阅读说明：本技术 测定蛋白质或肽浓度的方法及其用途 (Method for determining protein or peptide concentration and use thereof ) 是由 S·A·布兰得利 W·C·小杰克逊 W·F·韦斯四世 于 2019-08-14 设计创作，主要内容包括：本发明涉及用于测定蛋白质和/或肽浓度或分子参数(例如消光系数)的方法,及其用途。(The present invention relates to a method for determining protein and/or peptide concentration or molecular parameters (e.g. extinction coefficient) and uses thereof.)

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

a. denaturing the protein or optionally the peptide;

b. qNMR spectroscopy with diffusion filtering; and is

c. Protein or peptide concentrations were calculated using reference standards and reference techniques.

2. The method of claim 1, wherein the peptide is denatured.

3. A method according to claim 1 or claim 2 wherein the protein or peptide is denatured by a chaotropic agent.

4. The method of claim 3, wherein the chaotropic agent is guanidinium chloride-d₆Or urea-d₄。

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

6. The method of claim 5, wherein the external reference standard is a small molecule reference.

7. The method of claim 5, wherein the external reference standard is maleic acid.

8. The method of any one of claims 1-7, wherein the solution comprises D₂O。

9. The method of any one of claims 1-8, wherein the reference technique is PULCON.

10. The method of any one of claims 1-9, wherein the protein is an antibody.

11. The method of claim 10, wherein the antibody is a monoclonal antibody.

12. The method of claim 10, wherein the antibody is a bispecific antibody.

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

a. denaturing the protein or optionally the peptide;

b. qNMR spectroscopy with diffusion filtering;

c. calculating the concentration of the protein or peptide using reference standards and reference techniques; and is

d. Determining from the calculated concentration a molecular parameter of the protein or peptide.

14. The method of claim 13, wherein the peptide is denatured.

15. A method according to claim 13 or claim 14, wherein the molecular parameter is the extinction coefficient.

16. The method of claim 15, wherein the extinction coefficient is determined according to Beer-Lambert's law.

17. The method of any one of claims 13-16, wherein the protein or peptide is denatured with a chaotropic agent.

18. The method of claim 17, wherein the chaotropic agent is guanidinium chloride-d₆Or urea-d₄。

19. The method of any one of claims 13-18, wherein the reference standard is an external standard.

20. The method of claim 19, wherein the external reference standard is a small molecule reference or maleic acid.

21. The method of claim 20, wherein the external reference standard is a small molecule reference.

22. The method of claim 20, wherein the external reference standard is maleic acid.

23. The method of any one of claims 13-22, wherein the solution comprises D₂O。

24. The method of any one of claims 13-23, wherein the reference technique is PULCON.

25. The method of any one of claims 13, 15-24, wherein the protein is an antibody.

26. The method of claim 25, wherein the antibody is a monoclonal antibody.

27. The method of claim 25, wherein the antibody is a bispecific antibody.

28. A method of determining a molecular parameter of a protein or peptide in a reference batch, wherein the method comprises:

a. denaturing the protein or optionally the peptide;

b. qNMR spectroscopy with diffusion filtering;

c. calculating the concentration of the protein or peptide using reference standards and reference techniques; and is

d. Determining from the calculated concentration a molecular parameter of the protein or peptide.

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

30. The method of claim 28 or claim 29, wherein the molecular parameter is an extinction coefficient.

31. The method of claim 30, wherein the extinction coefficient is determined according to Beer-Lambert's law.

32. The method of any one of claims 28-31, wherein the peptide is denatured.

33. The method of any one of claims 28-32, wherein the protein or peptide is denatured with a chaotropic agent.

34. The method of claim 33, wherein the chaotropic agent is guanidinium chloride-d₆Or urea-d₄。

35. The method of any one of claims 28-34, wherein the reference standard is an external standard.

36. The method of claim 35, wherein the external reference standard is a small molecule reference.

37. The method of claim 35, wherein the external reference standard is maleic acid.

38. The method of any one of claims 28-37, wherein the solution comprises D₂O。

39. The method of any one of claims 28-38, wherein the reference technique is PULCON.

40. The method of any one of claims 28-31, 33-39, wherein the protein is an antibody.

41. The method of claim 40, wherein the antibody is a monoclonal antibody.

42. The method of claim 40, wherein the antibody is a bispecific antibody.

43. The method of any one of claims 13-42, wherein the method further comprises using the molecular parameters to determine the concentration of the protein or peptide when formulating the protein or peptide.

