DESC:FIELD OF INVENTION
The present invention relates to an analytical method for measuring protein and its subunits including structurally related variant entities in a therapeutic preparation using chromatography.
BACKGROUND OF INVENTION
Biotherapeutics are mostly protein-based therapeutically effective compositions derived through recombinant DNA technology. Monoclonal antibodies, fusion proteins, bispecific antibodies and antibody-drug conjugates (ADCs), among others, represent a rapidly growing class of biotherapeutics. The desired activity of a protein-based biotherapeutic preparation is determined by the specific tertiary structure of the protein. During drug development and production, some of these proteins can lose the tertiary structure, resulting in formation of undesired structurally variant entities such as, denatured protein, dissociated protein subunits or fragmented protein, among others. Monitoring and analysing these structurally variant entities is an essential aspect for ensuring the quality of the drug. Such analysis requires precise and fast methods that can determine the presence of intact proteins, key subunits, major modifications, and other impurities.
Existing literature teaches methods that help determine intact proteins and subunit proteins separately, among which, mass spectrometry is a commonly used method. For example, intact protein mass spectrometry can help elucidate whether the correct protein has been expressed with the expected amino acid sequence. It also offers insight into post-translational modifications by measuring the protein’s molecular weight accurately. Mass spectrometry with liquid chromatography (LC/MS) can provide information on relative abundances of different proteins, their isoforms, and impurities that are present in a sample. Protein analysis by LC/MS provides wide applications which includes measurement of intact protein and protein subunits including middle-up components such as light chain, Fab, Fd and Fc subunits.
: Reverse phase ultra-performance liquid chromatography
Reverse phase-Ultra-performance liquid chromatography (RP-UPLC) coupled with MS method has been widely used for characterizing monoclonal antibodies (mAbs) and fusion proteins. In reverse-phase chromatography, separation is based on relative hydrophobic differences of the proteins in solution. Due to its high resolving power and amenability to mass spectrometric (MS) detection, reverse phase chromatography is a frequently relied-upon methodology.
However, analysis of every type of the above-mentioned structural entities often requires a separate protocol or separate chromatography runs, thereby limiting the versatility of a given method in identifying different possible structurally variant entities in a single run. Thus, it is beneficial to have a robust protocol which employs a single chromatographic run that can enable separation and measurement of multiple structurally variant entities of the protein including intact protein and subunits (heavy chain, light chain, Fab, Fd and Fc) to save time, effort, sample size and cost.
SUMMARY OF THE INVENTION
Accordingly, present invention discloses a versatile analytical method to measure protein, its subunits and related structurally variant entities in any biotherapeutic preparation with high specificity in a single chromatographic run. Particularly, the method uses a multi-attribute approach to characterize the protein, to understand the sequence, identify post translational modifications, impurities and assign other critical quality attributes.
The disclosed method is versatile, and robust and can be used as a platform method to qualitatively and quantitatively determine related structurally variant entities of a protein in a biotherapeutic preparation in a single chromatography run with spectral clarity for mass spectrometry based deconvolution. The peak picking, integration and assignment of these entities through a defined informatics workflow provides seamless and high throughput process. The disclosed method uses a combination of polyphenyl based separation chemistries with optimized gradients by blending aqueous and organic solvents with suitable ion pairing agents along with defined data processing method to identify, screen and monitor the intact and subunit proteins. This harmonized and abridged multi-attribute approach provides protein-level monitoring to authenticate consistency of protein mass measurements cascading from intact to subunit levels.
