EA040889B1 - METHODS FOR DETERMINING PROTEIN OR PEPTIDE CONCENTRATION AND THEIR APPLICATION - Google Patents

Description

This invention relates to methods for determining the concentration and/or specific molecular parameters such as extinction coefficient, protein or peptide, and their use.

The concentration of a peptide or protein, such as an antibody, in solution is an important property for the development and commercialization of biological therapeutics, as well as in many other areas of research. For example, a reliable and accurate concentration is required to determine the effectiveness of a drug and combine the data into a single package for submission to regulatory authorities.

Currently, there are several approaches for determining the concentration of protein in solution. One such approach involves the determination of specific molecular parameters such as the UV extinction coefficient (ε) or the differential refractive index increment (dn/dc). Other approaches include (i) gravimetric analysis (Nozaki Y., Arch. Biochem. Biophys. 249 (2), 437-446 (1986)), (ii) chromogenic methods (Bradford M. M., 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 (AKA). . AAA) (Spackman D. H., et al., Anal. Chem. 30 (7): 1190-1206 (1958)). These methods, however, rely on experimentally derived properties of the protein, such as ligand binding, degradation or derivatization, as opposed to intrinsic properties of the protein, and may lead to unreliable and inaccurate results.

For example, in the method for determining the UV extinction coefficient (ε), ε is predicted based on empirical calculations; the result, however, is only an estimate and not an actual value (see, for example, Edelhoch H, Biochemistry 6 (7), 1948-1954 (1967)). Moreover, in gravimetric analysis of lyophilized proteins, for example, the sample may contain significant amounts of bound water, salts, and/or other composition components, which may lead to inaccurate concentration calculations. Chromogenic methods depend on protein composition and require calibration curves to obtain absolute concentrations. Problems and limitations also arise as a consequence of the use of stringent conditions in the determination of concentration by the AKA method, for example, which can lead to degradation of several key amino acids in the hydrolysis and derivatization steps, long assay times, and high variability. (Sittampalam G. S., et al, J. Assoc. of Official Anal. Chemists 71 (4), 833-838 (1988)). All of the aforementioned problems lead to a decrease in reliability and accuracy in calculating the concentration and specific molecular parameters of proteins or peptides, which can affect the efficiency, development and commercialization of the molecule.

Therefore, a new, accurate method for determining absolute protein concentration that is independent of protein structure and formulation components, does not rely on molecular interactions or calibration curves to obtain absolute concentrations, is more reliable and accurate, and avoids rigorous sample preparation. Such a method may be useful, for example, in determining the concentration of a protein or peptide in a protein or peptide preparation intended for therapeutic use.

Nuclear magnetic resonance spectroscopy (NMR spectroscopy) is a quantitative technique that is used to measure the concentration of compounds. However, the use of NMR spectroscopy to measure the absolute concentrations of large proteins in complex matrices such as therapeutic antibody formulations is both challenging and faces problems similar to those noted above. For example, one problem is that the NMR spectra are dominated by intense peaks from various salts, buffers, surfactants, tonicity regulators, and even water present in therapeutic compositions. Another such problem is that the high order structure of most proteins and antibodies creates unique magnetic environments around hydrogen atoms that cause large 1H chemical shift windows for a given amino acid. These problems, along with other inherent problems that arise when using NMR (for example, inherently wider linewidth for protein resonances), lead to a decrease in reliability and accuracy in calculating the concentration of proteins and their specific molecular parameters.

The present invention provides a novel quantitative nuclear magnetic resonance spectroscopy method for measuring the absolute concentration of a protein or peptide. The spectroscopic method of quantitative NMR (qNMR) according to this invention gives clean spectra of a protein or peptide with well resolved resonances, regardless of the complexity of the solution. The first step is the denaturation of the protein (for a small peptide this step is optional but recommended) in order to reduce the linewidth of protein resonances caused by secondary, tertiary and quaternary structure and ensure integration. The second stage is the acquisition of the NMR spectrum using a diffusion filter to eliminate the resonances of water and other components of the composition. Finally, this data is compared with a reference standard to accurately determine the concentration. This method is referred to herein as method A. The concentration determined using method A can then be used to determine a molecular parameter such as

- 1 040889 extinction coefficient, protein or peptide.

Thus, the present invention provides a method for determining the concentration of a protein in a solution, which method includes denaturing the protein, performing NMR spectroscopy with a diffusion filter, and calculating the protein concentration using a reference standard. The present invention also provides a method for determining the concentration of a peptide in a solution, which method includes performing NMR spectroscopy with a diffusion filter and calculating the concentration of the peptide using a reference standard. In some embodiments of this invention, the method further includes denaturing the peptide prior to NMR spectroscopy with a diffusion filter.

The present invention provides a method for obtaining the extinction coefficient of a protein in solution, wherein the extinction coefficient is determined by the Beer-Lambert law and the protein concentration used in the Beer-Lambert law is determined by a method including denaturing the protein, performing NMR spectroscopy with a diffusion filter, and calculation of protein concentration using a reference standard.

The present invention also provides a method for obtaining the extinction coefficient of a peptide in solution, wherein the extinction coefficient is determined by the Beer-Lambert law and the concentration of the peptide used in the Beer-Lambert law is determined by a method including the optional step of denaturing the peptide, followed by the implementation of the execution steps NMR spectroscopy with a diffusion filter and calculation of the peptide concentration using a reference standard. In some embodiments of this invention, the method includes denaturing the peptide prior to NMR spectroscopy with a diffusion filter.

The present invention provides a method for determining the concentration of a protein in the preparation of a drug substance or drug product, wherein said substance contains a protein in solution, and said method includes obtaining a molecular parameter of said protein. In some embodiments of the present invention, the molecular parameter is the extinction coefficient, and the extinction coefficient is determined by the Beer-Lambert law, and the protein concentration used in the Beer-Lambert law is determined by a method including denaturing the protein, performing NMR spectroscopy with a diffusion filter, and calculation of protein concentration using a reference standard.

The present invention provides a method for determining the concentration of a peptide in a drug substance or drug product preparation, said substance containing a peptide in solution, and said method comprising obtaining a molecular parameter of said peptide. In some embodiments of this invention, the molecular parameter is the extinction coefficient, and the extinction coefficient is determined by the Beer-Lambert law, and the concentration of the peptide used in the Beer-Lambert law is determined by a method including optionally denaturing the peptide, performing NMR spectroscopy with a diffusion filter and calculating the concentration of the peptide using the reference standard. In some embodiments of this invention, the method includes denaturing the peptide prior to NMR spectroscopy with a diffusion filter.

