DESC:FIELD OF INVENTION
The present invention relates to the field of analytical quantitation of proteins in a biotherapeutic preparation. More particularly, the invention relates to SE-UPLC (Size Exclusion-Ultra Performance Liquid Chromatography) based method for quantitation of various protein species in a biotherapeutic preparation.
BACKGROUND
Biotherapeutics are therapeutic products derived from recombinant DNA technology and include molecules ranging from simple peptides to complex multimeric proteins. While recombinant DNA technology has revolutionized the field of therapeutics, accompanying downstream processes that aid in attaining the acceptable levels of safety and efficacy of the final drug product are of utmost value as well. For example, in case of biotherapeutics that are active in dimeric or multimeric form, analytical methods have to be designed to detect and quantify monomers, which are in fact counted as ‘impurity’ in the drug preparation. Abatacept is an example of one such drug where the active, intact protein is a homodimer, wherein the two monomers are held together by covalent interactions.
Abatacept is a recombinant, homodimeric, soluble fusion protein of two identical subunits covalently linked by one disulphide bond. It acts as a co-stimulation modulator of the interaction of CD80 and CD86 on antigen presenting cells (APC) with CD28 on T-lymphocytes. Abatacept is an approved drug, for the treatment of Adult Rheumatoid Arthritis, Juvenile Idiopathic Arthritis and Psoriatic Arthritis in adults. The abatacept monomer subunit consists of extracellular domain of human cytotoxic T lymphocyte associated antigen 4 (CTLA-4) and the hinge CH2-CH3 region of the Fc domain of human IgG1, which has been modified to prevent complement fixation and antibody dependent cellular cytotoxicity (ADCC). An inter-chain disulphide bond between the monomer subunits in the CTLA-4 portion of the molecule creates a CTLA-4-IgG1 homodimer. This covalent homodimer is the intact active species in an abatacept drug preparation.
There are two predominant size variants reported in abatacept samples at a given stage of purification namely, monomer and high molecular weight (HMW) species. Polypeptide chains inter-linked by non-covalent interactions rather than disulphide bond form the HMW species. Thus, monomers and HMW species are impurities in an abatacept drug preparation.
The presence of any type of impurity in a therapeutic preparation is typically considered to be undesirable, due to the concern that the impurity may lead to an immunogenic reaction or may cause adverse events upon administration. As presence of impurities potentially impact drug performance (potency, pharmacokinetic, pharmacodynamics) and safety (immunogenicity, adverse effects), their levels in biopharmaceuticals has to be closely monitored as a critical quality attribute. Analytical quantification of impurities also aids in assessing the robustness of the purification process. Present invention relates to an analytical method to quantitate monomer impurities in abatacept drug preparations at any given time point of drug purification.
Monomeric, multimeric and HMW species in heterogeneous protein samples are usually quantified using liquid chromatographic methods, among which, size exclusion chromatography (SEC) is the most commonly employed method. A challenge most often faced while analyzing dynamically heterogeneous protein samples is the robustness of the method in clearly differentiating the peaks corresponding to the parameter(s) of interest. In the present invention, this specifically refers to the robustness of the analytical method to differentiate intact active protein (dimeric) from subunit protein (monomeric). In order to achieve the desired objective, several parameters need to be tailor-made as per the properties of the protein and dynamics in solution.
Thus, present invention describes a robust and effective method to differentiate intact active protein (dimeric) from subunit protein (monomeric) in abatacept drug preparation at any given stage of purification. The disclosed method facilitates rapid quantitation of monomer impurities at different stages of downstream protein processing, such as, neutralized protein-A eluate, in-process samples from downstream purification stages, drug substance (DS) and the final drug product (DP).
SUMMARY OF THE INVENTION
The present invention discloses an analytical method to differentiate and quantitate subunit (monomers) and intact (homodimers) proteins in a biotherapeutic preparation by size exclusion chromatography (SEC) using ultra performance liquid chromatography (UPLC). The method employs use of optimal flow rate based on the nature of the species in the sample in a time dependent manner during the process of chromatography. The method further provides rapid quantitation of undesired species with optimized flow rate and column conditions thereby reducing the time needed to analyze the sample.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1. Overlay of SE-UPLC chromatographic profiles of dimer and monomer (reduced) species in abatacept reference medicinal product (RMP) sample.