The method can be employed for analysis of a biotherapeutic protein such as, but not limited to monoclonal antibodies, fusion protein, bispecific antibodies or antibody-drug conjugates (ADCs) and can be helpful to understand multiple attributes such as the sequence, post translational modifications, impurities and other critical quality attributes. Also, heterogeneous or partially purified samples generated during early stage of development as well as end to end purified drug substance (DS) and drug product (DP) can be tested using this approach. The multi-attribute approach is harnessed for better protein component resolution, recovery, low injection to injection carryover, cost reduction and shorter acquisition/ analysis time. Thus, disclosed method is helpful in monitoring multiple product quality attributes upon single sample analysis compared to conventional characterization methods, thus reducing the development time span, reducing cost and increasing the quality of information obtained during drug development process
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1a: Separation of proteins using 1% Difluroacetic acid (DFA) as solvent in mobile phase
Figure 1b: Separation of proteins using 1% Trifluroacetic acid (TFA) as solvent in mobile phase
Figure 2: Separation of proteins using 1% Formic acid (FA) as solvent in mobile phase with 4 trials
Figure 3: Electrospray spectrum comparing Formic acid (FA), Trifluroacetic acid (TFA) and Formic acid +Trifluroacetic acid (FA+ TFA) used as solvent system

Figure 4: Intact protein, subunit protein and middle-up protein LC-MS analysis (UV chromatogram)
Figure 5: Intact protein, subunit protein and middle-up protein LC-MS analysis (Deconvoluted)
Figure 6: Comparison of polyphenol column with C4, C8, Phenyl and SEC columns for separation of intact, subunit and middle-up protein
DETAILED DESCRIPTION OF THE INVENTION
Separation and measurement of structurally variant entities of a protein is an important analytical aspect in drug development as such variants can elicit undesired immunogenic responses in the body. Measuring molecular mass of such structurally variant entities (arising as a by-product of development) that may be present as intact protein, subunit protein, middle-up protein, denatured protein, etc. often requires extensive methodology for each type of entity, thereby being costly and time-consuming. Disclosed invention describes a robust and effective platform method to separate and measure structurally variant entities of a protein including the protein and its subunits in a single chromatographic run, thereby eliminating the need of extensive methodology for such characterization.
The present invention discloses a multi-attribute approach employing RP-UPLC which uses a polyphenyl column to separate and measure the molecular mass of protein, its subunits and related structurally variant entities. Due to larger ligand surface for protein contact and the potential for pi-pi interactions, the polyphenyl column can resolve the abovementioned variants more effectively. The polyphenyl column has solid core particle which reduces the diffusion distances, facilitating higher throughput separation with enhanced selectivity. The multi-attribute approach further utilizes gradient mode of elution for the separation of proteins which is optimized by blending water and ACN with formic acid. The method is used for the characterization of biotherapeutics to understand its sequence, identify post translational modifications, impurities, and other critical quality attributes. This approach monitors the different product quality attributes using a single sample analysis run compared to conventional characterization methods, thus reducing the development time span and concurrently increasing the quantity of information processed.
More particularly, the claimed invention describes a rapid and sensitive chromatographic method for separation and measurement of structurally variant entities of a protein in a biotherapeutic composition, including but not limited to intact protein, subunit protein, middle-up protein components, denatured protein, wherein, the method involves separation and measurement of these entities in a single chromatographic run with spectral clarity for mass spectrometry based deconvolution.
In an embodiment the claimed invention discloses a rapid and sensitive chromatographic method for separation and analysis of a protein, its subunits and related structurally variant entities in a sample of a biotherapeutic preparation, wherein the method comprises:
a) preparing the samples for analysis;
b) loading the sample on a polyphenyl column;
c) separating the entities using Reverse Phase Ultra-Performance liquid Chromatography (RP-UPLC);
d) eluting the entities by gradient elution using mobile phase A, B and C; and
e) analyzing the entities using Electro Spray Ionisation-Mass Spectrometry (ESI-MS);
wherein, the structurally variant entities comprise intact protein, subunit protein, and middle-up components; and
wherein, the mobile phase A comprises water for injection (WFI), mobile phase B comprises 100% Acetonitrile and mobile phase C comprises 1.0 % formic acid in WFI.
In an embodiment, the sample preparation comprises treating the sample with one or more of di-sulfide bond reduction, peptide:N-glycosidase F (PNGaseF) treatment and Carboxypeptidase B (CpB) treatment;
In another embodiment, the middle-up components in case of antibody sample comprises light chain, Fab, Fc and Fd regions.
In yet another embodiment the protein present in a sample of the biotherapeutic preparation is a monoclonal antibody, a fusion protein, a bispecific antibody or an antibody-drug conjugate (ADC).
In another embodiment the injection volume of the sample is 1uL.
In another embodiment the temperature of the polyphenyl column is about 50? to about 90?.
In another embodiment the temperature of the polyphenyl column is preferably about 80?.
In yet another embodiment the flow rate of the sample in the chromatographic run is about 0.2mL/min.
Definitions
The term "biotherapeutic" used herein refers to 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, multi-specific antibodies and antibody fragments so long as they exhibit the desired biological activity.