The present invention further provides a method for obtaining the molecular parameter of a protein in solution, the method comprising denaturing the protein, performing NMR spectroscopy with a diffusion filter, calculating the concentration of the protein using a reference standard, and determining the molecular parameter of the protein based on the calculated concentration. In one embodiment of the present invention, the calculated protein concentration and the determined property of the system are used in an appropriate equation to determine the molecular parameter of the protein. In some embodiments of this invention, the molecular parameter is the extinction coefficient, and the corresponding equation is the Beer-Lambert law.

The present invention also provides a method for obtaining the molecular parameter of a peptide in solution, the method comprising optionally denaturing the peptide and then performing NMR spectroscopy with a diffusion filter, calculating the concentration of the peptide using a reference standard, and determining the molecular parameter of the protein based on the calculated concentration. In one embodiment of the present invention, the calculated peptide concentration and the determined property of the system are used in an appropriate equation to determine the molecular parameter of the peptide. In some embodiments of this invention, the molecular parameter is the extinction coefficient, and the corresponding equation is the Beer-Lambert law.

The present invention provides a method for determining a dose in the preparation of a drug substance or drug product, wherein said substance contains a protein in solution, the method comprising obtaining a molecular parameter of a protein from a control lot, wherein the molecular parameter is determined using a method that includes denaturing the protein, performing NMR spectroscopy with a diffusion filter, calculating the protein concentration using a reference standard, and determining the molecular parameter of the protein based on

- 2 040889 calculated concentration. In a further embodiment of the present invention, the method includes preparing a test lot of the drug substance or drug product, wherein said protein concentration in the test lot is determined based on a molecular parameter. In some such embodiments of the present invention, the protein concentration is determined using a molecular parameter and an appropriate property of the system in an appropriate equation.

The present invention provides a method for determining a dose in the preparation of a drug substance or drug product, wherein said substance contains a peptide in solution, the method comprising obtaining a molecular parameter of a peptide from a control lot, wherein the molecular parameter is determined using a method that includes include optionally denaturing the peptide, performing NMR spectroscopy with a diffusion filter, calculating the concentration of the peptide using a reference standard, and determining the molecular parameter of the peptide based on the calculated concentration. In an additional embodiment of this invention, the method includes preparing a test lot of a drug substance or drug product, wherein the specified concentration of the peptide in the test lot is determined by a molecular parameter. In some such embodiments of the present invention, the peptide concentration is determined using the molecular parameter and the corresponding property of the system in the appropriate equation.

The invention also provides a method for determining the concentration of a protein or peptide in the preparation of a control lot, wherein the control lot contains the protein or peptide in solution, and the method includes denaturing the protein or optionally denaturing the peptide, performing NMR spectroscopy with a diffusion filter , calculating the concentration of the protein or peptide using the reference standard and the reference method, and determining the molecular parameter of the protein or peptide based on the calculated concentration. In another embodiment of the invention, the method further comprises determining the concentration of a protein or peptide in a test lot preparation, wherein said peptide or protein concentration in the test lot is determined based on a molecular parameter.

This invention provides implementation options that are applicable to the methods described in this document. One such embodiment of the present invention includes a method in which a protein or peptide is denatured with a chaotropic agent. In some such embodiments of this invention, the chaotropic agent is urea-d ₄ . In other such embodiments of this invention, the chaotropic agent is guanidinium chloride-d ₆ . In some embodiments of this invention, the reference standard is an internal reference standard. In preferred embodiments of this invention, the reference standard is an external reference standard. In some such embodiments of this invention, the external reference standard is a low molecular weight primary standard. In a specific embodiment of this invention, the external reference standard is maleic acid. In some such embodiments of this invention, the external reference method is PULCON. In some embodiments of this invention, the protein is an antibody. In some such embodiments of this invention, the antibody is a monoclonal antibody. In other such embodiments of this invention, the antibody is a bispecific antibody. In some embodiments of this invention, the solution containing the measured protein or peptide contains D2O.

The invention also contemplates the use of Method A, or a molecular parameter determined from the concentration obtained in Method A, in a variety of applications. Such applications include the determination of toxicity, the determination of clinical and/or commercial dosage of a drug, the method of determining corporate or primary reference standards, the method of determining the concentration of a protein or peptide during a manufacturing process, the method of aliquoting a protein or peptide, the method of aliquoting a protein or peptide, at which the concentration of the aliquoted protein or peptide is similar to the desired concentration, a method including a batch release test, or a method for formulating a drug substance or drug product.

This invention contemplates that method A can be used for the purposes described herein relating to any protein or peptide, for example, proteins or peptides in the fields of therapeutics or the petroleum industry. By way of example (not intended to be limiting), method A can be implemented in applications relating to any or all of the following, including any of their biomimetics, wherein the protein or peptide is the active ingredient in the following, or any of the following biomimetics: dulaglutide (sold as Trulicity® in some countries), ixekizumab (sold as Taltz® in some countries), ramucirumab (sold as Cyramza® in some countries), cetuximab (sold as Cyramza® in some countries).

- 3 040889 in other countries under the name Erbitux®), olaratumab (sold in some countries under the name Lartruvo®), necitumumab (sold under the name Portrazza ® in some countries), anti-CGRP antibodies such as galcanezumab (also known as LY2951742), antibodies anti-IL-23 such as mirikizumab (also known as LY3074828), solanezumab (also known as LY2062430), tanezumab, anti-N3pG-Ae antibodies, anti-tau antibodies, anti-Άβ42 antibodies, anti-BAFF/IL-17 bispecific antibodies, antibodies anti-CSF-1R, anti-CXCR1/2, anti-IL-21, bispecific anti-IL-23/CGRP, anti-IL-33, anti-PD-L1, and anti-TIM-3.

The invention also contemplates that method A: (i) is suitable for use as a basis for measuring other intrinsic parameters of a protein or peptide, or for converting any of the current relative concentration methods to an absolute concentration method, (ii) can be used to quantification of peptides that lack chromophore amino acids and are difficult to monitor with ultraviolet light, (iii) allows for simpler, faster and safer sample preparation and data collection than the current ΆΆΆ method, (iv) provides linearity, reliability and accuracy of the obtained concentrations , which are suitable for various proteins and compositions, and (v) can be used to quantify other types of macromolecules (eg polymers, surfactants, etc.) in the presence of small molecular weight impurities.

