Method for fingerprinting therapeutic proteins by two-dimensional (2D) nuclear magnetic resonance technology at natural abundance for formulated biopharmaceutical products

Abstract

Disclosed are methods for fingerprinting specific molecules in a composition using nuclear magnetic resonance (NMR). The disclosed NMR methods offer several modifications and improvements over existing NMR techniques. In some embodiments, the methods involve applying a cycle of signal processing steps, including applying a radio frequency (RF) pulse, applying a gradient pulse having a pulse length of 1000 μs or less, and applying a water suppression technique (WET). In some embodiments, the methods further involve repeating the cycle at least three times to obtain an enhanced composition signal. In some embodiments, the methods further involve fingerprinting specific molecules based on the enhanced composition signal.

Description

Technical Field

[0001] Sequence Listing This application is filed with a sequence listing in electronic format. The sequence listing submitted as a file named "041925-0924_SL.txt" was created on January 6, 2020, and is 265 KB in size. The information in the sequence listing in electronic format is hereby incorporated by reference in its entirety into this specification.

Background Art

[0002] Pharmaceutically active proteins such as antibodies, and recombinant therapeutic proteins (category "therapeutic proteins") are often formulated in liquid solutions for injection, etc. The pharmaceutical composition may contain, for example, agents for modifying, maintaining or protecting the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or permeability of the composition.

[0003] Generally, excipients can be classified based on the mechanism by which they stabilize proteins against various chemical and physical stresses. Some excipients mitigate the effects of specific stresses or modulate the specific susceptibilities of specific polypeptides. Other excipients have a more general effect on the physical and covalent stability of proteins. General excipients for pharmaceutical liquid protein formulations are described, for example, in Kamerzell TJ, Esfandiary R, Joshi SB, Middaugh CR, Volkin DB. 2011, Protein-excipient interactions: Mechanisms and biophysical characterization applied to protein formulation development, Adv Drug Deliv Rev 63:1118-59.

[0004] The higher-order structure (e.g., secondary structure, tertiary structure, and quaternary structure; HOS) of therapeutic proteins during the development, manufacture, and formulation of pharmaceutical preparations / compositions is evaluated to ensure the efficacy and safety of therapeutic proteins. This is because HOS is an important critical quality attribute (CQA) that can affect quality, stability, safety, and efficacy (when HOS changes over time, the likelihood of loss of function due to immunogenicity increases). CQA is a chemical, physical, or biological property that exists within a specific value or range of values. In therapeutic macromolecules of polypeptides, physical attributes and modifications of amino acids (the building blocks of polypeptides) are important CQAs and are monitored during and after manufacture (as well as during drug development). Similarly, although HOS is a CQA, excipients in formulations (e.g., sucrose and acetate) can strongly interfere with the methyl peaks of therapeutic proteins (such as antibodies or their fragments, or derivatives and their analogs), making it difficult to detect the HOS of formulated therapeutic proteins using, for example, nuclear magnetic resonance (NMR). NMR-based methods and techniques are useful for detecting the HOS of proteins, but can be difficult to perform when targeting the fingerprint of the target protein in a multi-component solution. In particular, in the generated NMR spectrum, there remains the problem of improving NMR technology to detect the signal from the target molecule (e.g., therapeutic protein) against signals from other molecules in the solution that generate signals in the same detection region as the signal generated by the therapeutic protein, especially. Therefore, an epoch-making approach to solve this problem is needed.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

Means for Solving the Problems

[0006] Exemplary methods are described herein for fingerprinting specific molecules in a composition using nuclear magnetic resonance (NMR). The method includes providing a composition having at least a first molecule having a first NMR signal, a second molecule having a second NMR signal, and a third molecule having a third NMR signal. In this method, each of the signals originates from each of the respective molecules having a non-zero nuclear spin. The method includes applying a cycle of signal processing steps. The cycle includes applying a radio frequency (RF) pulse, applying a gradient pulse having a pulse length of 1000 μs or less, and applying a water suppression technique (WET). In this method, the first NMR signal, the second NMR signal, and the third NMR signal are located in defined regions of the NMR spectrum. The method also includes repeating the cycle at least three times to obtain an enhanced signal of the composition. The method further includes fingerprinting specific molecules based on the enhanced signal of the composition.