The term “Fab” used herein refers to antigen-binding fragment and is a region on an antibody that binds to antigens.
The term “Fc” used herein refers to fragment crystallizable region which is the tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system.
The term “Fd” used herein refers to Fd region of the heavy chain of the Fab, i.e. approximately the first 220 amino acids from the N-terminus of the heavy chain comprised of the VH and CH1 regions.
The term “intact protein” used herein refers to a protein in its active and desired structure, whether it may be monoclonal antibody, fusion protein, bispecific antibody or antibody-drug conjugate.
The term "middle-up components" as used herein refers to protein subunits resulting from enzymatic digestion and/or reduction of the protein. This can be achieved by using structure-specific enzymes that are selective for only one or a handful of cleavage sites in the protein. For example, for studies of mAbs, IdeS and GingisKHAN allow cleavage in the lower and upper hinge region, respectively. Additionally, disulfide reduction can be employed to yield a limited number of relatively large fragments (either by itself or in combination with limited/restricted proteolysis, depending on the protein that is analyzed). "Middle-up components" in case of antibody sample include light chain, Fab, Fc and Fd regions, inclusive of both reduced and non-reduced conditions.
The term “platform method” used herein refers to the fact that the method can be employed for analysis of a larger sub-set of biotherapeutic preparations, rather than that limited to one molecule-type that comprises monoclonal antibodies, fusion proteins, bispecific antibodies or antibody-drug conjugates (ADCs). In addition, heterogeneous or partially purified samples generated during early stage of development as well as end to end purified drug substance (DS) and drug product (DP) can also be tested.
The term “RP-UPLC” used herein refers to Reversed Phase-Ultra Performance Liquid Chromatography, which is a chromatographic method used for separation and measurement of molecular mass of protein wherein the method employs a hydrophobic stationary phase.
The term “subunit protein” used herein refers to individual parts of the intact protein which result after enzyme treatment or reduction. For example, subunits of an immunoglobulin structure are heavy chain, light chain, Fab, Fc and Fd forms.
The term “structurally variant entities” used herein refers to the intact protein and its variants including but not limited to, subunits, heavy chain, light chain, Fab region, Fd region and Fc region.
Abbreviations
CpB : Carboxypeptidase B
DFA : Difluroacetic acid
FA : Formic acid
IP : Intact protein
IP+CpB : Intact protein treated with carboxypeptidase B
IPPF : Intact protein deglycosylated with PNGaseF
IPPF+CpB : Intact protein deglycosylated with PNGaseF and treated with CpB
LC-MS : Liquid chromatography mass spectrometry
MUNR : Middle up non reduced protein
MUR : Middle up reduced protein
PNGase : Peptide:N-glycosidase F
RP-UPLC : Reverse phase ultra-performance liquid chromatography
SP : Subunit protein
SP+CpB : Subunit protein treated with carboxypeptidaseB
SPPF : Subunit protein deglycosylated with PNGaseF
SPPF+CpB : Subunit protein glycosylated with PNGaseF and treated with CpB
TFA : Trifluroacetic acid
WFI : Water for injection

EXAMPLES
Example 1: Sample preparation
A. Intact protein and its variants
a. Intact protein (IP) mass sample was prepared by diluting biotherapeutic sample with WFI to a final concentration of 1 mg/mL.
b. CpB treated intact protein (IP+CpB) sample was prepared by mixing 1mg/mL IP sample with CpB (1 mg/mL) and incubated at 37°C.
c. Deglycosylated intact protein (IPPF) sample was prepared by mixing 1mg/mL IP sample with 20 µl of 5X Tris buffer, 2 µl of N-glycanase enzyme (PNGaseF) and incubated at 37°C for 4h or upto overnight.
d. Deglycosylated and Cp-B treated intact protein (IPPF+CpB): the IP sample was mixed with 20 µl of 5X Tris buffer, 2 µl of N-glycanase enzyme and incubated at 37°C for 4h up to overnight and then this deglycosylated protein sample was mixed and incubated with CpB (1 mg/mL) at 37°C for 2 h.
