Method for determining protein or peptide concentration and use thereof

Abstract

The present invention relates to methods for determining protein and / or peptide concentrations or molecular parameters (such as extinction coefficients), and uses thereof.

Description

[0001] The present invention relates to methods for determining the concentration and / or molecule-specific parameters (such as extinction coefficient) of proteins or peptides and uses thereof.

[0002] The concentration of a peptide or protein (e.g., antibody) in solution is an important property in the development and commercialization of biotherapeutics, as well as in many other areas of research. For example, accurate and precise concentrations are required to determine the effectiveness of a drug and to integrate the data into a cohesive package for regulatory submissions.

[0003] Currently, there are several methods for determining protein concentration in solution. One such method includes determining molecule-specific parameters such as UV extinction coefficient (ε) or differential refractive index increment (dn / dc). Other methods include (i) gravimetric analysis (Nozaki Y., Arch. Biochem. Biophys. 249 (2), 437-446 (1986)), (ii) chromogenic method (Bradford MM, Anal. Biochem. 72 (1-2), 248-254 (1976)), (iii) Kjeldahl nitrogen determination (Jaenicke L., Anal. Biochem. 61 (2), 623-627 (1974)) and (iv) amino acid analysis ("AAA") (Spackman DH et al., Anal. Chem. 30 (7): 1190-1206 (1958)). However, these approaches rely on experimentally derived protein properties such as ligand binding, degradation or derivatization compared to intrinsic properties of the protein and can lead to inaccurate and imprecise results.

[0004] For example, using the UV extinction coefficient (ε) method, ε is predicted from empirically calculated values; however, the results are only estimates, not actual values ​​(see, e.g., Edelhoch H., Biochemistry 6(7), 1948-1954 (1967)). In addition, for example, gravimetric analysis of lyophilized proteins may contain significant amounts of bound water, salts, and / or other formulation components that may result in inaccurate concentration calculations. Chromogenic methods depend on the composition of the protein and require a standard curve to provide absolute concentrations. Problems and limitations also arise from the use of harsh conditions when determining concentrations according to AAA, for example, harsh conditions that may cause degradation of several key amino acids during the hydrolysis step and the derivatization step, long run times, and high variability. (Sittampalam GS et al., J. Assoc. of Official Anal. Chemists 71(4), 833-838 (1988)). All of the above problems result in reduced accuracy and precision in calculating the concentration and molecule-specific parameters of proteins or peptides that may affect the effectiveness, developability, and commercialization of the molecule.

[0005] Therefore, there is a need for a new, accurate absolute protein concentration determination method that is not affected by protein structure and formulation components, does not rely on molecular interactions or standard curves to provide absolute concentrations, is more accurate and precise, and avoids harsh sample preparation processes. Such a method would be useful, for example, for determining the concentration of a protein or peptide when preparing a protein or peptide intended for therapeutic use.

[0006] Nuclear magnetic resonance (NMR) spectroscopy is a quantitative technique that has been used to measure the concentration of compounds. However, using NMR spectroscopy to measure the absolute concentration of larger proteins in complex matrices (such as therapeutic antibody formulations) is both challenging and poses problems similar to those shown above. For example, one challenge is that the NMR spectrum is dominated by strong peaks from the various salts, buffers, surfactants, tonicity agents, and even water present in the therapeutic formulation. Another such problem is that the higher-order structure of most proteins and antibodies creates a unique magnetic environment around hydrogen atoms that causes the concentration of a given amino acid to be distorted. 1 The H chemical shift window is larger. These issues, along with the inherent difficulties associated with using NMR (e.g., the line widths of protein resonances are inherently broader), result in reduced accuracy and precision when calculating protein concentrations and molecule-specific parameters.

[0007] The present invention provides a new quantitative nuclear magnetic resonance spectroscopy method for measuring the absolute concentration of proteins or peptides. The qNMR spectroscopy method of the present invention produces clear spectra of proteins or peptides with adequate resonance resolution, regardless of solution complexity. The first step is to denature the protein (optional but recommended for small peptides) to reduce the line width of the protein resonances due to secondary, tertiary and quaternary structures and to enable integration. The second step is to collect the qNMR spectrum using a diffusion filter to eliminate the resonances of water and other formulation components. Finally, the data is compared with a reference standard to accurately determine the concentration. This method is referred to herein as Method A. The concentration determined from Method A can then be used to determine molecular parameters of the protein or peptide, such as the extinction coefficient.

[0008] The present invention thus provides a method for obtaining the concentration of a protein in a solution, wherein the method comprises denaturing the protein, performing NMR spectroscopy with diffusion filtering, and calculating the concentration of the protein using a reference standard. The present invention also provides a method for obtaining the concentration of a peptide in a solution, wherein the method comprises performing NMR spectroscopy with diffusion filtering, and calculating the concentration of the peptide using a reference standard. In some embodiments, the method further comprises denaturing the peptide, followed by performing NMR spectroscopy with diffusion filtering.

[0009] The present invention provides a method for obtaining the extinction coefficient of a protein in a solution, wherein the extinction coefficient is determined according to the Beer-Lambert law, and wherein the concentration of the protein used in the Beer-Lambert law is determined in such a method, the method comprising denaturing the protein, performing NMR spectroscopy with diffusion filtering and calculating the concentration of the protein using a reference standard.

[0010] The present invention also provides a method for obtaining the extinction coefficient of a peptide in a solution, wherein the extinction coefficient is determined according to the Beer-Lambert law, and wherein the concentration of the peptide used in the Beer-Lambert law is determined in such a method, the method comprising the optional step of denaturing the peptide, followed by the step of performing NMR spectroscopy with diffusion filtering and calculating the concentration of the peptide using a reference standard. In some embodiments, the method comprises denaturing the peptide, followed by performing NMR spectroscopy with diffusion filtering.

[0011] The present invention provides a method for determining protein concentration in the preparation of a bulk drug or a drug product, wherein the bulk drug comprises a protein in a solution, and wherein the method comprises obtaining a molecular parameter of the protein. In some embodiments, the molecular parameter is an extinction coefficient, and the extinction coefficient is determined according to the Beer-Lambert law, and the protein concentration used in the Beer-Lambert law is determined in such a method, the method comprising denaturing the protein, performing NMR spectroscopy with diffusion filtering, and calculating the concentration of the protein using a reference standard.