[0007] Another exemplary method of fingerprinting specific molecules in a composition using NMR is described herein. The method includes providing a composition having at least a first molecule having a first NMR signal, a second molecule having a second NMR signal, and a third molecule having a third NMR signal. In this method, each of the signals originates from each of the respective molecules having a non-zero nuclear spin. The method includes applying a cycle of signal processing steps. The cycle includes applying an RF pulse and applying a gradient pulse. In this method, the first NMR signal, the second NMR signal, and the third NMR signal are located in the region of an NMR spectral window of about 5 ppm to about 150 ppm. The method also includes repeating the cycle at least three times to obtain an enhanced signal of the composition. The method further includes fingerprinting specific molecules based on the enhanced signal of the composition.

[0008] Furthermore, another exemplary method of fingerprinting specific molecules in a composition using NMR is described herein. The method includes providing a composition having at least a first molecule having a first NMR signal, a second molecule having a second NMR signal, and a third molecule having a third NMR signal. In this method, each of the signals originates from each of the respective molecules having a non-zero nuclear spin. The method includes applying an RF pulse to the composition to excite the first NMR signal while suppressing the second NMR signal. The RF pulse includes at least one of a Refocusing Band-Selective Pulse with Uniform Response and Phase (Reburp) pulse, a broadband inversion pulse (BIP), and a Gaussian (G3) inversion pulse, and an asymmetric adiabatic pulse. The method also includes applying a gradient pulse having a pulse length of 1000 μs or less and applying a WET sequence to suppress the third NMR signal. The method also includes repeating the cycle at least three times to obtain an enhanced signal of the composition. The method further includes fingerprinting specific molecules based on the enhanced signal of the composition.

[0009] These and other aspects and implementations are described in detail below. The foregoing information and the following detailed description of the invention include examples of various aspects and implementations and provide an overview or mechanism for understanding the nature and characteristics of the disclosed aspects and implementations. The drawings provide illustrations and further understanding of the various aspects and implementations and are incorporated in and constitute a part of this specification.