Figure 2. Representative SE-UPLC chromatographic profile of abatacept test sample showing peaks corresponding to monomer, dimer and “other species”. Monomer and dimer peaks are assigned based on retention time of system suitability sample.
DETAILED DESCRIPTION
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. Whereas the term “monomer” or “monomers” refers to the subunits of dimeric or multimeric proteins. Any methods and materials similar or equivalent to the scope of the disclosed invention described herein can be used in the practice of testing of the present invention.
Various embodiments of the disclosed invention provide a rapid and sensitive SE-UPLC (Size Exclusion-Ultra Performance Liquid Chromatography) based method for quantitation of monomer content in a biotherapeutic protein sample.
In an embodiment the claimed invention describes a rapid and sensitive SE-UPLC (Size Exclusion-Ultra Performance Liquid Chromatography) based chromatographic method to quantitate monomeric species from a heterogeneous biotherapeutic protein sample.
In another embodiment the heterogeneous composition comprises monomers, dimers and high molecular weight species.
In yet another embodiment monomers, high molecular weight species and aggregates are impurities.
In yet another embodiment the impurities are separated by varying the flow rate in size exclusion chromatography (SEC) column.
In yet another embodiment the flow rate of SEC column is increased gradually to achieve the desired flow rate.
In yet another embodiment the flow rate of SEC column is gradually increased by not more than 0.1 mL/min at a time.
In yet another embodiment the flow rate of SEC column is increased by about 0.35 to 0.5 mL/minute.
In one embodiment the column is cleaned after setting up using 0.5M Na2SO4 pH 2.5 ± 0.2 or 20% methanol with Milli Q water / filtered WFI before use.
In another embodiment the analysis of test sample is carried out by injecting the sample volume to maintain 10 µg on-column load.
In yet another embodiment the injection volume is 5 µL to 20 µL.
Definitions
“Biotherapeutics” or “drug” are interchangeably used in this document and refer to protein-based therapeutics derived from recombinant DNA technology. Some examples of such proteins are growth factors (e.g. insulin), monoclonal antibodies and fusion proteins (e.g. abatacept). “Biotherapeutic preparation” refers to the drug substance at a given stage of downstream processing or purification.
“Monomer” or “monomers” refers to subunits of dimeric or multimeric proteins. It can refer to monomeric CTLA-4-Fc fusion subunit of abatacept. Whereas “dimer” or “homodimer” refers to CTLA-4-Fc fusion subunits that are covalently bound together by disulfide bond(s).
The term “HMW” herein this invention refers to two or more polypeptide chains inter-linked by non-covalent interactions rather than disulfide bond.
“SE-UPLC” herein refers to Size Exclusion-Ultra Performance Liquid Chromatography.
“RMP” refers to reference medicinal product.
EXAMPLES
Example 1. Preparation of mobile phase
300 mL of LCMS grade or equivalent grade acetonitrile (ACN) was added to 700 mL of Milli Q water/filtered WFI and transferred to a dry and clean 1 liter Schott Duran bottle and mixed thoroughly. 2 mL of the mixture was removed and 1 mL each of trifluoroacetic acid and formic acid was added under elephant trunk exhaust system and immediately mixed thoroughly.
Example 2. Preparation of system suitability and test sample
A 1:3 (w/w) mix of non-reduced RMP and reduced and alkylated RMP was treated as system suitability standard.
For preparing the reduced and alkylated RMP standard, RMP was diluted to 1mg/ml in 1X PBS buffer in presence of 10 mM DTT and incubated at 37°C for 25 ± 5 min. Following DTT treatment, IAM was added to the reaction mixture such that final concentration is 10 mM and further incubated in the dark at RT for 40 min. The sample was immediately used for analysis. Non-reduced RMP sample was diluted to 1 mg/ml in 1X PBS buffer and mixed with reduced and alkylated RMP in a ratio of 1:3 (w/w). The mixture was transferred to UPLC sample vial and 10 µl of the mix was injected such that on-column load was 10 µg.