[0012] The present invention provides a method for determining the concentration of a peptide in the preparation of a bulk drug or a drug product, wherein the bulk drug comprises a peptide in solution, and wherein the method comprises obtaining a molecular parameter of the peptide. In some embodiments, the molecular parameter is an extinction coefficient, and the extinction coefficient is determined according to the Beer-Lambert law, and the concentration of the peptide used in the Beer-Lambert law is determined in such a method, the method comprising optionally denaturing the peptide, performing NMR spectroscopy with diffusion filtering, and calculating the concentration of the peptide using a reference standard. In some embodiments, the method comprises denaturing the peptide, followed by performing NMR spectroscopy with diffusion filtering.

[0013] The present invention also provides a method for obtaining molecular parameters of proteins in solution, wherein the method comprises denaturing the protein, performing NMR spectroscopy with diffusion filtering, calculating the concentration of the protein using a reference standard and determining the molecular parameters of the protein from the calculated concentration. In one embodiment, the calculated protein concentration and the determined system properties are used in a corresponding equation to determine the molecular parameters of the protein. In some embodiments, the molecular parameter is the extinction coefficient and the corresponding equation is the Beer-Lambert law.

[0014] The present invention also provides a method for obtaining the molecular parameters of a peptide in solution, wherein the method comprises optionally denaturing the peptide, and then performing NMR spectroscopy with diffusion filtering, calculating the concentration of the peptide using a reference standard, and determining the molecular parameters of the peptide from the calculated concentration. In one embodiment, the calculated peptide concentration and the determined system properties are used in the corresponding equation to determine the molecular parameters of the peptide. In some embodiments, the molecular parameter is the extinction coefficient and the corresponding equation is the Beer-Lambert law.

[0015] The present invention provides a method for determining dosage when preparing a bulk drug or drug product, wherein the bulk drug product comprises a protein in a solution, wherein the method comprises obtaining a molecular parameter of the protein from a reference batch, wherein the molecular parameter is determined in such a method, wherein the method comprises denaturing the protein, performing NMR spectroscopy with diffusion filtering, calculating the concentration of the protein using a reference standard, and determining the molecular parameter of the protein from the calculated concentration. In yet another embodiment, the method comprises preparing a test batch of bulk drug or drug product, wherein the protein concentration described in the test batch is determined from the molecular parameter. In some such embodiments, the protein concentration is determined using the molecular parameter and the corresponding system property in the corresponding equation.

[0016] The present invention provides a method for determining dosage when preparing a bulk drug or drug product, wherein the substance includes a peptide in a solution, wherein the method includes obtaining a molecular parameter of the peptide from a reference batch, wherein the molecular parameter is determined in such a method, the method includes optionally denaturing the peptide, performing NMR spectroscopy with diffusion filtering, calculating the concentration of the peptide using a reference standard, and determining the molecular parameter of the peptide from the calculated concentration. In another embodiment, the method includes preparing a test batch of bulk drug or drug product, wherein the peptide concentration described in the test batch is determined from the molecular parameter. In some such embodiments, the peptide concentration is determined using the molecular parameter and the corresponding system attribute in the corresponding equation.

[0017] The present invention also provides a method for determining the concentration of a protein or peptide when preparing a reference batch, wherein the reference batch comprises the protein or peptide in solution, and wherein the method comprises denaturing the protein or optionally the peptide, performing qNMR spectroscopy with diffusion filtering, calculating the concentration of the protein or peptide using a reference standard and a reference technique and determining a molecular parameter of the protein or peptide from the calculated concentration. In yet another embodiment, the method further comprises determining the concentration of the protein or peptide in a work-in-progress test batch, wherein the peptide or protein concentration in the test batch is determined from the molecular parameter.

[0018] The present invention provides embodiments suitable for the methods described herein. Such an embodiment includes a method in which a protein or peptide is denatured by a chaotropic agent. In some such embodiments, the chaotropic agent is urea-d4. In other such embodiments, the chaotropic agent is guanidine chloride-d6. In some embodiments, the reference standard is an internal standard. In preferred embodiments, the reference standard is an external reference standard. In some such embodiments, the external reference standard is a small molecule reference substance (primary standard). In a specific embodiment, the external reference standard is maleic acid. In some such embodiments, the external reference technology is PULCON. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a monoclonal antibody. In other such embodiments, the antibody is a bispecific antibody. In some embodiments, the solution containing the protein or peptide to be measured contains D2O.

[0019] The present invention also contemplates the use of method A or a molecular parameter determined from the concentrations obtained in method A in a variety of applications. Such applications include methods for determining dosages for toxicology, clinical and / or commercial drug products, methods for determining corporate or primary reference standards, methods for determining the concentration of a protein or peptide during production, methods for aliquoting a protein or peptide, methods for aliquoting a protein or peptide wherein the concentration of the aliquoted protein or peptide is similar to the desired concentration of the protein or peptide, methods for batch release testing, or methods for formulating a drug substance or drug product.

[0020] The present invention contemplates that method A can be used for the purposes described herein involving any protein or peptide (e.g., a protein or peptide in the field of therapeutic drugs or oils). By way of example (not intended to be limiting), method A can be implemented in applications involving any or all of the following (including any such biosimilars thereof), wherein the protein or peptide is the active ingredient in the following or any such biosimilars of the following: dulaglutide (known in some countries as Sold as ixekizumab (in some countries ), ramucirumab (sold in some countries as cetuximab (sold in some countries as Olaratumab (sold in some countries as Necituzumab (sold in some countries as sold), anti-CGRP antibodies such as galcanezumab (also known as LY2951742), anti-IL-23 antibodies such as mirikizumab (also known as LY3074828), solanezumab (also known as LY2062430), tanezumab, anti-N3pG-Aβ antibodies, anti-Tau antibodies, anti-Aβ42 antibodies, anti-BAFF / IL-17 bispecific antibodies, anti-CSF-1R antibodies, anti-CXCR1 / 2 antibodies, anti-IL-21 antibodies, anti-IL-23 / CGRP bispecific antibodies, anti-IL-33 antibodies, anti-PD-L1 antibodies, and anti-TIM-3 antibodies.

[0021] The present invention also contemplates that Method A (i) is suitable for serving as a basis for measuring other intrinsic parameters of proteins or peptides or for converting any of the current relative concentration methods into an absolute method, (ii) can be used to quantify peptides that lack chromogenic amino acids and are difficult to monitor using UV, (iii) allows for easier, faster, and safer sample preparation and data acquisition than current AAA methods, (iv) provides linearity, precision, and accuracy of the resulting concentrations that are suitable for a variety of proteins and formulations, and (v) can be used to quantify other types of macromolecules (e.g., polymers, surfactants, etc.) in the presence of small molecule contaminants.

[0022] The present invention contemplates that the methods described herein can utilize the mathematical equations also described herein. For example, the protein concentration c in grams / liter can be calculated from the qNMR spectrum using the following equation: P ,

[0023]

[0024] For example, this article describes a detailed description of this equation.