[0010] The accompanying drawings are not intended to be drawn to scale. Like reference numerals and designations in different drawings indicate like components. It is not possible to label all components in all drawings for clarity. In an embodiment of the present invention, for example, the following items are provided. (Item 1) A method of fingerprinting specific molecules in a composition using nuclear magnetic resonance (NMR), providing the composition comprising at least a first molecule having a first NMR signal, a second molecule having a second NMR signal, and a third molecule having a third NMR signal, wherein each of the signals originates from each of the respective molecules having a non-zero nuclear spin; applying a cycle of a signal processing step, the cycle comprising: applying a radio frequency (RF) pulse; applying a gradient pulse having a pulse length of 1000 μs or less; applying a water suppression technique (WET), wherein the first NMR signal, the second NMR signal, and the third NMR signal 13 are located in a region of the NMR spectrum within a defined ppm range of C methyl signals; applying a cycle of a signal processing step; repeating the cycle at least 3 times to obtain an enhanced signal of the composition; fingerprinting the specific molecule based on the enhanced signal of the composition; A method comprising. (Item 2) The method according to item 1, wherein the region of the NMR spectrum comprises an NMR spectrum window of about 5 ppm to about 150 ppm. (Item 3) The method according to item 1, wherein the region of the NMR spectrum comprises an NMR spectrum window of about 5 ppm to about 100 ppm. (Item 4) The method according to item 1, wherein the region of the NMR spectrum comprises an NMR spectrum window of about 5 ppm to about 50 ppm. (Item 5) The method according to item 1, wherein the region of the NMR spectrum comprises an NMR spectrum window of about 7 ppm to about 35 ppm. (Item 6) The method according to item 1, wherein the RF pulse comprises at least one of a Reburp pulse, a combination of a broadband inversion pulse (BIP) and a Gaussian (G3) inversion pulse, or an asymmetric adiabatic pulse. (Item 7) The method according to item 6, wherein the Reburp pulse excites the first NMR signal. (Item 8) The method according to item 6, wherein the broadband inversion pulse excites each of the NMR signals and the G3 inversion pulse suppresses the second NMR signal. (Item 9) The method according to item 6, wherein the asymmetric adiabatic pulse excites the first NMR signal while suppressing the second NMR signal. (Item 10) The first NMR signal is 13 an NMR signal related to 13C methyl, and the second NMR signal is 13 a signal related to 13C sucrose, and the third NMR signal is 1 a signal related to at least the 13C NMR signal from another excipient consisting of one of H acetate, or glutamate, proline, arginine, or mannitol. 1 H / 13 The method according to item 1, wherein the signal is (Item 11) The method according to item 1, wherein the method for using NMR is performed in a frequency range of about 100 MHz to about 2000 MHz. (Item 12) The method according to item 6, wherein the Reburp pulse has a pulse length of about 500 μs to about 1000 μs. (Item 13) The method according to item 6, wherein the Reburp pulse has a pulse length of about 600 μs to about 900 μs. (Item 14) The method according to item 6, wherein the Reburp pulse has a pulse length of about 600 μs to about 800 μs. (Item 15) The method according to item 6, wherein the combination of the BIP and the G3 inversion pulse has a pulse length of about 200 μs to about 2500 μs. (Item 16) The method according to item 6, wherein the combination of the BIP and the G3 inversion pulse has a pulse length of about 200 μs to about 2000 μs. (Item 17) The method according to item 6, wherein the combination of the BIP and the G3 inversion pulse has a pulse length of about 200 μs to about 1500 μs. (Item 18) The method according to item 6, wherein the combination of the BIP and the G3 inversion pulse has a pulse length of about 250 μs to about 1000 μs. (Item 19) The method according to item 6, wherein the combination of the BIP and the G3 inversion pulse has a pulse length of about 250 μs to about 750 μs. (Item 20) The method according to item 6, wherein the combination of the BIP and the G3 inversion pulse has a pulse length of about 620 μs to 660 μs. (Item 21) The method according to item 20, wherein the BIP has a pulse length of about 120 μs to 160 μs and the G3 inversion pulse has a pulse length of about 500 μs. (Item 22) The method according to item 6, wherein the asymmetric adiabatic pulse has a pulse length of about 50 μs to about 2500 μs. (Item 23) The method according to item 6, wherein the asymmetric adiabatic pulse has a pulse length of about 50 μs to about 2000 μs. (Item 24) The method according to item 6, wherein the asymmetric adiabatic pulse has a pulse length of about 50 μs to about 1500 μs. (Item 25) The method according to item 6, wherein the asymmetric adiabatic pulse has a pulse length of about 50 μs to about 1000 μs. (Item 26) The method according to item 6, wherein the asymmetric adiabatic pulse has a pulse length of about 100 μs to about 800 μs. (Item 27) The method according to item 1, wherein the gradient pulse has a pulse length range of about 50 μs to about 990 μs. (Item 28) The method according to item 1, wherein the gradient pulse has a pulse length range of about 50 μs to about 900 μs. (Item 29) The method according to item 1, wherein the gradient pulse has a pulse length range of about 50 μs to about 800 μs. (Item 30) The method according to item 1, wherein the gradient pulse has a pulse length range of about 50 μs to about 700 μs. (Item 31) The method according to item 1, wherein the gradient pulse has a pulse length range of about 50 μs to about 600 μs. (Item 32) The method according to item 1, wherein the gradient pulse has a pulse length range of about 50 μs to about 500 μs. (Item 33) The method according to item 1, wherein the gradient pulse has a pulse length range of about 50 μs to about 400 μs. (Item 34) The method according to item 1, wherein the gradient pulse has a pulse length range of about 50 μs to about 300 μs. (Item 35) The method according