The present invention contemplates that the methods described herein may use the mathematical equations also described herein. For example, the protein concentration, c _P , in grams per liter can be calculated from the qNMR spectrum using the following equation

ARCH ^C R ^{= C} 5 d and fERfoiifu (Lv. 1).

This document provides a detailed description of this equation as an example.

One of ordinary skill in the art will appreciate that while examples of specific software are provided herein, other programs may be used to obtain comparable results.

Brief description of graphic materials

Fig. 1. Pulse train bppste for Agilent spectrometers (also known as Dbppste) and the definition of various delays.

Fig. 2. 1H NMR spectra for a standardized monoclonal antibody from NIST - US National Institute of Standards and Technology (NIST mAb). ^1H NMR spectrum of NIST mAbs (Ά) in native form with standard single pulse sequence, (B) same as Ά with 5000x vertical scale, (C) in native form with diffusion filter, and (D) in denatured form with diffusion filter. Peaks marked with an asterisk are from histidine buffer. The number of scans was 1024 for A - C and 64 for D.

Fig. 3. D-measurement for NIST mat. Fig. 3a: NMR spectra for standardized denatured mAb obtained with bppste pulse train. Inset: Peak decomposition for I/L/V resonances. Inset: An enlarged image of the specified area. Fig. 3b, left: Pure NMR spectra for various diffusible particles determined using the DECRA algorithm. Fig. 3b, right: Steiskal-Tanner plot and diffusion coefficients from the DECRA algorithm.

Fig. 4. Tj and T2 measurements for NIST mat. Fig. 4a: Representative Tj and T2 measurements using the bppste pulse train. Left: NMR spectra and bppste pulse train settings for Ti measurements. Right: NMR spectra and bppste pulse train settings for T2 measurement. Fig. 4b: Plot for Tj and T2 calculations.

Fig. Fig. 5. NMR spectra with a diffusion filter (sNMR-DF, eng. DF-qNMR) for 14 samples of denatured BSA (bovine serum albumin) from NIST (NIST BSA, eng. NIST BSA). Inset: An enlarged image of the specified area.

Fig. 6. Concentrations of NIST BSA Samples: Method A vs. Gravimetric Measurement.

Fig. 7. NIST BSA Partial Specific Volume: sample density versus BSA concentration.

Fig. 8. DP-sNMR spectra for 14 denatured bispecific antibody samples. DP-sNMR spectra for 14 denatured bispecific antibody samples. The residual water peak is marked with an asterisk. Inset: An enlarged image of the specified area.

Fig. 9. Bispecific antibody sample concentrations: Method A versus gravimetric measurement.

Definitions

The term nuclear magnetic resonance spectroscopy (also referred to as NMR spectroscopy), as used herein, refers to a spectroscopic technique known to one of ordinary skill in the art, in which nuclear magnetic resonance (NMR) spectra can be used to study the structure of organic molecules. Term

Quantitative nuclear magnetic resonance spectroscopy (also referred to as qNMR spectroscopy), as used herein, refers to obtaining quantitative information about the purity or concentration of a sample from one or more NMR spectra. Such spectra are referred to herein as quantitative nuclear magnetic resonance (QNMR) spectra. NMR spectroscopy may include the use of a diffusion filter, which can be used to selectively attenuate the signal from smaller molecules in a mixture depending on their size (see, for example, Stilbs P., Prog. Nucl. Magn. Reson. Spectrosc. 19 (1): 1-45 (1987)). To balance the attenuation of the matrix peaks and the protein peaks, the strength of the diffusion filter can be determined by one of ordinary skill in the art based on the context of each experiment. For example, if the balance is too weak, then the diffusion filter is of limited value. If the balance is too strong, then the diffusion filter begins to suppress protein signals, which reduces the signal-to-noise ratio of the measurement. This decrease can lead to an increase in the time of the experiment. As another example, if one of the matrix components is not completely removed and is an interfering peak, then it will contribute to the protein peak area and must be subtracted to obtain the most accurate results. This can be done by preparing a blank sample containing the matrix without protein, passing the blank sample with a diffusion filter, determining the area of the remaining peak of the matrix, and subtracting this number from the area obtained for the protein sample.

The term reference standard, as used herein, refers to a compound whose ¹ H-NMR spectrum provides at least one distinctive peak representing a known number of protons, and whose purity and concentration are known with a high degree of certainty. The reference standard may be an internal reference standard, with the reference standard present in the solution containing the protein or peptide of interest, or the reference standard may be external, with the reference standard separated from the solution containing the protein or peptide of interest. The reference standard may be a low molecular weight primary reference standard, which is a material that is not calibrated to another standard but is instead defined by qualities such as its weight. Certified low molecular weight reference samples commonly used as internal ¹ H qNMR standards are readily available and can also be used as external standards [see e.g. Rigger et al., M. I AOAC International, 2017, 100, 1365-1375] .

The reference method is a mathematical algorithm that uses data from a reference standard. For example, an external reference method is a mathematical algorithm that uses data from an external reference standard. One example of this is the technique for determining the concentration by pulse duration (PULCON method) (Wider G. and Dreier L. I, Am. Chem. Soc. 128 (8), 2571 - 2576 (2006)).

The term DP-sNMR spectra, as used herein, refers to NMR spectra to which a diffusion filter has been applied and from which quantitative information can be obtained on the purity or concentration of a protein or peptide of interest when compared to a reference standard.

The term molecular parameter, as used in the context of this document, means a scalar value that refers to how some system property (e.g. absorbance, physical density, refractive index) changes in response to changes in other system properties (e.g. temperature, pressure). or compositions). The equation (corresponding equation) used to determine the molecular parameter may vary depending on which property or properties of the system is known. A molecular parameter refers to an intrinsic property of a protein or peptide.

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 absorbing particles and the length of the light path. The extinction coefficient of a protein or peptide [mL/(mg-cm)] can be determined using a combination of ultraviolet and visible (UV-visible) spectroscopy and method A as follows: A=εcl (Beer-Lambert Law), where A represents is the optical density of the protein or peptide solution, l is the path length [cm], and c is the absolute concentration of the protein or peptide [mg/mL]. As another example, the apparent partial specific volume (y) [mL/g] of a protein or peptide can be determined using a combination of densitometry and method A as follows:

where ρ is the physical density of the protein or peptide solution [g/ml], p ₀ is the physical density of the protein or peptide solution matrix [g/ml], and ^c is the absolute concentration of the protein or peptide [mg/ml]. As a third example, the (differential) refractive index increment (dn/dc) of a protein or peptide [ml/mg] can be determined using a combination of (differential) refractometry and method A as follows:

- 5 040889 η = η ₀ + (dn/dc^c where n is the refractive index of the protein or peptide solution, n ₀ is the refractive index of the protein or peptide solution matrix, and c is the absolute concentration of the protein or peptide [mg/ml ].