B. Subunit protein and its variants sample preparation
a. Subunit protein sample (SP): To reduce the disulfide bonds, 1 µL of 0.5 M DTT was added to 100 µg of biotherapeutic protein (1 mg/mL), mixed gently and incubated at 37°C for 30 minutes. After reduction, the solution was incubated at room temperature.
b. CpB treated SP sample (SP+CpB): 100 µg of biotherapeutic was mixed with 5.76 µL of CpB (1 mg/ml) and up the volume was adjusted with WFI to get final concentration of biotherapeutic 100µg/100mL (~1 mg\mL) and incubated at 37°C for 2 h. 1 µL of 0.5 M DTT was added to this and the solution was mixed gently. The solution was incubated at 37°C for 30 minutes for reduction.
c. Deglycosylated SP sample (SPPF): To 100 µg of biotherapeutic, 20 µL of 5X Reaction buffer, 2 µL of PNGase F enzyme was added and the volume was adjusted with WFI to get final concentration of biotherapeutic ( ~1 mg/mL), mixed and incubated at 37°C for 4 h (or incubated overnight). 1 µL of 0.5 M DTT (5 mM final conc.; 5-10 mM DTT) was added to this and the solution was mixed gently. The solution was incubated at 37°C for 30 minutes for reduction.
d. Deglycosylated and CpB treated SP sample: To 100 µg of biotherapeutic, 20 µL of 5X reaction buffer, 2 µL of PNGase F enzyme was added and incubated at 37 °C for 4 h. Then, 5.76 µL of CP-B (5 mg/ml) was mixed with 100 µg of the deglycosylated biotherapeutic (100 µL) and the volume was adjusted with WFI to get final concentration of biotherapeutic ( ~1 mg/mL) and incubated at 37°C for 2 h. 1 µL of 0.5 M DTT was added to this and the solution was mixed gently. The solution was incubated at 37°C for 30 minutes for reduction.
C. Middle-up protein (partially proteolyzed protein subunits) sample preparation
a. Middle-up non reduced protein sample (MUNR): The biotherapeutic protein sample was treated with IdeS/FabRICATOR at 1:1 protein to enzyme ratio and incubated at 37°C for 1 h.
b. Middle-up reduced protein sample (MUR): The biotherapeutic protein sample was treated with IdeS/FabRICATOR at 1:1 protein to enzyme ratio and incubated at 37°C for 1 h. Then the solution is reduced to break the inter chain disulfide linkages using DTT (final concentration of 10 mM) and incubated for 10-30 minutes at 37 °C.
Example 2: RP- UPLC method and polyphenyl column parameters
Waters Acquity UPLC H-Class bio instrument was used for ultra-performance liquid chromatography. Flow rate of the sample into chromatography column was adjusted to 0.2 mL/min which can be changed with the gradient. The column used in UPLC is Water Bioresolve reverse phase mAB polyphenyl column with 2.1 mm ? 50 mm dimensions, 2.7µm particle size and 450Å pore size. This polyphenyl bonding was both high in coverage (upto 6 µmol phenyl moiety/m2) and comprised of rigidly constrained carbons. The column temperature used for chromatography was 80°C. The sample was injected with 1 µL volume and detected at 214 nm and 280 nm wavelength.
Example 3: Optimization of buffer system for gradient elution in polyphenyl column
Gradient mode of elution has been used for RP-UPLC. Formic acid (FA), Difluroacetic acid (DFA) and Trifluroacetic acid (TFA) have been used for method optimization.
1. Trial with Difluroacetic acid (DFA)
For trial with DFA as a solvent, mobile phase A was prepared using 1%DFA in WFI and mobile phase B using 1%DFA in ACN. The gradient used for DFA is shown in Table 1 and the chromatogram is shown in Figure 1a. Although separation of protein component was observed using DFA, a smear of the protein sample was also observed. As a result, use of DFA was not pursued further.

Table 1: Gradient using 1% DFA as a solvent in mobile phase.
2. Trial with Tifluroacetic acid (TFA)
For trial with TFA as a solvent, mobile phase A was WFI, mobile phase B was 100% ACN and mobile phase C was prepared adding 1% TFA in WFI. The gradient used for TFA is detailed in Table 2 and the chromatogram is shown in Figure 1b.

Table 2: Gradient using 1%TFA as a solvent in mobile phase.