to item 1, wherein the gradient pulse has a pulse length range of about 50 μs to about 250 μs. (Item 36) The method according to item 1, wherein the gradient pulse has a pulse length range of about 50 μs to about 200 μs. (Item 37) The method according to item 1, wherein the gradient pulse has a pulse length range of about 50 μs to about 150 μs. (Item 38) The method according to item 1, wherein the gradient pulse has a pulse length range of about 50 μs to about 100 μs. (Item 39) The method according to any one of items 27 to 38, wherein at least one reverse gradient pulse having the same pulse length range follows the gradient pulse. (Item 40) The method according to item 39, wherein another gradient pulse having the same pulse length range follows the at least one reverse gradient pulse. (Item 41) The method according to item 1, wherein repeating the cycle at least 3 times includes a delay in the repetition in the range of about 10 μs to about 990 μs. (Item 42) The method according to item 41, wherein the delay is from about 30 μs to about 900 μs, from about 50 μs to about 800 μs, from about 50 μs to about 700 μs, from about 100 μs to about 600 μs, from about 150 μs to about 500 μs, or from about 200 μs to about 300 μs. (Item 43) A method for fingerprinting specific molecules in a composition using nuclear magnetic resonance (NMR), comprising providing the composition comprising at least a first molecule having a first NMR signal, a second molecule having a second NMR signal, and a third molecule having a third NMR signal, wherein each of said signals results from each of said respective molecules having a non-zero nuclear spin; applying a cycle of a signal processing step, said cycle comprising: applying a radio frequency (RF) pulse; applying a gradient pulse; wherein the first NMR signal, the second NMR signal, and the third NMR signal are located in a region of an NMR spectral window of about 5 ppm to about 150 ppm; applying a cycle of a signal processing step; repeating said cycle at least 3 times to obtain an enhanced signal of said composition; fingerprinting said specific molecules based on said enhanced signal of said composition. A method comprising. (Item 44) wherein said cycle further comprises: applying a water suppression technique (WET) sequence to suppress said third NMR signal. The method according to item 43. (Item 45) The method according to item 43, wherein said region of the NMR spectrum comprises an NMR spectral window of about 5 ppm to about 100 ppm, about 5 ppm to about 50 ppm, or about 7 ppm to about 35 ppm. (Item 46) The method according to item 43, wherein said RF pulse comprises at least one of a combination of a Reburp pulse, a broadband inversion pulse (BIP) and a Gaussian (G3) inversion pulse, and an asymmetric adiabatic pulse. (Item 47) The method according to item 46, wherein said Reburp pulse excites said first NMR signal. (Item 48) The method according to item 46, wherein said broadband inversion pulse excites a wide range of NMR signals and said G3 inversion pulse suppresses said second NMR signal. (Item 49) The method according to item 46, wherein the asymmetric adiabatic pulse excites the first NMR signal while suppressing the second NMR signal. (Item 50) The first NMR signal is 13 an NMR signal related to C methyl, and the second NMR signal is 13 a signal related to an NMR signal related to C sucrose, and the third NMR signal is 1 at least related to a C NMR signal from one of H acetate, or glutamate, proline, arginine, or mannitol 13 The method according to item 43. (Item 51) The method for using NMR is performed in a frequency range of about 100 MHz to about 2000 MHz. The method according to item 43. (Item 52) The Reburp pulse has a pulse length of about 500 μs to about 1000 μs, about 600 μs to about 900 μs, or about 600 μs to about 800 μs. The method according to item 46. (Item 53) The combination of the BIP and the G3 inversion pulse has a pulse length of about 200 μs to about 2500 μs, about 200 μs to about 2000 μs, about 200 μs to about 1500 μs, about 250 μs to about 1000 μs, or about 250 μs to about 750 μs. The method according to item 46. (Item 54) The combination of the BIP and the G3 inversion pulse has a pulse length of about 620 μs to 660 μs. The method according to item 46. (Item 55) The BIP has a pulse length of about 120 μs to 160 μs, and the G3 inversion pulse has a pulse length of about 500 μs. The method according to item 54. (Item 56) The asymmetric adiabatic pulse has a pulse length of about 50 μs to about 2500 μs, about 50 μs to about 2000 μs, about 50 μs to about 1500 μs, about 50 μs to about 1000 μs, or about 100 μs to about 800 μs. The method according to item 46. (Item 57) The gradient pulse has a pulse length of 1000 μs or less. The method according to item 43. (Item 58) The method according to item 43, wherein the gradient pulse has a pulse length range of about 50 μs to about 1000 μs, about 50 μs to about 900 μs, about 50 μs to about 800 μs, about 50 μs to about 700 μs, about 50 μs to about 600 μs, about 50 μs to about 500 μs, about 50 μs to about 400 μs, about 50 μs to about 300 μs, about 50 μs to about 250 μs, about 50 μs to about 200 μs, about 50 μs to about 150 μs, or about 50 μs to about 100 μs. (Item 59) The method according to any one of items 57 or 58, wherein at least one inversion gradient pulse having the same pulse length or the same pulse length range follows the gradient pulse. (Item 60) The method according to item 59, wherein another gradient pulse having the same pulse length range follows the at least one inversion gradient pulse. (Item 61) The method according to item 43, wherein repeating the cycle at least three times includes a delay in the repetition in the range of about 10 μs to about 990 μs, about 30 μs to about 900 μs, about 50 μs to about 800 μs, about 50 μs to about 700 μs, about 100 μs to about 600 μs, about 150 μs to about 500 μs, or about 200 μs to about 300 μs. (Item 62) A method for fingerprinting specific molecules in a composition using nuclear magnetic resonance (NMR), providing the composition comprising at least a first molecule having a first NMR signal, a second molecule having a second NMR signal, and a third molecule having a third NMR signal, wherein each of the signals originates from each of the respective molecules having a non-zero nuclear spin; applying a radio frequency (RF) pulse to the composition to excite the first NMR signal while suppressing the second NMR signal, the RF pulse including at least one of a combination of a Reburp pulse, a broadband inversion pulse, and a Gaussian inversion pulse, and an asymmetric adiabatic pulse; applying a gradient pulse having a pulse length of 1000 μs or less; applying a water suppression technique (WET) sequence to suppress the third NMR signal; acquiring the enhanced signal of the composition; fingerprinting the specific molecule based on the enhanced signal of the composition; A method comprising the above steps. (Item 63) The method according to item 62, wherein the first NMR signal, the second NMR signal, and the third NMR signal are located in a region of the NMR spectrum near the C methyl signal. 