One of ordinary skill in the art can determine the molecular parameter of interest by linear regression of several optical density/physical density/refractive index measurements as a function of concentration (concentration determined by Method A). A molecular parameter, such as the extinction coefficient, can be used to determine drug concentration by measuring the corresponding property of the system, such as absorbance in the UV spectrum, and solving the appropriate equation that correlates them (eg, the Beer-Lambert law).

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) that are cross-linked via intra- and inter-chain disulfide bonds. The variable regions of each heavy chain-light chain pair are associated to form binding sites. The heavy chain variable region (V _H ) and the light chain variable region (V _L ) can be subdivided into regions of hypervariability called complementarity determining regions (CDR) interspersed with more conserved regions called framework regions (FR). Each V _H and V _L consists of three CDRs and four FRs arranged in the amino to carboxy direction in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDR regions contain most of the residues that provide specific interactions with the antigen.

The term bispecific antibody, when used in the context of this document, refers to a bivalent antibody construct that includes, but is not limited to, the IgGocFv format (as specified in PCT/US2015/058719) and the bivalent IgG formats (as described in US 2018/ 0009908). The invention also contemplates that any human engineered protein or antibody, regardless of tertiary or quaternary structure, can also be used in the methods described herein. Examples of such human engineered proteins or antibodies include trispecific or tetraspecific antibodies and fusion proteins.

In the context of this document, the peptide contains a polymer chain of amino acids. These amino acids may be natural or synthetic amino acids, including modified amino acids. Peptides may have a less pronounced secondary and tertiary structure, but are prone to aggregation in solution; therefore, the peptide can also be denatured with a chaotropic agent. Denaturation of the peptide can lead to an increase in the signal-to-noise ratio in NMR spectroscopy, thereby facilitating integration. Peptide denaturation can also allow more concentrated samples to be tested, reducing experiment time. For the purposes of the methods described herein, the determination of peptide concentration may be more accurate if the peptide is denatured. 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 one of skill in the art.

One of ordinary skill in the art understands that a protein contains one or more polymeric peptide chains of amino acids. These amino acids may be natural or synthetic amino acids, including modified amino acids. The protein may be a recombinant protein. The primary structure of a protein consists of its linear sequence of monomeric amino acid subunits. The secondary structure of a protein contains a hydrogen bonding structure giving rise to three-dimensional structural features of local amino acid chain segments such as α-helices and β-sheets. The tertiary structure of a protein describes the overall shape of the protein as determined by the three-dimensional coordinates of the atoms. Quaternary structure refers to the arrangement of two or more protein subunits in a complex. A protein can be denatured when a solution containing the protein is exposed to an external stress or chaotropic agent and the protein becomes unfolded. Thus, the denatured protein loses most of its secondary, tertiary, and quaternary structural features. Examples of chaotropic agents include urea-d ₄ and guanidinium chloride-d ₆ .

In addition, the term solution, when used in the context of this document, refers to a peptide or protein in a man-made aqueous mixture that contains components other than water. The term batch refers to a specific quantity of a drug or other material that is a protein or peptide, and must be of uniform characteristics and quality within certain limits, and produced according to one production order during the same production cycle. The term lot refers to a lot or a specific identified part of a lot that has uniform characteristics and quality within certain limits; or, in the case of a drug product manufactured in a continuous process, the batch is

- 6 040889 a certain identified quantity produced per unit of time or per unit of quantity in such a way as to guarantee its uniform characteristics and quality within certain limits. The term control lot refers to an established and properly characterized internal reference material, said material containing a protein or peptide, and said material is from a batch(es) representative of manufacturing 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 solution in a control lot. The molecular parameter, such as the extinction coefficient obtained from the control lot, is then used to determine the concentration of the protein or peptide in the subsequent lot (referred to herein as the test lot).

The term drug product, when used in the context of this document, means a finished dosage form, for example, a tablet, capsule or solution, which (which) contains a drug substance, usually, but not necessarily, in combination with one or more other ingredients. A drug substance is an active ingredient that is intended to provide pharmacological activity or other direct effect in the diagnosis, rehabilitation, alleviation, treatment or prevention of a disease, or to affect the structure or any function of the human body, but does not contain intermediates used in the synthesis of such ingredient. When used in the context of this document, the drug substance contains a protein or peptide. Method A can be used for various purposes associated with a drug product or drug substance containing a protein or peptide, for example, in the manufacturing process of a drug product or drug substance.

The term dose refers to the amount of protein or peptide in the matrix that will elicit the desired biological or medical response. The substance may be lyophilized or in aqueous solution. For the preparation of a substance intended for therapeutic use, one skilled in the art understands that a reliable and accurate concentration of a protein or peptide is required. The methods described herein provide a novel means of obtaining the concentration of a protein or peptide by determining the extinction coefficient or other molecular parameter of a protein or peptide in a control lot and then using that extinction coefficient or other molecular parameter to determine the protein or peptide concentration in subsequent test lots.

Aliquoting means removing part of a larger volume from one vessel and adding it to a separate vessel. The container can be any item such as a container, vial, flask, or device that is suitable for storing a protein or peptide in lyophilized form or in solution.

Batch release testing refers to the appropriate laboratory determination of satisfactory compliance with the final specifications for the medicinal product (containing a protein or peptide), including the strength (concentration) of each active ingredient, prior to release (release by the manufacturer of the medicinal product to the market). The batch release test may be, for example, a UV test at batch release. The concentration of a protein or peptide can be determined based on the extinction coefficient of the protein or peptide, with the extinction coefficient determined using Method A on a control lot.

The following examples further illustrate the invention and provide exemplary methods and procedures for performing various specific embodiments of the invention. However, it should be understood that the following examples are provided by way of illustration and not limitation, and that various modifications may be made by those skilled in the art.

Examples

Sample preparation.