44. The method of any one of claims 13-42, wherein the method further comprises determining the concentration of the protein or peptide in a batch release assay using the molecular parameters.

45. The method of claim 44, wherein the batch release inspection is a batch release UV inspection.

46. The method of any one of claims 13-42, wherein the method further comprises determining the concentration of the protein or peptide at the time of batch preparation using the molecular parameters.

47. The method of any one of claims 13-42, wherein the method further comprises using the molecular parameters to determine the concentration of the protein or peptide when determining the dosage of the protein or peptide.

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

49. The method of any one of claims 1-48, wherein the protein or peptide is an active ingredient in dolacilin, eculizumab, ramucirumab, cetuximab, olaratumab, nixituzumab, natalizumab, or mirikizumab.

Brief Description of Drawings

FIG. 1. definition of bppste pulse sequence and various delays for an Agilent (also known as Dbppste) spectrometer.

FIG. 2. of NIST mAb¹H NMR spectrum. Of NIST mAbs in native form (A), with a standard monopulse sequence, with ordinate 5000x identical to A (B), native with diffusion filtering (C) and denatured with diffusion filtering (D)¹H NMR spectrum. The asterisked peaks are from histidine buffer. The number of scans A-C and D was 1024 and 64.

Figure 3D magnitude on NIST mAb. FIG. 3 a: NMR spectra of denatured mAb standards collected using bppste pulse sequences. Illustration is shown: the spread of the peaks of the I/L/V resonance. Illustration is shown: a magnified image of the indicated area. FIG. 3 b: left: pure NMR spectra of different diffusive species as determined by the DECRA algorithm. And (3) right: Stejkal-Tanner plots from the DECRA algorithm and diffusion coefficients.

FIG. 4T on NIST mAb₁Magnitude sum T₂Magnitude. FIG. 4 a: from T using bppste pulse sequence₁Magnitude sum T₂Representative data of the magnitude. Left: from measurement T₁NMR spectrum of bppste pulse sequence settings and parameters of the settings, right: from measurement T₂NMR spectra of the bppste pulse sequence set up and a parametric map of the set up. 4 b: calculating T₁And calculating T₂The figure (a).

FIG. 5.14 DF-qNMR spectra of denatured NIST BSA samples. Illustration is shown: a magnified image of the indicated area.

FIG. 6 NIST BSA sample concentrations: method A and gravimetric measurement.

FIG. 7 shows NIST BSA partial hematocrit (partial specific volume): sample density and BSA concentration.

FIG. 8.14 DF-qNMR spectra of denatured bispecific antibody samples.

DF-qNMR spectra of 14 samples of denatured bispecific antibody. The peak of residual water is marked with an asterisk. Illustration is shown: a magnified image of the indicated area.

Figure 9 bispecific antibody sample concentrations: method A and gravimetric measurement.

Definition of

As used herein, "nuclear magnetic resonance spectroscopy" (also referred to as "NMR spectroscopy") refers to spectroscopic techniques known to those of ordinary skill in the art, wherein Nuclear Magnetic Resonance (NMR) spectroscopy 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 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 incorporate the use of "diffusion filtering" that may be used to selectively attenuate the signal based on the size of smaller molecules in the mixture (see, e.g., Stilbs p., prog.nucl.magn.reson.spectrosc.19(1):1-45 (1987)). To balance the attenuation effects of the matrix and protein peaks, the intensity of the diffusion filter can be determined by one of ordinary skill based on the contents of each experiment. For example, if the balance is too weak, the diffusion filter has a finite value. If the equilibrium is too strong, the diffusion filtering starts to suppress the protein signal, which reduces the signal-to-noise ratio of the measurement. This reduction may then lead to longer experimental times. As another example, if one of the matrix components is not completely eliminated and it appears as an interference peak, it will contribute to the protein peak area and it must be subtracted to get the most accurate result. This can be done by: a blank sample containing matrix protein-free was prepared, run with diffusion filtering, determine the area of the remaining matrix peak and subtract this value from the area obtained for the protein sample.