[0025] One of ordinary skill will recognize that although specific software programs are exemplified herein, other programs may be used to achieve comparable results. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Definition of the bppste pulse sequence and various delays for Agilent (also known as Dbppste) spectrometers.

[0028] Figure 2 .NIST mAb 1 H NMR spectra of NIST mAb in native form (A), same as A at ordinate 5000x (B), native with diffusion filtering (C), and denatured with diffusion filtering (D) with standard single pulse sequence. 1H NMR spectra. The peaks marked with asterisks are from histidine buffer. The scan numbers for AC and D are 1024 and 64.

[0029] Figure 3. D magnitude on NIST mAb. Figure 3a : NMR spectrum of a denatured mAb standard acquired with the bppste pulse sequence. Inset: expansion of the peaks for the I / L / V resonances. Inset: magnified image of the indicated area. Figure 3b : Left: Pure NMR spectra of different diffusive species as determined by the DECRA algorithm. Right: Stejkal-Tanner plots and diffusion coefficients from the DECRA algorithm.

[0030] Figure 4. T1 and T2 measurements on NIST mAb. Figure 4a : Representative data from T1 measurements and T2 measurements using the bppste pulse sequence. Left: NMR spectrum from the bppste pulse sequence setup measuring T1 and the parameters of this setup, right: NMR spectrum from the bppste pulse sequence setup measuring T2 and the parameters of this setup. 4b: Graph of calculated T1 and calculated T2.

[0031] Figure 5 .DF-qNMR spectra of 14 denatured NIST BSA samples. Inset: magnified image of the indicated area.

[0032] Figure 6 .NIST BSA sample concentration: Method A with gravimetric measurement.

[0033] Figure 7 .NIST BSA partial specific volume: sample density and BSA concentration.

[0034] Figure 8 DF-qNMR spectra of 14 denatured bispecific antibody samples.

[0035] DF-qNMR spectra of 14 denatured bispecific antibody samples. The peaks of residual water are marked with asterisks. Inset: magnified image of the indicated area.

[0036] Fig. 9 . Bispecific antibody sample concentration: Method A and gravimetric measurement.

[0037] definition

[0038] As used herein, "nuclear magnetic resonance spectroscopy" (also referred to as "NMR spectroscopy") refers to a spectroscopic technique known to those of ordinary skill in the art, in which nuclear magnetic resonance (NMR) spectra can be used to study the structure of organic molecules. As used herein, "quantitative nuclear magnetic resonance spectroscopy" (also referred to as "qNMR spectroscopy") refers to obtaining quantitative information about sample purity or concentration from one or more NMR spectra. Such spectra are referred to herein as quantitative nuclear magnetic resonance (qNMR) spectroscopy. NMR spectroscopy can incorporate the use of "diffusion filtering", which can be used to selectively attenuate the signal of smaller molecules in a mixture based on their size (see, e.g., Stilbs P., Prog. Nucl. Magn. Reson. Spectrosc. 19(1): 1-45 (1987)). In order to balance the attenuating effects of matrix peaks and protein peaks, the strength of the diffusion filter can be determined by a person of ordinary skill based on the context of each experiment. For example, if the balance is too weak, the diffusion filter has a limited value. If the balance is too strong, the diffusion filter begins to suppress the protein signal, which reduces the signal-to-noise ratio of the measurement. This reduction can then result in longer experimental times. As another example, if one of the matrix components is not completely eliminated and it appears as an interfering peak, it will contribute to the protein peak area and must be subtracted to get the most accurate results. This can be done by preparing a blank sample containing matrix without protein, running the blank sample with diffusion filtering, determining the area of ​​the remaining matrix peak and subtracting that value from the area obtained for the protein sample.

[0039] As used herein, a "reference standard" refers to a compound that 1 The H-NMR spectrum provides at least one distinct peak representing a known number of protons and its purity and concentration are known with a high degree of certainty. The reference standard can be an internal reference standard, where the reference standard is present in a solution containing the protein or peptide of interest, or the reference standard can be external, where the reference standard is independent of the solution containing the protein or peptide of interest. The reference standard can be a small molecule benchmark reference standard, which is a material that is not calibrated against another standard and is instead defined in terms of properties, such as its mass. Commonly used 1 Small molecule reference substances for H qNMR internal standards are readily available and can also be used as external standards [see, e.g., Rigger et al., MJAOAC International, 2017, 100, 1365-1375].

[0040] Reference techniques are mathematical algorithms that use reference standard data. For example, external reference techniques are mathematical algorithms that use external reference standard data. An example of this type is the pulse length-based concentration determination (PULCON) technique (Wider G. and Dreier LJ, Am. Chem. Soc. 128 (8), 2571-2576 (2006)).

[0041] As used herein, a "DF-qNMR" spectrum refers to an NMR spectrum to which diffusion filtering has been applied and from which quantitative information about the purity or concentration of a protein or peptide of interest can be obtained when compared to a reference standard.

[0042] As used herein, "molecular parameters" means scalar values ​​that relate the way some system property (e.g., absorbance, density, refractive index) changes in response to changes in other system properties (e.g., temperature, pressure, or composition). The equations used to determine molecular parameters ("corresponding equations") can vary depending on which system property or properties are known. Molecular parameters refer to intrinsic properties of a protein or peptide.

[0043] For example, the extinction coefficient (ε) is a molecular parameter that describes how the absorbance of a solution changes in response to changes in the concentration of the absorbing species and the optical path length traveled by the light. The extinction coefficient [mL / (mg·cm)] of a protein or peptide can be determined by a combination of ultraviolet-visible (UV-Vis) spectroscopy and Method A as follows: A=εcl (Beer-Lambert's law), where A is the absorbance of the protein or peptide solution, l is the optical path length [cm], and c is the absolute protein or peptide concentration [mg / mL]. As another example, the apparent microvolume of a protein or peptide can be determined by a combination of densitometry and Method A as follows [mL / g]: Wherein ρ is the density of the protein or peptide solution [g / mL], ρ0 is the density of the protein or peptide solution matrix [g / mL], and c is the absolute protein or peptide concentration [mg / mL]. As a third example, the (differential) refractive index increment (dn / dc) [mL / mg] of a protein or peptide can be determined by a combination of (differential) refractometry and method A as follows: n=n0+(dn / dc)c, where n is the refractive index of the protein or peptide solution, n0 is the refractive index of the protein or peptide solution matrix, and c is the absolute protein or peptide concentration [mg / mL].