Reference Standard: Any compound with a 1H-NMR spectrum that provides at least one characteristic peak that is representative of a known number of protons, of known purity and concentration with a high degree of confidence, that is chemically and physically stable in solution, can be used to provide an absolute concentration calculation according to the DFQNMR spectra. For example, maleic acid can be used as an external reference standard for quantitation. 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 aliquot is transferred to a tube, such as a Wilmad 435 precision 4 mm NMR tube, such that the height of the sample is 40 mm.

Protein samples: guanidinium chloride-d ₆ (0.6 g, 6 mmol) is placed in a 1 ml volumetric flask. The flask is placed on a balance and 400 µl of the protein solution is added. The solution is then gently sonicated and vortexed until all solids are dissolved. Add D2O to reach final volume. An aliquot is transferred to a tube, such as a Wilmad 435 precision 4 mm NMR tube, such that the height of the sample is 40 mm.

- 7 040889

Peptide samples: MO4eeuHy-d ₄ (0.13 g, 2 mmol) are placed in a 1 ml volumetric flask. The flask is placed on a balance and ~1 ml of the protein solution is added. The solution is then gently sonicated and vortexed until all solids are dissolved. Add D ₂ O to reach the final volume. An aliquot is transferred to a tube, such as a Wilmad 435 precision 4 mm NMR tube, such that the height of the sample is 40 mm.

Calculations.

The protein concentration, c _P , in grams per liter is calculated from the DP-qNMR spectrum using the following equation:

^C P ^{= Cs} A H f ^DF f ^ER ^Ilfu (Eq. 1) where c _S is the concentration of the reference standard, AP is the area of the selected protein peak in the DPhNMR spectrum (usually the area of the methyl groups of valine, isoleucine and leucine in the range of 1.0 -1.4 ppm (parts per million, but can be any peak with sufficient resolution), HS is the number of protons contributing to the reference standard peak, A _S is the area of the reference standard peak, _HP is the number of protons contributing to the peak of the protein, and the various f are functions that depend on the specific experimental conditions. The parameter f _DF takes into account the attenuation of the protein peak area due to the diffusion filter and is derived from the Steiskal-Tanner equation (Johnson, C. S. et al., Concepts in Magnetic Resonance Part A 2012, 40A, 39-65). For the pulse sequence bppste (Pelta, M. D et al., Magn. Reson. Chem. 1998, 36, 706-714) used in this document, this parameter is given by the following equation:

f _DF = 2 eA in 5 p - ₃ - ₂ ] ( _ur where γ is the gyromagnetic ratio for 1H, g is the strength of the pulsed field gradient, δ is the duration of the pulsed field gradient, Δ is the diffusion delay, T1 is the spin-lattice nuclear relaxation time for the protein protons of interest, _T2 is the nuclear spin-spin relaxation time for the protein protons of interest, D is the protein translational diffusion coefficient, τ1 is the time between the second and third 90-degree pulses in the bppste pulse sequence (see Fig. 1), τ2 is the total time between the first and second 90-degree pulses (and between the third 90-degree pulse and capture) and τ ₃ is the total time between pulsed field gradient pulses of pairs of bipolar pulses. the following instrument parameters:

Δ δ 46p 2t _rg (Eq. 3) ⁼ ^(δ I 2tg _S II 2t _rg ) (Eq. _{4 )} ^τ 3 ^— tgs “I + t _rg (y _p the receiver pulse preceding the pulse and Θ is the width of the 90-degree pulse.For other diffusion pulse sequences, the expression for f _DF will be different.The variable f _ER contains the instrument parameters needed for external referencing.In its simplest form:

g _ ^S S ^G S

Sp G _? (Eq. 6) where S and G are the number of scans and receiver gain, respectively, used to collect the individual NMR spectra of the protein and reference standard. When using the PULCON technique, this variable takes the following form:

,. _ 7p θρ S _s G _s “ T _S 0 _S S _P G _{p (} y _p . ₇₎ where T is the sample temperature at the time of NMR acquisition. f _DIL takes into account the dilution of the original protein sample to the final NMR sample. If the final volume is obtained by a volumetric method, and an aliquot of the original sample is obtained by a gravimetric method, then this coefficient is determined as follows:

vokfniL = ---Wt _t (Eq. 8) where vol _F is the volume of the final solution (ie the NMR sample solution) and wtI and dI are the mass and physical density of an aliquot of the original protein solution. Alternatively, if the dilution is done by gravimetric addition of both the protein stock solution and D2O, then:

_=i ! d _s ^D2C ' (LEV 9) where wt _D2O and d _D2O are the mass and physical density of D2O. Finally, fU converts the concentration units of the quantification reference standard, usually millimolar, to typical

- 8 040889 units - grams per liter (or, equivalently, milligrams per milliliter). In this case:

_ M _R ^Ti 1000 (Eq. 10) where MP is the molecular weight of the protein.

The first exponential term in equation 2 describes the attenuation of the signal due to nuclear relaxation T1, the second - due to nuclear relaxation T ₂ and the third - due to molecular diffusion. Of all the terms in this equation, these three quantities (T1, T2 and D) are the only unknowns; all other quantities are pulse train parameters/instrument parameters or a physical constant (γ). T1, _T2 and D depend on the state of the molecule and the conditions of the solution and must be measured for each unique sample. This can be done with three separate experiments using the same pulse train as in the diffusion filter. The D measurement is a well-known experiment in which multiple spectra are obtained with increasing gradient strength (Stejskal, E. O. and Tanner, JE, J. Chem. Phys., 1965, 42, 288-292). This keeps the T1 and _T2 terms in Equation 2 constant, making the signal attenuation dependent solely on D. D is then computed using a suitable algorithm such as DECRA (Windig, W.; Antalek, B. Chemom. Intel. Lab. Syst. , 1997, 37, 241-254). To measure T1 with an Agilent bppste pulse train, several spectra are obtained using combinations of diffusion delay and gradient strength (gzlvl1) such that:

gzlvll„ = αζίνίΐι X — (Eq. 11) where Δ' is the actual diffusion delay defined as follows:

(Lvl 12)

This keeps the T ₂ and D terms of equation 2 constant, making the signal decay due solely to the relaxation of T ₁ . The effective T ₁ value for protons of the selected protein peak is then calculated based on the mono-exponential correspondence of AP vs. τ 1 .

To measure T ₂ with an Agilent bppste pulse train, several spectra are acquired using combinations of gradient stabilization delay, diffusion delay, and gradient strength such that:

Ai - 41 + (1 _g&p - t _rg ,i) (Ur ,

a gzlvl1 _n is defined as shown above. This keeps the terms T1 and D of this equation constant, making the signal decay due solely to the relaxation of T ₂ . The effective T ₂ value for the protons of a selected protein peak can be calculated from the mono-exponential dependence of _AP versus τ ₂ .