As used herein, "reference standard" refers to a compound, which is¹The 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 may be an internal reference standard, wherein the reference standard is present in a solution containing the protein or peptide of interest, or the reference standard may be external, wherein the reference standard is independent of the solution containing the protein or peptide of interest. The reference standard may be a small molecule 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). Is often used as¹Small molecule reference substance of H qNMR internal standard substanceAre readily available and can also be used as external markers [ see, e.g., Rigger et al, M.J. AOAC International,2017,100,1365-]。

The reference technique is a mathematical algorithm using reference standard data. For example, the external reference technique is a mathematical algorithm that uses externally referenced standard data. An example of this is the pulse length based concentration determination (PULCON) technique (wire g. and Dreier l.j., am. chem. soc.128(8), 2571-.

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

As used herein, "molecular parameters" means scalar values that relate the manner in which some system properties (e.g., absorbance, density, refractive index) change in response to changes in other system properties (e.g., temperature, pressure, or composition). The equation used to determine the molecular parameters ("the corresponding equation") may vary depending on which system attribute or attributes are known. Molecular parameters refer to the intrinsic properties 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 species and the 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]: where A is the absorbance of the protein or peptide solution and l is the path length [ cm ] ε cl (Beer-Lambert's law)]And c is the absolute concentration of protein or peptide [ mg/mL]. As another example, the apparent partial hematocrit of a protein or peptide can be determined by a combination of densitometry and method A as follows[mL/g]：Where ρ is the density of the protein or peptide solution [ g/mL]，ρ₀Is the density [ g/m ] of the protein or peptide solution matrixL]And c is the absolute concentration of protein or peptide [ 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, n₀Is the refractive index of the protein or peptide solution matrix, and c is the absolute protein or peptide concentration [ mg/mL]。

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

The general structure of a "monoclonal antibody" is known. IgG antibodies are heterotetramers 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-light chain pair associate to form a binding site. The heavy chain variable region (VH) and the light chain variable region (VL) may be subdivided into hypervariable regions, known as complementarity determining regions ("CDRs"), interspersed with more conserved regions, known as framework regions ("FRs"). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The CDRs contain a large proportion of residues that form specific interactions with the antigen.

As used herein, "bispecific antibody" refers to bivalent antibody constructs including, but not limited to, the IgG-scFv format (as reported in PCT/US 2015/058719) and the bivalent IgG format (as disclosed in US 2018/0009908). The present invention also contemplates that any engineered protein or antibody, whether tertiary or quaternary, 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.

As used herein, a peptide includes a polymeric chain of amino acids. These amino acids may be natural or synthetic amino acids, including modified amino acids. Peptides may have fewer secondary and tertiary structures, but tend to aggregate in solution; thus, the peptide may also be denatured by chaotropic agents. Denatured peptides can lead to increased signal-to-noise ratio in NMR spectroscopy, thus making integration easier. Denatured peptides may also allow for testing of more concentrated samples, thus resulting in shorter experimental times. For the purposes of the methods described herein, peptide concentration determinations may be more accurate when the peptide is denatured. For denaturation with chaotropic agents, the preferred chaotropic agent will depend on the specific requirements of each peptide and can be determined experimentally by one of ordinary skill in the art.

One of ordinary skill in the art will recognize that a protein includes 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 comprises a linear sequence of its monomeric amino acid subunits. The secondary structure of proteins includes hydrogen bonding patterns that create three-dimensional structural features of partial segments of the amino acid chain, such as alpha-helices and beta-sheets. 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. Proteins may be "denatured" in that an external stress or chaotrope is added to a solution containing the protein and the protein unfolds. The denatured protein has therefore lost most of its secondary, tertiary and quaternary structural features. Examples of chaotropic agents include urea-d₄And guanidinium chloride-d₆。

Also as used herein, "solution" refers to a artificially engineered aqueous mixture of peptides or proteins containing components other than water. "batch" refers to the amount of 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 having uniform characteristics and properties within specified limits; or, in the case of pharmaceutical products produced by continuous processes, in specifically determined quantities per unit time or quantity, in such a way as to ensure uniform characteristics and properties within the specified limits. "reference batch" refers to an established and appropriately characterized self-contained reference standard substance, wherein the substance comprises a protein or peptide, and wherein the substance is prepared from batches representative of production materials and clinical materials. Method a is used to determine the concentration of protein or peptide in solution and a molecular parameter, such as extinction coefficient, in a reference batch. The molecular parameters, such as extinction coefficient, obtained from the reference batch are then used to determine the concentration of the protein or peptide in subsequent batches (referred to herein as "test batches").