13 (Item 64) The method according to item 62, wherein the first NMR signal, the second NMR signal, and the third NMR signal are located in an NMR spectrum window of about 5 ppm to about 150 ppm. (Item 65) The method according to item 62, wherein the first NMR signal, the second NMR signal, and the third NMR signal are located in an NMR spectrum window of about 5 ppm to about 100 ppm, about 5 ppm to about 50 ppm, or about 7 ppm to about 35 ppm. (Item 66) The method according to item 62, wherein the method for using NMR is performed in a frequency range of about 100 MHz to about 2000 MHz. (Item 67) The method according to item 62, wherein the Reburp pulse has a pulse length of about 500 μs to about 1000 μs, about 600 μs to about 900 μs, or about 600 μs to about 800 μs. (Item 68) The method according to item 62, wherein the combination of the BIP and the G3 inversion pulse has a pulse length of about 200 μs to about 2500 μs, about 200 μs to about 2000 μs, about 200 μs to about 1500 μs, about 250 μs to about 1000 μs, or about 250 μs to about 750 μs. (Item 69) The method according to item 62, wherein the combination of the BIP and the G3 inversion pulse has a pulse length of about 620 μs to 660 μs. (Item 70) The method according to item 69, wherein the BIP has a pulse length of about 120 μs to 160 μs and the G3 inversion pulse has a pulse length of about 500 μs. (Item 71) The method according to item 62, wherein the asymmetric adiabatic pulse has a pulse length of about 50 μs to about 2500 μs, about 50 μs to about 2000 μs, about 50 μs to about 1500 μs, about 50 μs to about 1000 μs, or about 100 μs to about 800 μs. (Item 72) (Item 72) The method according to item 62, wherein the gradient pulse has a pulse length range of about 50 μs to about 990 μs, about 50 μs to about 900 μs, about 50 μs to about 800 μs, about 50 μs to about 700 μs, about 50 μs to about 600 μs, about 50 μs to about 500 μs, about 50 μs to about 400 μs, about 50 μs to about 300 μs, about 50 μs to about 250 μs, about 50 μs to about 200 μs, about 50 μs to about 150 μs, or about 50 μs to about 100 μs. (Item 73) The method according to item 72, wherein at least one inversion gradient pulse having the same pulse length range follows the gradient pulse. (Item 74) The method according to item 73, wherein another gradient pulse having the same pulse length range follows the at least one inversion gradient pulse. (Item 75) Applying the RF pulse, the gradient pulse, and the WET sequence constitutes a cycle of a signal processing step, and the method The method according to item 62, further comprising repeating the cycle at least 3 times to obtain the signal of the enhanced composition. (Item 76) The method according to item 75, wherein repeating the cycle at least 3 times includes a delay in the repetition in the range of about 10 μs to about 990 μs, about 30 μs to about 900 μs, about 50 μs to about 800 μs, about 50 μs to about 700 μs, about 100 μs to about 600 μs, about 150 μs to about 500 μs, or about 200 μs to about 300 μs. (Item 77) 13 The first NMR signal related to C-methyl is a BiTE® molecule selected from the group consisting of anti-CD33 and anti-CD3 BiTE molecules, anti-BCMA and anti-CD3 BiTE molecules, anti-FLT3 and anti-CD3 BiTE, anti-CD19 and anti-CD3 BiTE, anti-EGFRvIII and anti-CD3 BiTE molecules, anti-DLL3 and anti-CD3 BiTE, BLINCYTO (blinatumomab), and solitomab; an antibody selected from the group consisting of adalimumab, bevacizumab, blinatumomab, cetuximab, conatumumab, denosumab, eculizumab, elotuzumab, evolocumab, infliximab, natalizumab, panitumumab, rilotumumab, rituximab, romosozumab, and trastuzumab, and an antibody selected from Table A; and a protein selected from the group consisting of combinations thereof, according to the method of item 10 or 50. (Item 78) The first NMR signal is 13 an NMR signal related to C methyl, and the second NMR signal is 13 a signal related to an NMR signal related to C sucrose, and the third NMR signal is 1 at least related to a C NMR signal from one of H acetate, or glutamate, proline, arginine, or mannitol 1 H / 13 The method according to item 62. (Item 79) The method for using NMR according to any one of items 11, 51, or 66, wherein the method is executed in a frequency range of about 500 MHz to about 2000 MHz. (Item 80) The method for using NMR according to any one of items 11, 51, or 66, wherein the method is executed in a frequency range of about 500 MHz to about 1000 MHz. (Item 81) The method for using NMR according to any one of items 11, 51, or 66, wherein the method is executed in a frequency range of about 900 MHz. (Item 82) The method for using NMR according to any one of items 11, 51, or 66, wherein the method is executed in a frequency range of about 800 MHz. (Item 83) The method for using NMR according to any one of items 11, 51, or 66, wherein the method is executed in a frequency range of about 700 MHz. (Item 84) The method for using NMR according to any one of items 11, 51, or 66, wherein the method is executed in a frequency range of about 600 MHz. (Item 85) The method for using NMR according to any one of items 11, 51, or 66, wherein the method is executed in a frequency range of about 500 MHz. (Item 86) The method according to any one of items 10, 50, or 78, wherein the third NMR signal is related to glutamate or proline.

