of protein. 25
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Reversible aggregates are non-covalent in nature and revert to native monomer. The equilibrium of this reaction can be effectively altered by controlling any one of the aforementioned parameters. Altering the temperature of a protein in solution would alter vibrational motion and diffusion of protein molecules, which is a necessary step for physical protein 5 aggregation. Similarly pH of the solution affect protein folding and intermolecular protein-protein interactions due to the distribution of surface charges that will lead to formation of varied aggregate content. Increasing concentration of a protein in a solution could potentially result in the varying consequences such as decreased aggregation due to crowding effect, 10 increased aggregation due to increased chance of association, and precipitation due to solubility limit. Bevacizumab (rhuMAb VEGF) is a humanized anti-vascular endothelial growth factor (VEGF) antibody used for treatment of various diseases including glioma, renal cancer and lung cancer. The unique characteristic of rhuMAb VEGF (Ig G1 15 antibody) is formation of reversible aggregates. It exists predominantly as monomer at pH 5.5 but as the pH, and temperature are increased it forms non covalent, reversible aggregates. The formation of aggregates is predominantly observed to be concentration dependent and hence dilution leads to disruption of these reversible higher order forms. 20 Aggregates ranging from dimer to visible particles that are hundreds of micrometers in size have been recognized for their potential to elicit immune responses to therapeutic protein products. Hence, it is one of the recommendations from regulatory agency to measure possible types of aggregates present in a protein sample that includes reversible aggregates/dimers. 25 As discussed above, various factors such as pH, temperature and concentration of the protein directly influences the equilibrium between monomer and reversible
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aggregates and lead to varying amount of reversible aggregate content present in the sample. Additionally, it is also observed that amount of reversible aggregate in protein sample varies with time when stored at a temperature between 2 -8°C. Hence, it is essential to develop a robust method for estimation of reversible aggregate content in a protein where influential parameters such as pH, temperature 5 and concentration is optimized in such a way that it does not vary the amount of aggregate content present in a sample over a period of time. Further, the developed conditions for the said method should mimic physiological conditions of a patient administered with said protein. The primary objective of the invention is to provide, a sample treatment method 10 for estimation of reversible aggregate content present in bevacizumab protein sample prior to subjecting it to a chromatographic technique, wherein the said chromatographic technique is a size-exclusion chromatography. Further objective of the invention is to provide, a method for optimizing conditions such as pH and temperature prior to subjecting a sample to size exclusion 15 chromatography wherein said optimized conditions help in estimation of reversible aggregate content present in a protein sample containing bevacizumab.
SUMMARY OF THE INVENTION
The present invention discloses a method for estimation of reversible aggregate content present in bevacizumab, wherein the said sample is incubated for a period 20 of time under optimal conditions of pH, temperature and concentration prior to subjecting to a chromatographic technique, where in the said chromatographic technique is size exclusion chromatography.
Further, the invention discloses optimized conditions for treatment of a protein sample comprising bevacizumab for estimation of basal reversible aggregate 25 content that does not vary over a period of time for the same sample under identical conditions.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1a: Illustrates a chromatographic profile of unprocessed (neat) bevacizumab sample in size exclusion chromatography.
Figure 1b: Illustrates a chromatographic profile of processed bevacizumab sample in size exclusion chromatography. 5
DETAILED DESCRIPTION OF THE INVENTION
Various factors which include pH, concentration and temperature govern the type and degree of protein aggregation. Aggregates include reversible and irreversible aggregates. Reversible aggregates are non-covalent in nature and get converted to native form of monomer up on dilution. 10
Dimerization is not a common phenomenon for genetically engineered Ig G1 type of antibodies. The unique characteristic of bevacizumab is formation of reversible aggregate content compared to other Ig G1 antibodies.
Estimation of reversible aggregate content is influenced by various parameters such as protein concentration, pH and temperature. 15
Hence, there is a need to provide an optimized condition for sample treatment method for estimation of reversible aggregates in bevacizumab, wherein the said method does not result in varying amount of reversible aggregate for a period of time. Hence, the said method can be used to report reversible aggregate content present in a protein sample. 20
The disclosed method estimates both total aggregate content present in unprocessed/neat sample and irreversible aggregate content in a processed sample.