As used herein, a "drug product" is a finished dosage form (e.g., a tablet, capsule, or solution) that contains a drug substance, usually, 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, mitigation, treatment or prevention of 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 a drug or drug substance comprising a protein or peptide, such as during the entire production of the drug or drug substance.

"dose" refers to the amount of protein or peptide in the matrix that will elicit the desired biological or medical response. The material may be lyophilized or in an aqueous solution. To prepare a substance 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 novel means of obtaining protein or peptide concentrations by: the extinction coefficient or other molecular parameter of the protein or peptide in the reference batch is determined and then used to determine the concentration of the protein or peptide in subsequent test batches.

Aliquoting means taking a portion of a certain larger amount from one vessel and adding it to a separate vessel. The vessel may be any item suitable for containing the protein or peptide lyophilized or in solution, such as a container, vial, bottle, or device.

The batch release test (test at lot release) refers to a suitable laboratory determination before release (released by the manufacturer for marketing) that the final quality criteria (including the intensity (concentration) of each active ingredient) of the pharmaceutical product (containing the protein or peptide) are satisfactorily met. The batch release test may be, for example, a batch release UV test. 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.

The following examples further illustrate the invention and provide general methods and procedures for practicing various embodiments of the invention. It is to be understood, however, that the embodiments are set forth 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: having provision for at least one distinct peak representing a known number of protons¹Any compound whose 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 the calculation of absolute concentrations from DF-qNMR spectra. For example, maleic acid may be used as an external quantitative reference standard. To formulate maleic acid as an external reference standard, certified maleic acid (11.183mg, 0.096347mmol) was placed in a 5mL volumetric flask and dissolved in D₂And (4) in O. Aliquots are transferred to a tube, such as a Wilmad 435 precision 4mm NMR tube, so that the sample height is 40 mm.

Protein sample: mixing guanidinium chloride-d₆(0.6g, 6mmol) was placed in a 1mL volumetric flask. The vial was placed on a balance and 400 μ L of protein solution was added. The solution was then gently sonicated and vortex mixed until all solids were dissolved. Addition of D₂O to achieve the final volume. Aliquots are transferred to a tube, such as a Wilmad 435 precision 4mm NMR tube, so that the sample height in the tube is 40 mm.

Peptide samples: reacting urea-d₄(0.13g, 2mmol) was placed in a 1mL volumetric flask. The vial was placed on a balance and about 1mL of protein solution was added. The solution was then gently sonicated and vortex mixed until all solids were dissolved. Addition of D₂O to achieve the final volume. Will be provided withAliquots are transferred to a tube, such as a Wilmad 435 precision 4mm NMR tube, so that the sample height in the tube is 40 mm.

Computing

Protein concentration c in grams/liter was calculated from the DF-qNMR spectrum using the following equation_P

Wherein c is_SIs the concentration of the reference standard, A_PIs the area of the selected protein peak in the DF-qNMR spectrum (typically the peak for valine, isoleucine and leucine methyl, between 1.0 and 1.4ppm, but can be any distinguishable peak), H_SIs the number of protons contributing to the peak of the reference standard, A_SIs based on the area of the standard peak, H_PIs the number of protons contributing to the protein peak and the various f's are functions that depend on the specific experimental conditions. f. of_DFExplaining the reduction in protein peak area due to diffusion filter (diffusion filter), it was derived from the Stejskal-Tanner equation (Johnson, C.S. et al, Concepts in Magnetic Resonance part A2012, 40A, 39-65). For the bppste pulse sequence used herein (Pelta, M.D. et al, Magn. Reson. chem.1998,36,706-