[0044] One of ordinary skill can determine a molecular parameter of interest by linear regression of multiple absorbance / density / refractive index measurements as a function of concentration, such as that determined from Method A. Molecular parameters, such as the extinction coefficient, can be used to determine the concentration of the drug by measuring the corresponding system property (such as UV absorbance) and solving the corresponding equation relating them (e.g., Beer-Lambert's law).

[0045] The general structure of a "monoclonal antibody" is known. An IgG antibody is a heterotetramer of four polypeptide chains (two identical "heavy chains" and two identical "light chains") cross-linked by intra- and inter-chain disulfide bonds. The variable regions of each heavy chain-light chain pair associate to form a binding site. The heavy chain variable region (VH) and light chain variable region (VL) can be subdivided into hypervariable regions called complementarity determining regions ("CDRs"), interspersed with more conserved regions called framework regions ("FRs"). Each VH and VL consists of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs contain most of the residues that form specific interactions with the antigen.

[0046] As used herein, "bispecific antibodies" refer to bivalent antibody constructs including, but not limited to, IgG-scFv patterns (as reported in PCT / US2015 / 058719) and bivalent IgG patterns (as disclosed in US 2018 / 0009908). The present invention also contemplates that any human engineered protein or antibody, whether or not having a tertiary or quaternary structure, may also be used in the methods described herein. Examples of such human engineered proteins or antibodies include trispecific or tetraspecific antibodies and fusion proteins.

[0047] As used herein, peptides include polymeric chains of amino acids. These amino acids can be natural or synthetic amino acids, including modified amino acids. Peptides can have less secondary and tertiary structure, but are prone to aggregation in solution; therefore, peptides can also be denatured by chaotropes. Denatured peptides can lead to an increase in the signal-to-noise ratio in NMR spectroscopy, thereby making integration easier. Denatured peptides may also allow for the testing of more concentrated samples, thereby resulting in shorter experimental times. For the purposes of the methods described herein, when the peptides are denatured, the peptide concentration determination may be more accurate. For denaturation with a chaotropic agent, the preferred chaotropic agent will depend on the specific requirements of each peptide and can be determined experimentally by a person of ordinary skill in the art.

[0048] Those of ordinary skill in the art will recognize that proteins include one or more polymeric peptide chains of amino acids. These amino acids can be natural or synthetic amino acids, including modified amino acids. Proteins can be recombinant proteins. The primary structure of a protein comprises a linear sequence of its monomeric amino acid subunits. The secondary structure of a protein includes three-dimensional structural features such as α-helices and β-folds that are generated by hydrogen bonding patterns over local segments of the amino acid chain. The tertiary structure of a protein describes the overall shape of the protein as defined by three-dimensional atomic coordinates. Quaternary structure refers to the arrangement of two or more protein subunits in a complex. A protein can be "denatured," wherein an external stress or a chaotropic agent is added to a solution containing the protein and the protein unfolds. Denatured proteins have therefore lost most of their secondary, tertiary, and quaternary structural features. Examples of chaotropic agents include urea-d4 and guanidine chloride-d6.

[0049] As also used herein, "solution" refers to a human engineered aqueous mixture of a peptide or protein containing components other than water. "Batch" refers to the amount of a drug or other substance that is a protein or peptide and is intended to have uniform characteristics and properties within specified limits, and is produced according to a single production order during the same production cycle. "Batch" refers to a batch or a specific, determined portion of a batch that has uniform characteristics and properties within specified limits; or, in the case of a drug produced by a continuous process, it is a specific, determined amount produced per unit time or quantity in a manner that ensures that it has uniform characteristics and properties within specified limits. "Reference batch" refers to an established and properly characterized proprietary reference standard material, wherein the material contains a protein or peptide, and wherein the material is prepared from a batch representing production materials and clinical materials. Method A is used to determine the concentration and molecular parameters, such as the extinction coefficient, of a protein or peptide in a solution in a reference batch. Molecular parameters, such as the extinction coefficient, obtained from a reference batch are then used to determine the concentration of a protein or peptide in a subsequent batch (referred to herein as a "test batch").

[0050] As used herein, a "drug" is a finished dosage form, e.g., a tablet, capsule, or solution, containing a drug substance, typically but not necessarily in combination with one or more other ingredients. A "drug substance" is an active ingredient intended to provide pharmacological activity or other direct effect in the diagnosis, cure, alleviation, treatment, or prevention of a disease or intended to affect the structure or any function of the human body, but does not include intermediates used in the synthesis of such ingredients. As used herein, a drug substance includes a protein or peptide. Method A can be used for a variety of purposes related to drugs or drug substances comprising proteins or peptides, such as use during the entire drug or drug substance production period.

[0051] "Dose" refers to the amount of protein or peptide in a matrix that will stimulate a desired biological or medical response. The material can be lyophilized or in aqueous solution. To prepare a material intended for therapeutic use, one skilled in the art will appreciate that accurate and precise protein or peptide concentrations are required. The methods described herein provide a new means of obtaining protein or peptide concentrations by determining the extinction coefficient or other molecular parameter of a protein or peptide in a reference batch and then using this extinction coefficient or other molecular parameter to determine the concentration of the protein or peptide in a subsequent test batch.

[0052] Aliquoting means taking a portion of a larger amount in one vessel and adding it to a separate vessel. A vessel can be anything suitable for holding a protein or peptide, lyophilized or in solution, such as a container, vial, bottle or device.

[0053] Batch release testing (test at lot release) refers to the appropriate laboratory determination of satisfactory compliance with the final specifications of the drug product (including the strength (concentration) of each active ingredient) before release (release by the manufacturer to the market). Batch release testing can be, for example, batch release UV testing. The concentration of the protein or peptide can be determined from the extinction coefficient of the protein or peptide, wherein the extinction coefficient is determined from method A in a reference batch.

[0054] The following examples further illustrate the present invention and provide common methods and procedures to implement various specific embodiments of the present invention. However, it will be appreciated that the examples are described by way of illustration and not limitation and that various modifications may be made by those skilled in the art. Example

[0055] Sample preparation:

[0056] Reference Standards : having at least one distinct peak representing a known number of protons 1 Any compound for which the H-NMR spectrum, purity and concentration are known with a high degree of certainty and which is chemically and physically stable in solution can be used to facilitate calculation of absolute concentrations from the DF-qNMR spectrum. For example, maleic acid can be used as an external quantitative reference standard. To prepare maleic acid as an external reference standard, certified maleic acid (11.183 mg, 0.096347 mmol) is placed in a 5 mL volumetric flask and dissolved in D2O. An equal portion is transferred to a tube, such as a Wilmad 435 precision 4 mm NMR tube, so that the sample height is 40 mm.