Method A: data collection.

The Agilent DD2 600 MHz NMR spectrometer is equipped with an Agilent 1H- ¹⁹ F/ ¹⁵ N- ³¹ P PFG OneProbe module. Probe temperature gradients and pulsed field gradients are calibrated with ethylene glycol and 1% H ₂ O in D2O samples, respectively. The NMR spectrum was obtained at 30.0°C using a bipolar pulse pair stimulated echo (bppste) pulse train. The number of scans is 64. Acquisition parameters include spectral width - 20 ppm, acquisition time - 1.363 s, relaxation delay - 30 s, diffusion delay - 150 ms, gradient pulses with a duration of 1.4 ms and with a force of 0.569 T/ m (92% of the maximum), and a stabilization gradient delay of 1 ms.

For translational diffusion coefficient (D) measurements, acquisition parameters include a relaxation delay of 1 s, a diffusion delay of 200 ms, a diffusion gradient duration of 2.0 ms, a gradient stabilization delay of 0.5 ms, and seven gradient strengths in the range from 0.228 to 0.569 T/m (approx. 37-92% of maximum) with a logarithmic interval. The spin-lattice nuclear relaxation time (T ₁ ) is measured with a pulse train bppste using a spectral width of 20 ppm, an acquisition time of 1.363 s, a relaxation delay of 3.637 s, a diffusion gradient duration of 2.0 ms, and a gradient stabilization delay of 0.5 ms. Five pairs of values are 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) - diffusion delay and gradient strength.

Nuclear spin-spin relaxation time (T2) is also measured with a bppste pulse train using a spectral width of 20 ppm, an acquisition time of 1.363 s, a relaxation delay of 1 s, and a diffusion gradient duration of 1.4 ms. Six sets of parameters are used: gradient stabilization delay, diffusion delay and gradient strength - (1 ms, 150.001 ms, 0.570 T/m); (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).

- 9 040889

Data analysis.

DP-cNMR, _T1 and T2 data are processed with MNova software version 11.0 (Mestrelab Research, SL, Santiago de Compostela, Spain) and diffusion data are processed and analyzed with DOSYToolbox software version 2.5 (Nilsson, MJ Magn Reson 2009, 200, 296302). The FID parameters are reset once and multiplied by a 2.93 Hz exponential window function before Fourier transform. Diffusion coefficients are obtained using the DECRA algorithm available in the DOSYToolbox program. T1 and T2 values are computed from the bppste pulse train by regression analysis using MATLAB 2016b software (MathWorks, Inc., Natick, MA, USA). The peak areas of the methyl groups of leucine, isoleucine and/or valine are obtained by deconvolution using the line fitting procedure in the MNova or CRAFT software.

Using method A with NIST RM 8761 antibody.

To illustrate the challenges of quantifying large proteins in a formulation by NMR, NIST Sample RM 8761 (10 g/L antibody, 12.5 mM L-histidine HCl, pH 6.0, commercially available from the US National Institute of Standards and Technology (NIST), see e.g. Schiel et al., Anal. and Bioanal. Chem., 410(8): 2127-2139 (2018)) treated with 10% D2O and ^{a 1} H 1D NMR spectrum was obtained (Fig. 2) . The signal from the water is so intense and wide that it obscures the signal from the antibody. Peaks of the histidine excipient are also observed in this spectrum. To obtain a real protein spectrum, these dominant peaks must be eliminated. One NMR technique that is ideal for this application is the diffusion filter, in which peaks are excluded from the spectrum based on the diffusion coefficients and hence the size of the respective molecules. The 1H-NMR spectrum obtained on the same NIST mAb sample using a bipolar pulse pair stimulated echo (bppste) pulse train as a diffusion filter is also shown in FIG. 2. The diffusion filter is effective at removing unwanted signals from the composition and does not introduce baseline artifacts or phase distortion.

However, in most cases, the resolution is insufficient for accurate identification and integration of peaks due to the characteristic wide linewidth of protein resonances. This is primarily due to the higher order structure (HOS) of the protein. The same type of amino acid, which would otherwise have very similar chemical shifts, exhibits a wide distribution of chemical shift due to the unique magnetic environment resulting from the protein's secondary, tertiary, and quaternary structure. Therefore, SVP is removed by denaturing the protein. Adding a denaturing agent to a protein solution results in an increase in sample volume. For accurate quantitation, more solvent is added to achieve an accurate volume, for example with a volumetric flask. This is the basis for f _DIL in Equation 1.

1H-NMR after applying a diffusion filter to a NIST RM 8761 sample prepared using 6 M guanidinium-d ₆ is shown in FIG. 2. There is a decrease in the width of the spectral line and a subsequent increase in the signal-to-noise ratio. Groups of peaks between 1.0-1.4 ppm, which correspond to the methyl groups of isoleucine, leucine and/or valine, have a resolution close to the baseline, which allows them to be integrated for quantitative analysis.

The diffusion filter is effective in separating protein signals from the matrix; however, the diffusion filter results in an initially insufficient quantification of the measured NMR peak areas. Routine NMR experiments use a simple NMR pulse sequence that consists of three main elements: a nuclear relaxation delay, a single RF pulse, and a data acquisition period. When a sufficiently large nuclear relaxation delay is used, the resulting NMR peak area is exactly and reproducibly proportional to the number of nuclei observed; thus, the experiment is quantitative. All other NMR pulse sequences, including the diffusion sequence used in the context of this invention, require multiple RF pulses separated by different delays. Consequently, the resulting spectra are no longer quantitative in nature due to factors that attenuate the peaks from the equilibrium value. However, if these factors are known and the degree of attenuation can be calculated, the diffusion filter can be made quantitative. This is the basis for f _DF in Equation 1.

An external reference standard is preferred over an internal reference standard because the former avoids potential interactions between the two molecules and/or peak overlap in the NMR spectrum, although an internal standard can also be used. Several external standard methods have been described, including the PULCON technique (ie, determination of concentration based on pulse width). This method correlates the absolute areas of two separate spectra, one from the reference standard and the other from the analyte, even if the solution conditions and experimental parameters differ. Therefore, the external reference standard need not have the same characteristics as the analyte of interest, or even be a protein. In addition, instead of a standard for each amino acid, as in the case of AKA, for this

- 10 040889 only one standard is required.