In one embodiment, the said neat sample is processed in such optimum condition, wherein the equilibrium between monomers and aggregates are shifted such that it facilitates conversion of reversible aggregates to monomers. 25
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Further, the disclosed method estimates amount of reversible aggregate content present in a sample by estimating the irreversible aggregate content and the total aggregate content in the said sample.
In one embodiment the invention discloses a method for estimation of reversible aggregate content present in a protein sample comprising 5
a) measuring amount of total aggregate content present in the said unprocessed protein sample by subjecting it to size exclusion chromatography
b) measuring amount of irreversible aggregate content present in a processed protein sample by subjecting it to size-exclusion chromatography and 10
c) estimating reversible aggregate content present in the said protein sample by subtracting irreversible aggregate content from the total aggregate content.
In another embodiment of the invention, protein is a therapeutic protein molecules such as a monoclonal antibody. 15
In another embodiment of the invention, the antibody is a humanized antibody or more particularly is recombinant human VEGF antibody.
In yet another embodiment of the invention, unprocessed protein sample is a protein sample which is not subjected to any pretreatment conditions.
In another embodiment of the invention discloses, a method of processing said 20 sample comprises
a) diluting the said protein sample
b) incubating the sample obtained from step a) at a predetermined temperature for a predetermined period of time
In another embodiment of the invention, protein sample is diluted to required fold 25 such as 10 fold, 20 fold, 30 fold, 40 fold and 50 fold.
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In yet another embodiment of the invention, the diluted antibody sample is incubated at a temperature about 25 °C to 30 °C.
In yet another embodiment of the invention, the diluted antibody sample is incubated at said temperature for at least 4 hours.
In another embodiment of the invention, a method for estimation of reversible 5 aggregate content in a sample comprising anti-VEGF antibody, wherein the said comprises
a) Measuring amount of total aggregate content present in the said unprocessed containing anti-VEGF antibody by subjecting it to size exclusion chromatography 10
b) Processing the sample containing anti-VEGF antibody by diluting and incubating at 30°C for atleast 4 hours
c) Measuring amount of irreversible aggregate content present in said processed protein sample obtained in step (b) containing anti-VEGF antibody by subjecting it to size-exclusion chromatography 15
d) Estimation of reversible aggregate content present in the said anti-VEGF antibody by subtracting irreversible aggregate content from the total aggregate content.
The sample treatment method disclosed in the invention, is an optimized method to estimate basal reversible aggregate content and does not exhibit variability when 20 processed over time. Additionally the optimized conditions of dilution and temperature of the said method closely resemble physiological conditions during therapeutic administration of bevacizumab.
Definitions:
The term “dilution” as used herein for the preparation of processed sample is 25 defined as a process of reducing the concentration of a solute in solution, usually simply by mixing with more solvent. Further, dilution is a necessary step in order
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to bring the sample concentration to a level which allows movement of equilibrium between reversible aggregates and monomer towards right side or in other terms which will facilitate conversion of reversible aggregates to monomers.
Further the solvent used for dilution is but not limited to formulation buffer of respective protein or phosphate buffer, and 0.9% saline solution. Any other buffer 5 which is compatible with the protein does not impact physical property of the protein.
In addition, the fold of dilution of the protein sample to prepare a processed protein sample depends on nature of the protein as well as aggregate content present in the sample. 10
The term “reversible aggregates” as used herein include dimers and oligomers, and is restricted to aggregates that exist in equilibrium with the native monomeric subunit under specific solution conditions, where the disassociation of protein aggregates may be observed on the experimental timescale simply by returning to the original solution condition. Reversible protein aggregation typically results 15 from relatively weak non-covalent protein interactions and can be described thermodynamically which may get converted to monomers under favorable conditions such as pH, temperature and dilution.
The term “irreversible aggregates” as used herein are higher-molecular-weight species, which when formed cannot be dissociated short of the addition of 20 denaturants or reducing agents.
Certain specific aspects and embodiments of the invention are more fully described by reference to the following examples. However, these examples should not be construed as limiting the scope of the invention in any manner.
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Example 1: Sample preparation
Two set of samples (Processed and unprocessed) were prepared for the estimation of reversible dimer content present in a rhuMAB VEGF antibody.