[0057] Protein samplesGuanidine chloride-d6 (0.6 g, 6 mmol) was placed in a 1 mL volumetric flask. The flask was placed on a balance and 400 μL of the protein solution was added. The solution was then gently sonicated and vortexed until all solids were dissolved. D2O was added to achieve the final volume. An aliquot was transferred to a tube, such as a Wilmad 435 precision 4 mm NMR tube, so that the sample height in the tube was 40 mm.

[0058] Peptide samples Urea-d4 (0.13 g, 2 mmol) was placed in a 1 mL volumetric flask. The flask was placed on a balance and about 1 mL of protein solution was added. The solution was then gently sonicated and vortexed until all solids were dissolved. D2O was added to achieve the final volume. An equal portion was transferred to a tube, such as a Wilmad 435 precision 4 mm NMR tube, so that the sample height in the tube was 40 mm.

[0059] calculate

[0060] The protein concentration c in g / L was calculated from the DF-qNMR spectra using the following equation: P

[0061]

[0062] where c S is the concentration of the reference standard, A P is the area of ​​a selected protein peak in the DF-qNMR spectrum (typically the peaks of valine, isoleucine, and leucine methyl groups, between 1.0 and 1.4 ppm, but can be any resolvable peak), H S is the number of protons contributed to the reference standard peak, A S is the area of ​​the reference standard peak, H P is the number of protons contributing to the protein peak, and the various f's are functions that depend on the specific experimental conditions. DF Accounting for the reduction in protein peak area due to the diffusion filter, it is derived from the Stejskal-Tanner equation (Johnson, CS et al., Concepts in Magnetic Resonance Part A 2012, 40A, 39-65). For the bppste pulse sequence used herein (Pelta, MD et al., Magn. Reson. Chem. 1998, 36, 706-714), it is given by

[0063]

[0064] where γ is 1H is the magnetic gyroscopic ratio, g is the pulse field gradient strength, δ is the pulse field gradient length, Δ is the diffusion delay, T1 is the spin-lattice nuclear relaxation time of the target proton of the protein, T2 is the spin-spin nuclear relaxation time of the target proton of the protein, D is the translation diffusion coefficient of the protein, and τ1 is the time between the second and third 90° pulses in the bppste pulse sequence (see Figure 1 ), τ2 is the total time between the first and second 90° pulses (and between the third 90° pulse and acquisition), and τ3 is the total time between the pulsed field gradient pulses of the bipolar pulse pair. For the pulse sequence supplied on the Agilent instrument, these terms are defined by the following instrument parameters

[0065] τ1=Δ-δ-2t gs -4θ P -2t rg (Equation 3)

[0066] τ2=2(δ+2t gs +2θ P +2t rg ) (Equation 4)

[0067] τ3=t gs +2θ P +t rg (Equation 5)

[0068] where t gs is the gradient stabilization delay, t rg is the receiver gating time preceding the pulse, and Θ is the 90° pulse width. For other diffuse pulse sequences, f DF The expression of will be different. ER Contains the instrument parameters necessary for an external reference. In its most basic form

[0069]

[0070] where S and G are the scan number and receiver gain, respectively, used to acquire each NMR spectrum of the protein and the reference standard. When using the PULCON method, it becomes

[0071]

[0072] where T is the sample temperature during acquisition of the NMR spectrum. DIL accounts for the dilution of the initial protein sample into the final NMR sample. If the final volume is obtained by volume and initial sample aliquots are done gravimetrically, this factor is given by

[0073]

[0074] Among them, vol F is the volume of the final solution (ie, the NMR sample solution), and wt I and d I is the weight and density of the initial protein solution aliquot. Alternatively, if the dilution is done by gravimetric addition of both the initial protein solution and D2O, then

[0075]

[0076] where wt D2O and d D2O is the weight and density of D2O. Finally, f U Convert the concentration units (usually micromolar) from those quantitative reference standards to common units - grams per liter (or equivalently, mg per milliliter).

[0077]

[0078] Among them, M P is the molecular weight of the protein.

[0079] The first exponential term in Equation 2 describes the signal attenuation due to T1 nuclear relaxation, the second exponential term describes the signal attenuation due to T2 nuclear relaxation, and the third exponential term describes the signal attenuation due to molecular diffusion. Of all the terms in the equation, these three quantities (T1, T2, and D) are the only unknown quantities; all other quantities are pulse sequence / instrument parameters or physical constants (γ). T1, T2, and D depend on the molecule and solution conditions and must be measured for each unique sample. This can be done with three independent experiments using the same pulse sequence as the diffusion filter. Measuring D is a well-known experiment in which multiple spectra are obtained with increasing gradient intensity (Stejskal, EO and Tanner, JE, J. Chem. Phys., 1965, 42, 288-292). This keeps the T1 and T2 terms of Equation 2 constant, so that the signal attenuation is governed only by D. D is then calculated using a suitable algorithm such as DECRA (Windig, W.; Antalek, B. Chemom. Intell. Lab. Syst., 1997, 37, 241-254). To measure T1 with the Agilent bppste pulse sequence, a combination of diffusion delay and gradient intensity (gzlvl1) is used to collect multiple spectra, thereby

[0080]

[0081] where Δ' is the correct diffusion delay given by

[0082]

[0083] This keeps the T2 and D terms of Equation 2 constant, so that the signal decay is dominated by T1 relaxation alone. P The effective T1 of the protons of the selected protein peaks was calculated by a single exponential fit relative to τ1.

[0084] To measure T2 with the Agilent bppste pulse sequence, multiple spectra are collected using a combination of gradient stabilization delay, diffusion delay, and gradient strength, resulting in

[0085] Δ n =Δ1+(t rg，n -t rg，1 ) (Equation 13)

[0086] and gzlvl1 was determined as above n This keeps the T1 and D parts of the equation constant, so that the signal decay is dominated by T2 relaxation alone. P The effective T2 of the protons of the selected protein peaks was calculated by a single exponential fit relative to τ2.

[0087] Method A Data Collection

[0088] Agilent DD2 600MHz NMR spectrometer equipped with Agilent 1 H- 19 F / 15 N- 31 P PFG OneProbe. Probe temperature and pulsed field gradient were calibrated with ethylene glycol samples and 1% H2O in D2O samples, respectively. NMR spectra were acquired at 30.0°C using a bipolar pulse pair stimulated echo (bppste) pulse sequence. The number of scans was 64. Acquisition parameters included 20 ppm spectral width, 1.363 sec acquisition time, 30 sec relaxation delay, 150 ms diffusion delay, 1.4 ms gradient pulses with an intensity of 0.569 T / m (92% of the maximum value), and 1 ms gradient stabilization delay.