Brief Description of the Drawings

[0011]

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[0012] Generally, the present disclosure relates to a method of fingerprinting a therapeutic protein of a complex by two-dimensional (2D) nuclear magnetic resonance techniques for mapping the structure of a chemical composition.

[0013] In recent years, a 2D 13 C NMR methyl fingerprinting method has been introduced for mapping the structure of protein molecules such as monoclonal antibodies (mAbs), but the state-of-the-art NMR techniques or methods have not been applied to evaluate the HOS of formulated proteins that contain excipients such as sucrose and acetate at high concentrations. The application of these techniques is hindered by spectral interference from these excipients. This excipient interference can be particularly problematic for applications where the signal of the excipient is often on a much larger order than the signal of the target chemical composition such as a protein, introducing baseline distortion that can negatively affect quantitative chemical analysis or affect the fidelity of selected peak parameters in the vicinity of the excipient signal.

[0014] The disclosed NMR method brings about changes and improvements to existing NMR techniques and overcomes strong interference from sucrose and acetate signals regarding methyl peaks. Applicants have discovered that the interference problems described above have been overcome during various experiments on several samples and sample types to evaluate the effectiveness of using the described modified NMR technique.

[0015] Thus, it has been found that by changing the pulse profile, surprisingly, it is possible to significantly affect the signal-to-noise ratios in various NMR regions. For example, using a specific pulse profile, while suppressing the 13 C excipient signal such as that derived from sucrose, the 13 C methyl signal from the therapeutic molecule can be excited. By applying a shorter gradient pulse of less than 1 millisecond (ms), the signal can be further enhanced, 13 and the intensity of the

[0016] Subsequently, various special factors in the improved NMR method and various combinations of these specifically described factors are utilized to conduct considerations on the evaluation of related embodiments and verification of effectiveness.

[0017] According to related embodiments of the disclosed NMR method, the method may include applying at least one of a Refocusing Band-Selective Pulse with Uniform Response and Phase (Reburp) pulse, a broadband inversion pulse (BIP), and a Gaussian (G3) inversion pulse, and an asymmetric adiabatic pulse. By applying at least one of the three different pulses, signals such as the 13 C excipient signal are suppressed, while the 13 C methyl signal of the therapeutic molecule is excited. The method may also apply a water suppression technique (WET) sequence to suppress the 13 H acetate signal (and / or signals from other excipients) corresponding to the methyl region where the 1 C signal cannot be suppressed by at least one of the three different pulses (Reburp, BIP, G3, adiabatic). The method further includes applying a shorter gradient pulse to increase the intensity of the 13 C methyl signal of the therapeutic molecule. The application of the above-described pulses is for detecting specific compositions including peptides and proteins in pharmaceutical formulations, etc., in 2D13 This is possible in the disclosed NMR method, which can be used to perform a 13C NMR methyl fingerprint.