Example 3. Running of samples on chromatography column
Before loading of samples as prepared by Example 2 onto a suitable SEC column, the column was first equilibrated with respective buffers or mobile phase. For this, flow rate of mobile phase was first brought to about 0.2 mL/min, ensuring no air-bubbles are present in the column. Following this, flow rate was gradually increased to the required flow rate by not more than 0.1 mL/min at a time and it was ensured that the system pressure was stabilized.
For baseline check, WFI was injected into the column to ensure a stable baseline reading. As blank, 10 µL injection of DTT and IAM containing buffer was used.
To run the samples, 5-20 µL of mix of non-reduced RMP and reduced alkylated RMP at 1 mg/mL was injected into the column such that, the on-column load was about 10 µg. As intermediate checks on suitability of the chromatography system, reference standard was injected after every 10 samples. Reference standard was also injected at the end of a sample set to ensure there is no significant change in column performance during the run.
Example 4: Detection and analysis of data
Samples were analyzed using a photo diode array detector (Waters® 2998 Photodiode Array (PDA) detector) at 280 nm at a constant flow rate and isocratic gradient.
Peaks are assigned as ‘monomer’ or ‘dimer’ based on retention time of the system suitability standard (reduced alkylated RMP). As represented in Figure 1, the peak with lower intensity in the reduced alkylated RMP sample corresponds to the dimer and the one with the higher intensity corresponds to monomer species. As seen in Figure 2, any peaks (showing a protein spectrum with % area greater than 0.1) other than the aforementioned peaks, if present in test samples are integrated separately and reported as “other species”. The relative proportion of the dimer, monomer and other desired species present in the test sample(s) is captured as % total area of dimer, monomer and other species respectively.
Example 5. Acceptance criteria
The acceptance criteria for monomer and dimer peak separation are
a. retention time of the dimer peak and monomer peak for reduced-alkylated RMP should be at around 1.98 ± 0.02 minutes and 2.14 ± 0.02 minutes respectively.
b. the USP resolution between monomer and dimer peaks should be greater than 1.0.
,CLAIMS:CLAIMS
We claim,
1. A method for quantitation of variants in a biotherapeutic protein sample comprising:
a) pre-treating biotherapeutic protein sample;
b) loading the biotherapeutic protein sample onto size exclusion chromatography (SEC) column;
c) separating biotherapeutic protein sample using Waters® Ultra Performance Liquid Chromatography (UPLC); and
d) eluting the variants using isocratic gradient of mobile phase;
wherein the pre-treatment involves reduction and alkylation;
wherein the variants are monomeric species, dimeric species and high molecular weight (HMW) species of the protein; and
wherein the mobile phase comprises Trifluoroacetic Acid (TFA), Formic Acid (FA), and Acetonitrile (ACN).
2. The method as claimed in claim 1, wherein the pre-treatment comprises treating with 200mM DTT and 200mM IAM.
3. The method as claimed in claim 1, wherein the sample temperature is 5 ± 3°C.
4. The method as claimed in claim 1, wherein the SEC column is Protein BEH200 SEC (200Å, 1.7 µm, 4.6 mm ID × 150mm length).
5. The method as claimed in claim 1, wherein temperature of SEC column is between 25°C and 30ºC.
6. The method as claimed in claim 1, wherein the on-column load is 10µg.
7. The method as claimed in claim 1, wherein the flow rate of SEC column is increased gradually from 0.35 to 0.5 mL/minute.
8. The method as claimed in claim 1, wherein the flow rate of SEC column is preferably 0.46 mL/minute.
9. The method as claimed in claim 1, wherein the mobile phase comprises 0.1% TFA, 0.1% FA, and 30%ACN in filtered water for injection.
10. The method as claimed in claim 1, wherein the therapeutic protein is an anti CTLA4-Ig fusion protein.