3. Trial with Formic acid (FA) as a solvent in mobile phase
For trial with FA as a solvent, mobile phase A was WFI, mobile phase B was 100% ACN and mobile phase C was prepared adding 1% FA in WFI. The gradient optimized using FA and above mentioned mobile phases is shown in Table 3 and chromatogram with 3 trials is in Figure 2. Upon different trials and comparing the UV chromatogram and the electrospray spectrum (Figure 4) with FA, TFA and FA+ TFA, formic acid (FA) was selected as the solvent system.
Time(min) Flow Rate (ml/min) Composition A (%) Composition B (%) Composition C (%) Composition D (%) Curve
0.00 0.20 70.00 20.00 0.00 10.00 Initial
0.50 0.20 70.00 20.00 0.00 10.00 6.00
0.51 0.20 65.00 25.00 0.00 10.00 6.00
3.61 0.20 40.00 50.00 0.00 10.00 6.00
4.00 0.50 0.00 90.00 0.00 10.00 6.00
4.10 0.50 85.00 5.00 0.00 10.00 6.00
4.60 0.50 0.00 90.00 0.00 10.00 6.00
4.70 0.50 85.00 5.00 0.00 10.00 6.00
5.20 0.50 0.00 90.00 0.00 10.00 6.00
5.30 0.50 70.00 20.00 0.00 10.00 6.00
6.00 0.50 70.00 20.00 0.00 10.00 6.00
Table 3: Gradient using 1% FA as a solvent in mobile phase.
Example 5: ESI QToF MS parameter for intact, subunit and Middle-up protein mass analysis
Mass spectrometry used Xevo G2 XS MS QTOF instrument. The parameters used for molecular mass analysis of intact, subunit and middle-up protein are mentioned below in Table 4.
MS method parameters Intact protein Subunit protein Middle up protein
Optimal value Range Optimal value Range Optimal value Range
Capillary voltage (kV) 3 2.0-3.0 2 2.0-3.0 2 2.0-3.0
Collision energy ramp (V) NA NA NA NA NA NA
Cone voltage (V) 150 80-150 60 40-80 60 40-80
Desolvation gas flow (L/h) 600 200-1000 600 200-1000 600 200-1000
Desolvation temperature (°C) 300 100-600 300 100-600 300 100-600
Mass range 500-3995 500-4500 500-3000 500-3995 500-3000 500-3995
Resolution 10000 =< 8000 10000 =< 8000 10000 =< 8000
Source offset (V) 30 0-100 30 0-100 30 0-100
Source temperature (°C) 120 80-200 120 80-200 120 80-200
Table 4: MS method parameters for intact, subunit and Middle-up protein MS applications
,CLAIMS:CLAIMS:
We claim:
1. A method for separation and analysis of a protein, its subunits and related structurally variant entities in a sample of biotherapeutic preparation, wherein the method comprises:
f) preparing the samples for analysis;
g) loading the sample on a polyphenyl column;
h) separating the entities using Reverse Phase Ultra-Performance Liquid Chromatography (RP-UPLC) at a given flow rate;
i) eluting the entities by gradient elution using mobile phase A, B and C; and
j) analyzing the entities using Electro Spray Ionisation-Mass Spectrometry (ESI-MS);
wherein, the structurally variant entities comprise intact protein, subunit protein, and middle-up components; and
wherein, the mobile phase A comprises water for injection (WFI), mobile phase B comprises 100% Acetonitrile and mobile phase C comprises 1.0 % formic acid in WFI.
2. The method as claimed in claim 1 wherein, preparing the sample comprises treating the sample with one or more of di-sulfide bond reducing agent, peptide:N-glycosidase F (PNGaseF) and Carboxypeptidase B (CpB).
3. The method as claimed in claim 1 wherein, the middle-up components comprises immunoglobulin light chain, Fab, Fc and Fd regions.
4. The method as claimed in claim 1 wherein, the protein present in the sample is a monoclonal antibody, a fusion protein, a bispecific antibody or an antibody-drug conjugate (ADC).
5. The method as claimed in claim 1 wherein, the loading volume of sample is 1uL.
6. The method as claimed in claim 1 wherein, the temperature of the polyphenyl column is from about 50? to about 90?.
7. The method as claimed in claim 1 wherein, the temperature of the polyphenyl column is preferably about 80?.
8. The method as claimed in claim 1 wherein, flow rate of the sample in the chromatographic run is about 0.2mL/min.