Wherein gamma is¹Magnetic spin ratio of H, g is pulse field gradient strength, δ is pulse field gradient length, Δ is diffusion delay, T₁Is the spin-lattice nuclear relaxation time, T, of the protein's proton of interest₂Is the spin-spin nuclear relaxation time of the protein's target proton, D is the translational diffusion coefficient (tau) of the protein₁Is the time between the second and third 90 pulses in the bppste pulse sequence (see FIG. 1), τ₂Is the total time between the first and second 90 pulses (and between the third 90 pulse and the acquisition), and τ₃Is a pulse field gradient pulse in a bipolar pulse pairTotal time between bursts. For the pulse sequence supplied on an Agilent instrument, these terms are defined by the following instrument parameters

τ₁＝Δ-δ-2t_gs-4θ_P-2t_rg(equation 3)

τ₂＝2(δ+2t_gs+2θ_P+2t_rg) (equation 4)

τ₃＝t_gs+2θ_P+t_rg(equation 5)

Wherein t is_gsIs the gradient stabilization delay, t_rgIs the receiver gating time before the pulse and Θ is the 90 pulse width. For other diffusion pulse sequences, f_DFThe expression of (a) will be different. f. of_ERContains the instrument parameters necessary for external reference. In its most basic form

Where S and G are the scan number and receiver gain, respectively, used to acquire individual NMR spectra of the protein and reference standard. When the PULCON method is used, it becomes

Where T is the sample temperature during the acquisition of the NMR spectrum. f. of_DILThe dilution of the initial protein sample to the final NMR sample is explained. If the final volume is obtained by volume fixing and the aliquot of the initial sample is done in a gravimetric manner, this coefficient is given by

Wherein vol_FIs the volume of the final solution (i.e., NMR sample solution), and wt_IAnd d_IIs the weight and density of the initial protein solution aliquot. Alternatively, if the initial egg is added by weighingWhite matter solution and D₂O when both are diluted, then

In which wt_D2OAnd d_D2OIs D₂Weight and density of O. Finally, f_UThe concentration units (usually micromolar) from those quantitative reference standards are converted to the common unit-g/l (or equivalently mg/ml). In this case

Wherein M is_PIs the molecular weight of the protein.

The first exponential term in equation 2 describes T₁The signal attenuation due to nuclear relaxation, the second index describing T₂The signal decay due to nuclear relaxation and the third exponential term describes the signal decay due to molecular diffusion. Of all the terms in the equation, these three quantities (T)₁、T₂And D) is the only unknown quantity; all other quantities are pulse sequence/instrument parameters or physical constants (γ). T is₁、T₂And D depends on the molecular 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 filtering. Measurement D is a well-known experiment in which multiple spectra are taken with increasing gradient intensity (Stejskal, e.o. and Tanner, j.e., j.chem.phys.,1965,42, 288-. This results in T of equation 2₁Term and T₂The term remains constant so that signal attenuation is dominated only by D. D is then calculated by using a suitable algorithm such as DECRA (Windig, W.; Antalek, B.Chemom. Intell. Lab. Syst.,1997,37, 241-. To measure T with an Agilent bppste pulse sequence₁Multiple spectra were acquired using a combination of diffusion retardation and gradient intensity (gzlvl1) to obtain

Where Δ' is the correct diffusion delay given below

This results in T of equation 2₂The terms and D are held constant so that the signal attenuation is only by T₁And (4) relaxation governing. Then from A_PWith respect to tau₁Is calculated to obtain the effective T of the proton of the selected protein peak₁。

To measure T with an Agilent bppste pulse sequence₂Acquiring a plurality of spectra using a combination of gradient stabilization retardation, diffusion retardation and gradient intensity, thereby

Δ_n＝Δ₁+(t_rg，n-t_rg，1) (equation 13)

And gzlvl1 was measured as described above_n. This makes T of the equation₁The part and the D part are kept constant, so that the signal attenuation is only influenced by T₂And (4) relaxation governing. Can be selected from A_PWith respect to tau₂Is calculated to obtain the effective T of the proton of the selected protein peak₂。

Method A data acquisition

Agilent DD 2600 MHz NMR spectrometer outfitted with Agilent¹H-¹⁹F/¹⁵N-³¹P PFG OneProbe. With ethylene glycol samples and D, respectively₂1% H in O₂O sample calibration probe temperature and pulsed field gradient. NMR spectra were acquired at 30.0 ℃ with bipolar pulses for a stimulated echo (bppste) pulse sequence. The number of scans is 64. Acquisition parameters included a 20ppm spectral width, a 1.363 second acquisition time, a 30 second relaxation delay, a 150 millisecond diffusion delay, a 1.4 millisecond gradient pulse with an intensity of 0.569T/m (92% of maximum), and a 1 millisecond gradient stabilization delay.