[0018] Referring now to the figures, FIG. 1 shows, according to some embodiments, 1 H- 13 an exemplary NMR signal enhancement pulse profile 100 that uses a combination of a 1H-13C sensitivity enhanced HSQC experiment and additional signal processing steps. FIG. 2 shows, according to some embodiments, 1 H- 13 another example of an NMR signal enhancement pulse profile 200 based on a 1H-13C sensitivity enhanced HSQC experiment scheme. FIGS. 3A - 3F show, according to some embodiments, 13 exemplary excitation profiles 300a, 300b, and 300c of pulses having different shapes for suppressing the 13C sucrose signal. The exemplary NMR signal enhancement techniques shown in FIGS. 1, 2, and 3A - 3F are for illustrative purposes only.

[0019] FIG. 1 shows a specific set of signal processing steps applied to the 2D 13C NMR methyl fingerprint of an mAb, using an additional signal processing step in a state-of-the-art 1H-13C sensitivity enhanced HSQC experiment. As shown, the pulse profile 100 of FIG. 1, which is an RF pulse having a specific signal profile, is applied to induce proton ( 13 1H) magnetization, which then transfers to directly bonded carbon ( 1 13C) magnetization by an Insensitive Nuclei Enhanced by Polarization Transfer (INEPT) processing step. In FIG. 1, Δ = 1 / 2*J, δ = 1 / 8*J, where J is 145 Hz, φ 13 = 0.2; and φ 1 = 0,2; and φ 13 = 0,2; and φ 1 = 0,2; and φ recIt was set to 0.2. G1 = 80% at 1 ms, and G2 = 20.1% at 1 ms (or G1 = 80% at 250 μs, and G2 = 20.1% at 246 μs). G7 = -80% at 1 ms, G8 = -40% at 1 ms, G9 = -20% at 1 ms, G10 = -10% at 1 ms, G11 = 50% at 1 ms, G5 = 5% at 600 μs, G6 = -2% at 1 ms. The maximum gradient strength at 100% was approximately 53.5 G / cm (t1 and t2 are the periods for acquiring the respective time-domain data in the F1 (frequency 1 after Fourier transform of the t1 data points) and F2 (frequency 2 after Fourier transform of the t2 data points) dimensions).

[0020] When the INEPT processing step is applied, T 1 After the evolution period, the carbon frequency is encoded during carbon magnetization. Subsequently, the carbon magnetization is transferred back to the proton magnetization for detection by applying an inverse INEPT processing step with enhanced sensitivity. In various implementations, 1 H- 13 C magnetization coherence selection, (not NMR active) 12Suppression of proton magnetization coupled to C and the shape of the absorption lines in 2D data are achieved by using a combination of gradient pulses and echo / antiecho schemes as described in Davis, A. L.; Keeler, J.; Laue, E. D.; Moskau, D.; Experiments for recording pure-absorption heteronuclear correlation spectra using pulsed field gradients, J. Magn. Reson. 1992, 98, 207 - 216; Kay, L.; Keifer, P.; Saarinen, T.; Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity, J. Am. Chem. Soc. 1992, 114, 10663 - 10665; and J. Schleucher, J.; Schwendinger, M.; Sattler, M.; Schmidt, P.; Schedletzky, O.; Glaser, S. J.; Sorensen, O. W.; and Griesinger, O. W.; A general enhancement scheme in heteronuclear multidimensional NMR employing pulsed field gradients, J. Biomol. NMR 1994, 4, 301 - 306. 2D 13 In the current NIST protocol for 2D C NMR methyl fingerprints, the carbon bandwidth is set to 7 - 35 ppm and the transmitter frequency is 21 ppm. Since the carbon signals of sucrose are in the range of 60 - 103 ppm (as shown in Figure 3A), this signal causes aliasing in the HSQC spectrum in the range of 7 - 35 ppm. In some cases, the aliased sucrose signal cannot be properly phase - adjusted, resulting in signal dispersion in the terminal region of the F 2 domain. In some cases, these aliased signals interfere with the methyl peak analysis, as will be described in more detail with respect to Figure 6A.