To demonstrate the validity and accuracy of Method A, three independent replicates of the NIST RM 8761 sample were evaluated. Sufficient signal-to-noise ratio for this 10 g/L protein formulation was achieved using only 64 scans and a total acquisition time of 37 minutes. The group of signals for methyl groups from I/L/V (isoleucine/leucine/valine) in this protein (1.0-1.4 ppm), which corresponds to 1476 protons, had a resolution close to the baseline. These results provide precise integration for quantitative analysis. To facilitate concentration calculation, T1, T2 and D were also measured for this group of peaks using the bppste pulse train as described above, and the data acquisition and analysis performed are shown in FIG. 3 and 4.

The total experiment time for all four NMR experiments in one repetition was 110 min. The concentration of each replicate was then calculated using the formula from Eq. 9 using intact non-glycosylated molecular weight (148041 Da) and a dilution factor based on aliquot weight and measured sample density. The mean concentration was 10.02 g/l with a relative standard deviation (RSD) of 0.55%. This corresponds to the labeled value of 10 g/l.

These data demonstrate that method A can be used to reliably and accurately determine antibody concentration.

Using method A with NIST bovine serum albumin (NIST BSA).

Standard reference material 927e from NIST (BSA) (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 US technologies) were gravimetrically diluted to four additional protein concentrations (Table 1, samples B1-B4). A total of fourteen samples were analyzed, including six replicates for the average concentration (B3), three replicates for each of the maximum (B5) and minimum (B1) concentrations, and single replicates for intermediate concentrations (B2 and B4). An NMR sample for each replicate was prepared as described above. Data collection and analysis according to method A was performed as described above. The fourteen DPQNMR spectra obtained are shown superimposed on each other in FIG. 5.

These data demonstrate that the water peak is almost eliminated, the baseline is intact, and the I/L/V methyl group signals in the protein have near baseline resolution. The concentration for each sample was calculated using the formula from Eq. 1 using a molecular weight of 66398 Da, and the results are shown in table. 1. For all five concentrations, the value measured by method A is the same as the value calculated from the gravimetric dilution. As shown below in Table. 1, RSD is less than 1% for all three replicate sets.

Table 1. Results for NIST BSA samples.

Sample Method A, conc. (g/l) OSO gravimetric method, conc. (g/l) Difference IN 1 9.297 0.61% 9.525 2.39% AT 2 23.90 -- 23.91 0.04% VZ 37.53 0.81% 38.11 1.54% AT 4 52.50 -- 52.82 0.60% AT 5 66.18 0.82% 67.38 1.78%

As shown in FIG. 6, a plot of Method A concentrations versus gravimetric concentrations has a regression line with R ² = 0.9997, indicating linearity over the entire concentration range studied.

These data demonstrate that method A can be used to reliably and accurately determine the concentration of a medium sized protein over a wide range of concentrations.

In FIG. 7 is a plot of sample physical density versus protein concentration determined using method A. The equation relating the two quantities is

as defined above. Based on the slope of the regression line, the partial specific volume (V) of BSA is determined, which was 0.718 ml/g.

Using method A with a bispecific antibody.

Samples of the bispecific antibody (defined by sequences from Table 1 of US Pat. No. 9,718,884) at five different concentrations and a total of 14 samples were prepared as described above. Method A data collection and analysis was performed as described above. In accordance with the procedures essentially described above, the following data were obtained.

In FIG. 8 shows the resulting DP-qNMR spectra, which are similar in general appearance and quality to the BSA spectra (shown above). Concentrations were calculated using the formula from Eq. 1, with is- 11 040889

using the theoretical molecular weight of an intact non-glycosylated protein (~ 200,000 Da), and summarized in Table. 2 below. Table 2. Results for the bispecific antibody. Sample Method A, conc. (g/l) OSO Estimated conc. (g/l) Difference C1 9.427 0.25% 9.277 -1.61% C2 24.59 - - 24.61 0.09% NW 41.62 1.25% 39.67 -4.92% C4 54.43 -- 55.02 1.07% C5 78.37 0.85% 74.01 -5.89%

As shown in FIG. 9, a plot of concentrations obtained by method A versus concentrations obtained by the gravimetric method has a regression line with R ² = 0.996.

These data demonstrate that method A can be used to determine the concentration of a bispecific antibody over a very wide range of concentrations.

Using method A with peptide ~ 5000 Da.

Diluted small peptide samples (ca. 5000 Da; sequenced in Example 4 of PCT/US 2017/041922) are prepared with and without a chaotropic agent as described above. A lower concentration of the denaturing agent (2 M) is acceptable because the peptide in native form is a random helix and the concentration is low. Basic conditions are required for sample stability; hence, urea-d ₄ is used. Method A data collection and analysis was performed as described above, except that 624 scans were performed for each data collection instead of 64 scans. Following the procedures essentially as described above, the following data were obtained. A slight phase distortion was observed for the residual water signal, but this did not interfere with data analysis. The addition of a chaotropic agent resulted in better water suppression, more distinct peaks and consequently greater resolution and sensitivity. The concentration of each replicate was calculated using the formula from Eq. 1. Average total protein concentration was 0.65 g/l with an RSD of 3.6%. This value was similar to the value obtained using the mass balance method (0.63 g/l). These data demonstrate that method A can be used with a diluted small peptide sample.

Exemplary Embodiments of the Invention

The following is a list containing illustrative embodiments of the present invention in accordance with this disclosure, which are various embodiments of the present invention in accordance with this disclosure. These illustrative embodiments of this invention are not intended to be exhaustive or to limit this disclosure to the precise forms disclosed, but instead, these illustrative embodiments of this invention are provided to assist in further describing this disclosure so that others skilled in the art can use the ideas they provide.

1. A method for determining the concentration of a protein or peptide in a solution, wherein said method includes:

(i) protein denaturation or optionally peptide denaturation;

(ii) performing qNMR spectroscopy with a diffusion filter; and (iii) calculating the concentration of the protein or peptide using the reference standard and the reference method.

2. The method according to the implementation variant of the present invention 1, characterized in that the said peptide is denatured.

3. The method according to embodiment of the present invention 1 or according to the embodiment of the present invention 2, characterized in that said protein or peptide is denatured with a chaotropic agent.

4. Method according to embodiment 3 of the present invention, characterized in that said chaotropic agent is guanidinium chloride-d ₆ or urea-b ₄ .