[0089] To measure the translational diffusion coefficient (D), the acquisition parameters included a 1 second relaxation delay, a 200 millisecond diffusion delay, a 2.0 millisecond diffusion gradient duration, a 0.5 millisecond gradient stabilization delay, and seven logarithmically spaced gradient strength values ​​ranging from 0.228 to 0.569 T / m (approximately 37-92% of the maximum). Spin-lattice nuclear relaxation (T1) was measured with the bppste pulse sequence using a 20 ppm spectral width, a 1.363 second acquisition time, a 3.637 second relaxation delay, a 2.0 millisecond diffusion gradient duration, and a 0.5 millisecond gradient stabilization delay. Five pairs of diffusion delays and gradient intensities were used—(100 ms, 0.569 T / m); (203 ms, 0.400 T / m); (408 ms, 0.281 T / m); (804 ms, 0.200 T / m); and (1202 ms, 0.164 T / m).

[0090] Spin-spin nuclear relaxation (T2) was also measured with a bppste pulse sequence using a 20 ppm spectral width, 1.363 sec acquisition time, 1 sec relaxation delay, 1.4 ms diffusion gradient duration. Six sets of gradient stabilization delays, diffusion delays, and gradient intensities were used—(1 ms, 150.001 ms, 0.570); (2 ms, 150.002 ms, 0.571 T / m); (4 ms, 150.004 ms, 0.573 T / m); (8 ms, 150.008 ms, 0.577 T / m); (12 ms, 150.012 ms, 0.581 T / m); and (16 ms, 150.016 ms, 0.575 T / m).

[0091] Data analysis

[0092] DF-qNMR, T1 and T2 data were processed in MNova version 11.0 (Mestrelab Research, SL, Santiago de Compostela, Spain), and diffusion data were processed and analyzed with DOSYToolbox version 2.5 (Nilsson, MJ Magn. Reson. 2009, 200, 296-302). Before Fourier transform, FID was zeroed once and multiplied by a 2.93 Hz exponential window function. Using the DECRA algorithm available in DOSYToolbox, diffusion coefficients were obtained. Using MATLAB 2016b (MathWorks, Inc., Natick, Ma), T1 and T2 values ​​were calculated from the bppste pulse sequence by regression analysis. The peak areas of leucine methyl, isoleucine methyl and / or valine methyl were obtained by deconvolution using a straight line fitting path in MNova and CRAFT.

[0093] Method A for NIST antibody RM8761

[0094] To illustrate the challenges of quantifying large, formulated proteins by NMR, a NIST RM 8761 sample (10 g / L antibody, 12.5 mM L-histidine HCl, pH 6.0, commercially available from the National Institute of Standards and Technology (NIST); see, e.g., Schiel et al., Anal. and Bioanal. Chem., 410(8):2127-2139 (2018)) was treated with 10% D2O and obtained. 1 H 1D NMR spectrum ( Figure 2 ). The water signal is so strong and broad that it obscures the signal of the antibody. A histidine excipient peak is also observed in the spectrum. In order to obtain a valid protein spectrum, these dominant peaks must be eliminated. One NMR technique ideal for this application is "diffusion filtering", in which peaks are eliminated from the spectrum based on the diffusion coefficient of the corresponding molecule and therefore its size. Figure 2 The figure also shows the use of a bipolar pulse pair stimulated echo (bppste) diffusion pulse sequence as a diffusion filter for the same NIST mAb sample. 1 H-NMR Spectra. Diffusion filtering effectively removes unwanted signals of the formulation without introducing baseline artifacts or phase distortion.

[0095] However, in most cases, the resolution for accurate peak identification and integration is insufficient due to the characteristic broad line widths of the protein resonances. This is primarily due to the higher order structure (HOS) of the protein. A single type of amino acid that should have very similar chemical shifts shows a broad distribution of chemical shifts due to the unique magnetic environment caused by the secondary, tertiary and quaternary structures of the protein. Therefore, HOS is eliminated by denaturing the protein. Adding denaturants to the protein solution causes the sample volume to expand. For true quantitation, more solvent is added to achieve an exact volume, such as using a volumetric flask. This is the f in Equation 1. DIL basis.

[0096] Figure 2 Figure 2 shows the diffusion filter of the NIST RM 8761 sample prepared with 6M guanidine chloride-d6. 1 H-NMR. A decrease in line width and a subsequent increase in the signal-to-noise ratio were observed. The group of peaks between 1.0-1.4 ppm corresponding to the methyl groups of isoleucine, leucine and / or valine had near baseline resolution, which made it possible to integrate them for quantitative analysis.

[0097] Diffusion filtering effectively separates protein signals from the matrix; however, diffusion filtering results in a lack of intrinsic quantification of the measured NMR peak areas. Conventional qNMR experiments use a pure NMR pulse sequence, which consists of three basic elements: a nuclear relaxation delay, a radio frequency pulse, and data acquisition time. When a sufficiently long nuclear relaxation delay is used, the resulting NMR peak areas are accurately and reproducibly proportional to the number of observed nuclei; therefore, the experiment is quantitative. All other NMR pulse sequences, including the diffusion sequence used here, require multiple radio frequency pulses separated by various delays. Therefore, the resulting spectrum is no longer intrinsically quantitative due to the factors that attenuate the peaks from the equilibrium value. However, if these factors are known and the degree of attenuation can be calculated, diffusion filtering can be made quantitative. This is the f in Equation 1. DF basis.

[0098] External reference standards are preferred over internal reference standards because the former avoid potential interactions and / or overlapping peaks between two molecules in the NMR spectrum, although internal standards can be used. Several external standard methods have been reported, including the PULCON technique (e.g., pulse length-based concentration determination). This technique allows the absolute areas from two independent spectra (one for the reference standard and the other for the analyte) to be correlated, even if solution conditions and experimental parameters are different. Therefore, the external reference standard does not need to have similar characteristics to the analyte of interest or even be a protein. In addition, rather than requiring a standard for each amino acid as with AAA, this method requires only a single standard.

[0099] To demonstrate the accuracy and precision of Method A, three independent replicates of the NIST RM 8761 sample were evaluated. Using only 64 scans and a total acquisition time of 37 minutes, sufficient signal-to-noise ratio was obtained for this 10 g / L protein preparation. The signal group (1.0–1.4 ppm) of the methyl groups of I / L / V in the protein, which corresponds to 1,476 protons, has a resolution close to the baseline. These results make it possible to accurately integrate for quantification. To facilitate concentration calculations, the T1, T2, and D of this peak group were also measured using the bppste pulse sequence as described above, and the data acquisition and analysis performed are shown in Figures 3 and 4.