To measure the translational diffusion coefficient (D), 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 spacedGradient intensity values ranging from 0.228 to 0.569T/m (about 37-92% of maximum). Spin-lattice nuclear relaxation (T) was measured in bppste pulse sequence using a 20ppm 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 delay₁). Five pairs of diffusion delays and gradient strengths were used- (100ms, 0.569T/m); (203ms, 0.400T/m); (408ms, 0.281T/m); (804ms, 0.200T/m); and (1202ms, 0.164T/m).

Spin-spin nuclear relaxation (T) was also measured in bppste pulse sequence using a 20ppm spectral width, 1.363 sec acquisition time, 1 sec relaxation delay, 1.4 msec diffusion gradient duration₂). Six sets of gradient stabilization delay, diffusion delay, and gradient strength were used- - (-1 ms,150.001ms, 0.570); (2ms,150.002ms, 0.571T/m); (4ms,150.004ms, 0.573T/m); (8ms,150.008ms, 0.577T/m); (12ms,150.012ms, 0.581T/m); and (16ms,150.016ms, 0.575T/m).

Data analysis

DF-qNMR, T.sub.qNMR, was treated in MNova version 11.0(Mestrelab Research, S.L., Santiago de Compstela, Spain)₁And T₂Data, and diffusion data were processed and analyzed with DOSYToolbox version 2.5(Nilsson, m.j.magn.reson.2009,200, 296-302). Before the Fourier transform, the FID is zero-padded once and multiplied by a 2.93Hz exponential window function. The diffusion coefficients were taken using the DECRA algorithm available in DOSYToolbox. T was calculated from bppste pulse sequences by regression analysis using MATLAB 2016b (MathWorks, Inc., Natick, Ma)₁Value sum T₂The value is obtained. The peak areas for leucine methyl, isoleucine methyl and/or valine methyl groups were obtained by deconvolution using a straight line fit path in MNova and CRAFT.

Method A of use for NIST antibody RM8761

To illustrate the difficulty in quantifying large, formulated proteins by NMR, a NIST RM8761 sample (10g/L antibody, 12.5mM 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):2127-₂O treatment and obtaining¹H1D NMR spectrum (fig. 2). The water signal is so strong and broadSo that it obscures the signal of the antibody. Histidine excipient peaks were also observed in the spectra. To obtain an efficient protein spectrum, these dominant peaks must be eliminated. One NMR technique that is ideal for use in 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. Also shown in FIG. 2 is the collection of the same NIST mAb sample using bipolar pulse-to-stimulated echo (bppste) diffusion pulse sequence as diffusion filtering¹H-NMR spectrum. Diffusion filtering effectively removes the adverse signal of the agent while not introducing baseline artifacts or phase distortion.

However, in most cases, the resolution for accurate peak identification and integration is insufficient due to the characteristic broad line width of protein resonances. This is mainly due to the Higher Order Structure (HOS) of the protein. Single types of amino acids that would have very similar chemical shifts, however, exhibit a broad chemical shift distribution due to the unique magnetic environment resulting from the secondary, tertiary, and quaternary structure of the protein. Thus, HOS is eliminated by denaturing the protein. Addition of denaturants to the protein solution causes the sample volume to expand. For true dosing, more solvent is added to achieve precise volumes, for example using volumetric flasks. This is f in equation 1_DILThe basis of (1).

In FIG. 2, the use of 6M guanidinium chloride-d is shown₆Diffusion filtering of formulated NIST RM8761 samples¹H-NMR. A decrease in linewidth and a subsequent increase in signal-to-noise ratio are observed. The group of peaks between 1.0 and 1.4ppm corresponding to the methyl groups of isoleucine, leucine and/or valine has a resolution close to the baseline, which makes it possible to integrate them for quantitative analysis.