5. The method of any one of embodiments 1-4 of this invention, wherein the reference standard is an external reference standard.

6. The method of embodiment 5, wherein said external reference standard is a low molecular weight primary standard.

7. The method of embodiment 5, wherein said external reference standard is maleic acid.

8. The method according to any of the embodiments of the present invention 1-7, characterized in that the solution contains D2O.

- 12 040889

9. The method according to any one of the embodiments of the present invention 1-8, characterized in that the reference method is the PULCON method.

10. The method according to any of the embodiments of the present invention 1-9, characterized in that said protein is an antibody.

11. The method of embodiment 10, wherein said antibody is a monoclonal antibody.

12. The method of embodiment 10, wherein said antibody is a bispecific antibody.

13. A method for determining the molecular parameter of a protein or peptide in solution, wherein said method includes:

(i) protein denaturation or optionally peptide denaturation;

(ii) performing qNMR spectroscopy with a diffusion filter;

(iii) calculating the concentration of the protein or peptide using the reference standard and the reference method; and (iv) determining the molecular parameter of the protein or peptide based on the calculated concentration.

14. The method according to the embodiment of the present invention 13, characterized in that said peptide is denatured.

15. The method according to the embodiment of the present invention 13 or according to the embodiment of the present invention 14, characterized in that said molecular parameter is an extinction coefficient.

16. The method according to the embodiment of the present invention 15, characterized in that the said extinction coefficient is determined by the Beer-Lambert law.

17. The method according to any of the embodiments of the present invention 13-16, characterized in that said protein or peptide is denatured with a chaotropic agent.

18. The method according to the embodiment of the present invention 17, characterized in that said chaotropic agent is guanidinium chloride-d ₆ or urea ^ ₄ .

19. The method according to any of the embodiments of the present invention 13-18, characterized in that said reference standard is an external reference standard.

20. The method of embodiment 19 wherein said external reference standard is a low molecular weight primary standard or maleic acid.

21. The method of embodiment 20 wherein said external reference standard is a low molecular weight primary standard.

22. The method of embodiment 20 wherein said external reference standard is maleic acid.

23. The method according to any of the embodiments of this invention 13-22, characterized in that the solution contains D ₂ O.

24. The method according to any of the embodiments of the present invention 13-23, characterized in that the reference method is the PULCON method.

25. The method according to any of the embodiments of the present invention 13, 15-24, characterized in that said protein is an antibody.

26. The method of embodiment 25 of the present invention, wherein said antibody is a monoclonal antibody.

27. The method of embodiment 25 of the present invention, wherein said antibody is a bispecific antibody.

28. A method for determining the molecular parameter of a protein or peptide in a control lot, said method comprising:

a) protein denaturation or, optionally, peptide denaturation;

b) performing NMR spectroscopy with a diffusion filter;

c) calculating the concentration of the protein or peptide using the reference standard and the reference method; And

d) determining the molecular parameter of the protein or peptide based on the calculated concentration.

29. The method of claim 28, further comprising determining the concentration of the protein or peptide in the test lot, wherein said concentration of the peptide or protein in the test lot is determined based on a molecular parameter.

30. The method according to the embodiment of the present invention 28 or according to the embodiment of the present invention 29, characterized in that said molecular parameter is an extinction coefficient.

31. The method according to an embodiment of the present invention 30, characterized in that the specified extinction coefficient is determined according to the Beer-Lambert law.

32. The method according to any of the embodiments of this invention 28-31, characterized in that

-

Claims (4)

said peptide is denatured. 33. The method according to any of the embodiments of the present invention 28-32, characterized in that said protein or peptide is denatured with a chaotropic agent. 34. The method of embodiment 33, wherein said chaotropic agent is guanidinium chloride-d ₆ or urea ₄ . 35. The method of any of the embodiments of the present invention 28-34, wherein the reference standard is an external reference standard. 36. The method of embodiment 35 wherein said external reference standard is a low molecular weight primary standard. 37. The method of embodiment 35 wherein said external reference standard is maleic acid. 38. The method according to any of the embodiments of this invention 28-37, characterized in that the solution contains D ₂ O. 39. The method according to any of the embodiments of the present invention 28-38, characterized in that the reference method is the PULCON method. 40. The method according to any of the embodiments of the present invention 28-31, 33-39, characterized in that said protein is an antibody. 41. The method of embodiment 40, wherein said antibody is a monoclonal antibody. 42. The method of embodiment 40, wherein said antibody is a bispecific antibody. 43. The method according to any of the embodiments of the present invention 13-42, characterized in that said method further comprises using a molecular parameter to determine the concentration of the protein or peptide when formulating the protein or peptide. 44. The method of any of the embodiments of the present invention 13-42, wherein said method further comprises using a molecular parameter to determine the concentration of a protein or peptide in a batch release test. 45. The method of embodiment 44, wherein said batch release test is a UV batch release test. 46. The method according to any of the embodiments of the present invention 13-42, characterized in that said method further comprises using a molecular parameter to determine the concentration of a protein or peptide when preparing a batch. 47. The method of any of the embodiments of the present invention 13-42, wherein said method further comprises using a molecular parameter to determine the concentration of the protein or peptide when determining the dose of the protein or peptide. 48. The method according to any of the embodiments of the present invention 13-42, characterized in that said method further comprises using a molecular parameter to determine the concentration of a protein or peptide during the manufacturing process. 49. The method of any one of embodiments 1-48, wherein said protein or peptide is the active ingredient in dulaglutide, ixekizumab, ramucirumab, cetuximab, olaratumab, necitumumab, galkanezumab, or mirikizumab. CLAIM 1. A method for determining the absolute concentration of a protein in a protein solution, which includes the following steps: a) protein denaturation; b) performing qNMR spectroscopy of the denatured protein, said step of performing qNMR spectroscopy comprising the use of a diffusion filter; And c) calculation of the absolute protein concentration using the reference standard and the reference method. 2. A method for determining the molecular parameter of a protein in solution, which includes the following steps: a) protein denaturation; b) performing qNMR spectroscopy of the denatured protein, said step of performing qNMR spectroscopy comprising the use of a diffusion filter; c) calculation of absolute protein concentration, including the use of a reference standard and a reference method; And d) determining the molecular parameter of the protein based on the calculated protein concentration. 3. The method according to claim 2, characterized in that said molecular parameter of the protein includes extinction coefficient, refractive index or partial specific volume. 4. The method according to claim 3, characterized in that the specified extinction coefficient is determined by law -