[0100] The total experimental time for all four NMR experiments on one replicate was 110 minutes. The concentration of each replicate was then calculated from Equation 9 using the intact, non-glycosylated molecular weight (148,041 Da) and the dilution factor based on the weight of the aliquot and the measured sample density. The average concentration was determined to be 10.02 g / L with an RSD of 0.55%. This is consistent with the labeled value of 10 g / L.

[0101] These data show that Method A can be used to accurately and precisely determine antibody concentrations.

[0102] Using Method A for NIST Bovine Serum Albumin (BSA)

[0103] NIST (BSA) standard reference material 927e (67.38 g / L, 20 mM sodium chloride, pH adjusted to 6.5-6.8 with 1.0 mol / L sodium hydroxide, commercially available from the National Institute of Standards and Technology) was diluted gravimetrically to four additional protein concentrations (Table 1, samples B1-B4). A total of fourteen samples were analyzed, including six replicates of the middle concentration (B3), three replicates of each of the highest (B5) and lowest (B1) concentrations, and a single replicate of an intermediate concentration (B2 and B4). NMR samples for each replicate were prepared as described above. Method A data acquisition and analysis were performed as described above. Figure 5 The resulting fourteen DF-qNMR spectra are shown superimposed in FIG.

[0104] These data show that the peak from water has been almost eliminated, the baseline is not disturbed and the signal set of the methyl groups of I / L / V in the protein has near baseline resolution. The concentration of each sample was calculated from equation 1 using a molecular weight of 66,398 Da and reported in Table 1. For all five concentrations, the values ​​measured with method A were similar to the values ​​calculated from the gravimetric dilution. As shown in Table 1 below, the RSDs for all three sets of replicate samples were less than 1%.

[0105] Table 1 . NIST Results for BSA samples

[0106]

[0107]

[0108] like Figure 6 As shown in Figure 2, the concentration of method A versus the concentration by weight plot produces R 2 = 0.9997, indicating a linear concentration range across the concentration range studied.

[0109] These data show that Method A can be used to accurately and precisely determine the concentration of medium-sized proteins over a wide concentration range.

[0110] Figure 7 A graph showing the density of the samples as a function of protein concentration as determined by Method A. The equation relating the two quantities is as defined above The slope of the regression line yields the partial microvolume of BSA The latter was found to be 0.718 mL / g.

[0111] Using Method A for Bispecific Antibodies

[0112] Samples of bispecific antibodies (given in Table 1 of US Pat. No. 9,718,884) were prepared as described above, with five different concentrations and a total of 14 samples. Method A data acquisition and analysis were performed as described above. Following essentially the above procedure, the following data were obtained.

[0113] Figure 8 The resulting DF-qNMR spectrum is shown, which is similar to BSA (shown above) in overall appearance and properties. Using the theoretical molecular weight of intact non-glycosylated protein (approximately 200,000 Da), the concentrations were calculated using Equation 1 and are summarized in Table 2 below.

[0114] Table 2 . Results for bispecific antibodies

[0115]

[0116] like Fig. 9 As shown in Figure 2, the concentration of method A versus the concentration by weight plot produces R 2 =0.996 regression line.

[0117] These data show that Method A can be used to accurately and precisely determine the concentration of bispecific antibodies over a wide concentration range.

[0118] Method A was used for peptides of approximately 5000 Da

[0119] Dilute, small peptide samples (about 5,000 Da; given by the sequence in Example 4 of PCT / US2017 / 041922) were prepared with and without the chaotropic agent as described above. Since peptides naturally exist as random coils and at low concentrations, lower denaturant concentrations (2M) are acceptable. Sample stability requires alkaline conditions; therefore, urea-d4 was used. Method A data acquisition and analysis were performed as described above, with the exception that 624 scans were used instead of 64 scans for each acquisition. Following the procedure essentially as described above, the following data were obtained. Slight phase distortion of the residual water signal was observed, but this did not hinder data analysis. Adding a chaotropic agent produced better water repression, sharper peaks, and subsequently higher resolution and sensitivity. The concentration of each replicate sample was calculated from Equation 1. The average total protein concentration was determined to be 0.65 g / L with an RSD of 3.6%. This value is similar to the value obtained by mass balance (0.63 g / L). These data show that method A can be used for dilute samples of small peptides.

[0120] Illustrative Embodiments

[0121] Included below is a series of illustrative embodiments of the disclosure, which are representative of various embodiments of the disclosure.

[0122] These illustrative embodiments are not intended to be exhaustive or to limit the disclosure to the precise forms disclosed, but rather, these illustrative embodiments are provided to aid in further describing the disclosure so that others skilled in the art may utilize the teachings of these embodiments.

[0123] 1. A method for determining the concentration of a protein or peptide in a solution, wherein the method comprises:

[0124] (i) denaturing a protein or, optionally, a peptide;

[0125] (ii) performing qNMR spectroscopy using diffusion filtering; and

[0126] (iii) Calculate the concentration of the protein or peptide using reference standards and reference techniques.

[0127] 2. The method of embodiment 1, wherein the peptide is denatured.

[0128] 3. The method of embodiment 1 or embodiment 2, wherein the protein or peptide is denatured by a chaotropic agent.

[0129] 4. The method of embodiment 3, wherein the chaotropic agent is guanidine chloride-d6 or urea-d4.

[0130] 5. The method of any one of embodiments 1-4, wherein the reference standard is an external reference standard.

[0131] 6. The method of embodiment 5, wherein the external reference standard is a small molecule benchmark substance.

[0132] 7. The method of embodiment 5, wherein the external reference standard is maleic acid.

[0133] 8. The method of any one of embodiments 1 to 7, wherein the solution comprises D2O.

[0134] 9. The method according to any one of embodiments 1 to 8, wherein the reference technology is PULCON.

[0135] 10. The method of any one of embodiments 1-9, wherein the protein is an antibody.

[0136] 11. The method of embodiment 10, wherein the antibody is a monoclonal antibody.

[0137] 12. The method of embodiment 10, wherein the antibody is a bispecific antibody.

[0138] 13. A method for determining molecular parameters of a protein or peptide in a solution, wherein the method comprises:

[0139] (i) denaturing a protein or, optionally, a peptide;

[0140] (ii) qNMR spectroscopy using diffusion filtering;

[0141] (iii) calculating the concentration of the protein or peptide using a reference standard and reference technique; and

[0142] (iv) Determining molecular parameters of the protein or peptide from the calculated concentrations.

[0143] 14. The method of embodiment 13, wherein the peptide is denatured.