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: nuclear relaxation delay, one radio frequency pulse and data acquisition time. With sufficiently long nuclear relaxation delays, the resulting NMR peak area is accurate and reproducibly proportional to the number of observed nuclei; thus, the experiment was quantitative. All other NMR pulse sequences, including the diffusion sequences used herein, are requiredA plurality of radio frequency pulses separated by a plurality of delays. Thus, the resulting spectra are no longer inherently quantitative due to factors that clip the peak from the equilibrium value. However, if these factors are known and the degree of attenuation can be calculated, then the diffusion filtering can be made quantitative. This is f in equation 1_DFThe basis of (1).

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 may be used. Several external standard methods have been reported, including the PULCON technique (e.g., concentration determination based on pulse length). This technique allows absolute area correlation from two independent spectra (one of the reference standard and the other of the analyte) even if the solution conditions and experimental parameters are different. Thus, the external reference standard need not have similar characteristics to the analyte of interest or even need not be a protein. In addition, rather than requiring standards for each amino acid as in AAA, this method requires only a single standard.

To demonstrate the accuracy and precision of method a, three independent replicates of NIST RM8761 samples were evaluated. Sufficient signal to noise ratio was obtained for this 10g/L protein preparation using only 64 scans and a total acquisition time of 37 minutes. The signal set of I/L/V methyl groups in the protein (1.0-1.4 ppm), which corresponds to 1,476 protons, has near baseline resolution. These results make accurate integration possible for quantification. To facilitate concentration calculations, the T for this set of peaks was also measured using a bppste pulse sequence as described above₁、T₂And D, the data acquisition and analysis performed are shown in fig. 3 and 4.

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

These data show that method a can be used to accurately and precisely determine the concentration of the antibody.

Method A of use on NIST Bovine Serum Albumin (BSA)

NIST (BSA) standard reference 927e (67.38g/L, 20mM NaCl, pH adjusted to 6.5-6.8 with 1.0mol/L NaOH, commercially available from the national institute of standards and technology) was re-diluted to four additional protein concentrations (Table 1, samples B1-B4). A total of fourteen samples were analyzed, including six replicate samples of medium concentration (B3), three replicate samples of highest concentration (B5) and lowest concentration (B1), respectively, and a single replicate sample of intermediate concentration (B2 and B4). NMR samples were prepared as described above for each replicate. Method a data acquisition and analysis was performed as described above. The fourteen DF-qNMR spectra obtained are shown superimposed in FIG. 5.

These data show that: the peak from water has been almost eliminated, the baseline is undisturbed and the signal set of 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 66,398Da and reported in table 1. For all five concentrations, the values measured using method a were similar to the values calculated from the gravimetric dilutions. As shown in table 1 below, the RSD of all three replicate samples was less than 1%.

TABLE 1.NIST Results for BSA sample

As shown in FIG. 6, method A concentration vs. gravimetric concentration plot produces R²A linear concentration range across the concentration range studied is suggested as a regression line of 0.9997.

These data show that method a can be used to accurately and precisely determine the concentration of a medium-sized protein over a wide concentration range.

FIG. 7 shows samples as a function of protein concentration as determined by method AGraph of product density. The equation relating the two quantities is as defined aboveThe slope of the regression line yields the partial hematocrit of BSAThe latter was found to be 0.718 mL/g.

Methods of use for bispecific antibodies A

Samples of bispecific antibody (given according to the sequence in table 1 of us patent 9,718,884) were formulated as described above, with five different concentrations and a total of 14 samples. Method a data acquisition and analysis was performed as described above. According to the procedure substantially as described above, the following data were obtained.

Figure 8 shows the resulting DF-qNMR spectrum, which is similar to BSA (shown above) in overall appearance and properties. The concentration was calculated using equation 1 using the theoretical molecular weight of intact non-glycosylated protein (about 200,000Da) and is summarized in Table 2 below.

TABLE 2.Results for bispecific antibodies

As shown in FIG. 9, method A concentration vs. gravimetric concentration plot produces R²Regression line 0.996.