Processed sample was prepared by diluting rhuMAB VEGF antibody (25 mg/mL) in a suitable buffer to 10 folds. The diluted samples were incubated at 30°C for 4 5 hours to move the equilibrium between aggregate content and monomer towards right side for the conversion of reversible aggregate content to monomer. Whereas unprocessed sample was obtained without any treatment (i.e; rhuMAB VEGF antibody (25 mg/mL) without subjecting to dilution and incubation at a particular temperature). Further, the unprocessed sample was prepared before subjecting it to 10 size exclusion chromatography (SEC).
Sample Analysis by Size exclusion chromatography Both the samples unprocessed (which was prepared just before subjecting to SEC) and processed sample (after the 4 hrs of incubation) were subjected to SEC under isocratic conditions. Sample temperature was brought back to lower temperature 15 for example 10 °C but not limited to the said temperature. Amount of total aggregate content in unprocessed sample was measured at 280 nm whereas amount of irreversible aggregate content was measured at 214 nm using UV detector. (Representative chromatograms were given in figure 1a and figure 1b erspectively). 20 Reversible aggregate content present in rhuMab VEGF antibody was estimated by subtracting irreversible aggregate content from total aggregate content present in the sample and estimated reversible aggregate content was given in Table 1. The said content was calculated from the respective chromatogram.
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Sample Name In % Area Under the Curve (AUC) Monomer content in Total aggregate content Irreversible aggregate content Reversible aggregate content
Processed Sample
93.32
NA
5.65
1.6
Unprocessed Sample
91.47
7.25
NA
NA
Table 1: Represents amount of monomer, total aggregate and irreversible aggregate content of un-processed and processed bevacizumab samples.
From the above results, it is evident that there was an increase in monomer content for processed sample which indicated that said sample comprises reversible aggregate content. Further, optimized conditions of sample processing which 5 reflects therapeutic administration of bevacizumab has given the actual monomer content at the time of administration.
In addition, amount of monomer content in unprocessed sample was observed less as compared to processed sample.
Further, the amount of reversible dimer content was calculated by subtracting 10 irreversible aggregate content from irreversible aggregate content. ,CLAIMS:We claim:
1. A method for estimating reversible aggregate content present in a protein sample comprising
a) measuring an amount of total aggregate content present in the said 5 unprocessed protein sample by subjecting it to size exclusion chromatography
b) measuring an amount of irreversible aggregate content present in a processed protein sample by subjecting it to size-exclusion chromatography and 10
c) estimating reversible aggregate content present in the said protein sample by subtracting irreversible aggregate content from the total aggregate content.
2. The method according to claim 1 wherein, unprocessed protein sample is a protein sample which is not subjected to any pretreatment conditions. 15
3. The method according to claim 1 comprising
a) diluting the said protein sample
b) incubating the sample obtained from step a) at a predetermined temperature for a predetermined period of time.
4. The method according to claim 3 wherein, the protein sample is diluted to required 20 fold such as 10 fold, 20 fold, 30 fold, 40 fold or 50 fold.
5. The method according to claim 4 wherein, the diluted antibody sample is incubated at a temperature about 25 °C to 30 °C.
6. The method according to claim 5 wherein, the diluted antibody sample is incubated at said temperature for at least 4 hours. 25
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7. The method according to claim 1 wherein, the protein is a therapeutic protein molecule such as a monoclonal antibody.
8. The method according to claim 7 wherein, the antibody is a humanized antibody or more particularly is recombinant human anti-VEGF monoclonal antibody.
9. A method for estimating reversible aggregate content in a sample comprising 5 anti-VEGF antibody, wherein the said method comprises
a) measuring amount of total aggregate content present in the said unprocessed sample containing anti-VEGF antibody by subjecting it to size exclusion chromatography
b) processing the sample containing anti-VEGF antibody by diluting it 10 fold 10 and incubating at 30°C for atleast 4 hours
c) measuring amount of irreversible aggregate content present in said processed protein sample obtained in step (b) by subjecting it to size-exclusion chromatography
d) estimating reversible aggregate content present in the said sample 15 comprising anti-VEGF antibody by subtracting irreversible aggregate content as measured in step (c) from the total aggregate content as measured in step (a).