[0144] 15. The method of embodiment 13 or embodiment 14, wherein the molecular parameter is the extinction coefficient.

[0145] 16. The method of embodiment 15, wherein the extinction coefficient is determined according to the Beer-Lambert law.

[0146] 17. The method according to any one of embodiments 13 to 16, wherein the protein or peptide is denatured by a chaotropic agent.

[0147] 18. The method of embodiment 17, wherein the chaotropic agent is guanidine chloride-d6 or urea-d4.

[0148] 19. The method of any one of embodiments 13-18, wherein the reference standard is an external reference standard.

[0149] 20. The method of embodiment 19, wherein the external reference standard is a small molecule benchmark substance or maleic acid.

[0150] 21. The method of embodiment 20, wherein the external reference standard is a small molecule benchmark substance.

[0151] 22. The method of embodiment 20, wherein the external reference standard is maleic acid.

[0152] 23. The method according to any one of embodiments 13 to 22, wherein the solution comprises D2O.

[0153] 24. The method according to any one of embodiments 13 to 23, wherein the reference technology is PULCON.

[0154] 25. The method of any one of embodiments 13, 15-24, wherein the protein is an antibody.

[0155] 26. The method of embodiment 25, wherein the antibody is a monoclonal antibody.

[0156] 27. The method of embodiment 25, wherein the antibody is a bispecific antibody.

[0157] 28. A method for determining molecular parameters of a protein or peptide in a reference batch, wherein the method comprises:

[0158] a. denaturing a protein or optionally a peptide;

[0159] b. qNMR spectroscopy with diffusion filtering;

[0160] c. Calculate the concentration of the protein or peptide using reference standards and reference techniques; and

[0161] d. Determine the molecular parameters of the protein or peptide from the calculated concentrations.

[0162] 29. The method of claim 28, further comprising determining the concentration of a protein or peptide in the test batch, wherein the concentration of the peptide or protein in the test batch is determined from the molecular parameters.

[0163] 30. The method of embodiment 28 or embodiment 29, wherein the molecular parameter is the extinction coefficient.

[0164] 31. The method of embodiment 30, wherein the extinction coefficient is determined according to the Beer-Lambert law.

[0165] 32. The method according to any one of embodiments 28-31, wherein the peptide is denatured.

[0166] 33. The method according to any one of embodiments 28 to 32, wherein the protein or peptide is denatured by a chaotropic agent.

[0167] 34. The method of embodiment 33, wherein the chaotropic agent is guanidine chloride-d6 or urea-d4.

[0168] 35. The method according to any one of embodiments 28 to 34, wherein the reference standard is an external reference standard.

[0169] 36. The method of embodiment 35, wherein the external reference standard is a small molecule benchmark substance.

[0170] 37. The method of embodiment 35, wherein the external reference standard is maleic acid.

[0171] 38. The method according to any one of embodiments 28 to 37, wherein the solution comprises D2O.

[0172] 39. The method according to any one of embodiments 28 to 38, wherein the reference technology is PULCON.

[0173] 40. The method of any one of embodiments 28-31, 33-39, wherein the protein is an antibody.

[0174] 41. The method of embodiment 40, wherein the antibody is a monoclonal antibody.

[0175] 42. The method of embodiment 40, wherein the antibody is a bispecific antibody.

[0176] 43. The method of any one of embodiments 13-42, wherein the method further comprises determining the concentration of the protein or peptide when the protein or peptide is formulated using molecular parameters.

[0177] 44. The method of any one of embodiments 13-42, wherein the method further comprises determining the concentration of the protein or peptide at the time of batch release testing using molecular parameters.

[0178] 45. The method of embodiment 44, wherein the batch release inspection is a batch release UV inspection.

[0179] 46. ​​The method of any one of embodiments 13-42, wherein the method further comprises determining the concentration of the protein or peptide during batch preparation using molecular parameters.

[0180] 47. The method of any one of embodiments 13-42, wherein the method further comprises determining the concentration of the protein or peptide using molecular parameters when determining the dosage of the protein or peptide.

[0181] 48. The method of any one of embodiments 13-42, wherein the method further comprises determining the concentration of the protein or peptide during production using molecular parameters.

[0182] 49. The method of any one of embodiments 1-48, wherein the protein or peptide is the active ingredient in dulaglutide, ixekizumab, ramucirumab, cetuximab, olaratumab, necitumumab, galcanezumab or mirikizumab.

Claims

1. A method for determining the absolute protein concentration of a protein in a drug or drug product solution, wherein the drug or drug product contains a single protein, comprising the steps of: a. denaturing the protein, wherein the denaturation causes the protein to unfold and lose most of its secondary, tertiary and quaternary structural features; b. performing qNMR spectroscopy on the denatured protein, wherein the step of performing qNMR spectroscopy comprises using diffusion filtering; and c. Calculate the absolute protein concentration of the protein using a reference standard and a reference technique.

2. The method according to claim 1 further comprises the steps of: d. Determine the molecular parameters of the protein from the calculated concentration of the protein.

3. The method according to claim 2, wherein the molecular parameters of the protein include extinction coefficient, refractive index or partial microvolume. The method according to claim 3 , wherein the extinction coefficient is determined according to the Beer-Lambert law.

5. The method according to any one of claims 1 to 4, wherein the protein is denatured by a chaotropic agent.

6. The method of claim 5, wherein the chaotropic agent is guanidine chloride-d6 or urea-d4.

7. The method according to any one of claims 1 to 4, wherein the reference standard is an external standard. The method according to claim 7 , wherein the external reference standard is a small molecule benchmark substance.

9. The method of claim 7, wherein the external reference standard is maleic acid.

10. The method according to any one of claims 1 to 4, wherein the solution comprises D2O.

11. The method according to any one of claims 1 to 4, wherein the reference technology is PULCON.

12. The method according to claim 2 or 3, wherein the molecular parameters of the protein are the molecular parameters of the protein in a reference batch.

13. The method of claim 12, wherein the molecular parameters of the protein in the reference batch are used to determine the absolute protein concentration of the protein in the test batch.

14. The method of claim 2 or 3, wherein the method further comprises using molecular parameters of the protein to determine the protein concentration of the protein in the test batch.

15. The method according to claim 2 or 13, wherein the step of determining the molecular parameters of the protein further comprises ultraviolet spectroscopy.

16. The method of any one of claims 1-4, wherein the protein is an antibody.

17. The method of claim 16, wherein the antibody is a monoclonal antibody, a bispecific antibody, a trispecific antibody or a tetraspecific antibody.

18. The method of any one of claims 1-4, wherein the protein is a fusion protein.

19. The method of any one of claims 1-4, wherein the protein is a peptide.

Images