These data show that: method a can be used to accurately and precisely determine the concentration of bispecific antibody over a wide range of concentrations.

Method A for use with approximately 5000Da peptides

Dilute, small peptide samples (about 5,000 Da; given by the sequence in example 4 of PCT/US 2017/041922) were formulated with and without chaotropic agents as described above. Since the peptide is naturally present as random coil and at low concentrations, lower denaturant concentrations (2M) are acceptable. Sample stability requires alkaline conditions; therefore, urea-d 4 was used. Method a data acquisition and analysis was performed as described above, with the exception that 624 scans were taken per acquisition rather than 64 scans. Following the procedure substantially as described above, the following data were obtained. Slight phase distortion of the residual water signal was observed, but this did not hinder the data analysis. Addition of chaotropic agents results in better water suppression, sharper peaks and subsequent higher resolution and sensitivity. The concentration of each replicate sample was calculated from equation 1. The mean total protein concentration was determined to be 0.65g/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.

Illustrative embodiments

The following includes a series of illustrative embodiments of the present disclosure that represent various embodiments of the present disclosure.

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

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

(i) denaturing the protein or optionally the peptide;

(ii) qNMR spectroscopy with diffusion filtering; and is

(iii) Protein or peptide concentrations were calculated using reference standards and reference techniques.

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

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

4. The method of embodiment 3, wherein the chaotropic agent is guanidinium chloride-d₆Or urea-d₄。

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

6. The method of embodiment 5, wherein the external reference standard is a small molecule reference.

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

8. The method of any of embodiments 1-7, wherein the solution comprises D₂O。

9. The method of any one of embodiments 1-8, wherein the reference technique is PULCON.

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

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

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

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

(i) denaturing the protein or optionally the peptide;

(ii) qNMR spectroscopy with diffusion filtering;

(iii) calculating the concentration of the protein or peptide using reference standards and reference techniques; and is

(iv) Determining from the calculated concentration a molecular parameter of the protein or peptide.

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

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

16. The method of embodiment 15 wherein the extinction coefficient is determined according to Beer-Lambert's law.

17. The method of any of embodiments 13-16, wherein the protein or peptide is denatured with a chaotropic agent.

18. The method of embodiment 17, wherein the chaotropic agent is guanidinium chloride-d₆Or urea-d₄。

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

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

21. The method of embodiment 20, wherein the external reference standard is a small molecule reference.

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

23. The method of any of embodiments 13-22, wherein the solution comprises D₂O。

24. The method of any one of embodiments 13-23, wherein the reference technique is PULCON.

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

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

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

28. A method of determining a molecular parameter of a protein or peptide in a reference batch, wherein the method comprises:

a. denaturing the protein or optionally the peptide;

b. qNMR spectroscopy with diffusion filtering;

c. calculating the concentration of the protein or peptide using reference standards and reference techniques; and is

d. Determining from the calculated concentration a molecular parameter of the protein or peptide.

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

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

31. The method of embodiment 30 wherein the extinction coefficient is determined according to Beer-Lambert's law.

32. The method of any one of embodiments 28-31, wherein the peptide is denatured.

33. The method of any one of embodiments 28-32, wherein the protein or peptide is denatured by a chaotropic agent.

34. The method of embodiment 33, wherein the chaotropic agent is guanidinium chloride-d₆Or urea-d₄。

35. The method of any one of embodiments 28-34, wherein the reference standard is an external reference standard.

36. The method of embodiment 35, wherein the external reference standard is a small molecule reference.

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

38. The method of any of embodiments 28-37, wherein the solution comprises D₂O。

39. The method of any one of embodiments 28-38, wherein the reference technique is PULCON.

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

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

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

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

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 the batch release test using the molecular parameters.

45. The method of embodiment 44, wherein the batch release test is a batch release UV test.

46. 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 preparation using the molecular parameters.

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

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 the molecular parameters.

49. The method of any of embodiments 1-48, wherein the protein or peptide is an active ingredient in duravist, epratuzumab (ixekizumab), ramucirumab (ramucirumab), cetuximab (cetuximab), olaratumab (olaratumab), nimotuzumab (necitumumab), natalizumab (galcanezumab), or mirikizumab.