[0021] To solve the problem caused by the folding of the sucrose signal in FIG. 1, the disclosed NMR method involves improving the pulse design by a pulse profile modified to excite the 13 13C methyl signal while suppressing the 13 13C sucrose signal during the encoding period of the echo / antiecho scheme. In related embodiments, 13 the pulse profile can be designed to suppress the 1 13C sucrose signal. In related embodiments, since the carbon signal is more dispersed than the proton signal, 13 the suppression of the 1It may be easier than suppressing the H sucrose signal. Since the excitation band shown in FIG. 1 covers 7 ppm to 35 ppm and the suppression band exceeds 60 ppm, the transition band can be set, for example, to 60 to 35 ppm. Therefore, in the case of an NMR system operating at 600 MHz, the bandwidth of 25 ppm is 3772.5 Hz (150.9 Hz / ppm). However, the proton transition may be only about 1.5 ppm (900 Hz, 600 Hz / ppm) from 3.5 to 2 ppm or less. The bandwidth can be changed according to the NMR operating frequency and can be from 100 MHz to 2000 MHz. According to various embodiments, the operating frequency of the NMR can range from about 100 MHz to about 2000 MHz, from about 500 MHz to about 2000 MHz, from about 500 MHz to about 1000 MHz, from about 500 MHz to about 900 MHz, from about 600 MHz to about 800 MHz (including any frequency range therebetween). According to various embodiments, the NMR system can operate at frequencies of about 100 MHz, about 200 MHz, about 300 MHz, about 400 MHz, about 500 MHz, about 600 MHz, about 700 MHz, about 800 MHz, about 900 MHz, about 1000 MHz, about 1100 MHz, about 1200 MHz, about 1300 MHz, about 1400 MHz, about 1500 MHz, about 1600 MHz, about 1700 MHz, about 1800 MHz, about 1900 MHz, about 2000 MHz (including any frequency therebetween). For illustrative purposes, a 600 MHz NMR system was used in the experiments of Examples 1 and 2 described herein, and an 800 MHz NMR system was used in the experiment of Example 3. In the case of other magnetic field strengths, specific parameters of various pulses such as the lengths of Reburp and G3, and the position of the transmitter offset on the ppm scale for the asymmetric adiabatic pulse can be adjusted. Further, specific parameters of various pulses such as the length of G2 or G4 can be adjusted according to the operating frequency. For example, in 800 MHz NMR, the gradient pulse length can be 248 μs, G2 can be 40.00% to 40.50%, and G4 can be -40.00% to -40.50%. However, the performance of the asymmetric adiabatic pulse depends on the magnetic field strength.

[0022] In the example shown in FIG. 2, the disclosed NMR method involves using, for example, the CLUB sandwich technique as described in Mandelshtam, V.A.; Hu, H.; Shaka, A.J., Two-dimensional HSQC NMR spectra obtained using a self-compensating double pulsed field gradient and processed using the filter diagonalization method, Magn.Reson.Chem. 1998, 36, S17-S28; and Hu, H.; Shaka, A.J., Composite pulsed field gradients with refocused chemical shifts and short recovery time. J.Magn.Reson. 1999, 136, 54-62 during the encoding period of the echo / antiecho scheme. When designing a refocusing pulse using the double echo technique, the design process is simplified to examine the inversion profile of the elements used in the double echo sequence when the phase at the end of the double echo sequence is the same as the phase at the start of the sequence. By this technique, the refocusing profile becomes the probability of spin flips using squared inversion elements, as described, for example, in Hwang, T.-L.; Shaka, A.J., Water suppression that works. Excitation sculpting using arbitrary waveforms and pulsed field gradients. J.Magn.Reson.A 1995, 112, 275-279. This is different from the design of Reburp or similar refocusing pulses, which requires considering both the amplitude and phase responses of magnetization under the influence of RF pulses and offsets.

[0023] As described above, FIGS. 3A-3F are according to some embodiments 13Exemplary excitation profiles of pulses having different shapes are shown for suppressing the C sucrose signal. The sample used for the measurement is 1% water containing 0.1 mg / ml gadolinium chloride (GdCl 2 ) in heavy water (D 3 O). As described above, FIG. 3A shows the pulse profile 300a of the C signal in the sucrose and acetate signal regions. In this figure, the relative intensities of both the sucrose and acetate signals can be observed. 13

[0024] FIG. 3B shows the pulse profile 300b of the Reburp profile according to a related embodiment. In various embodiments, the disclosed NMR method replaces a conventional hard pulse with a Reburp refocusing pulse of 750 μs with a transmitter offset of 21 ppm that covers the excitation bandwidth of the methyl 13 C region to remove the sucrose signal, including the Reburp refocusing pulse 300b as shown in FIG. 3B. Although excited side lobes are present during the transition period, as shown in FIG. 3B, the intensity of the excited peak is small and is in the vicinity of the 60 ppm region.

[0025] FIG. 3C shows the pulse profile 300c combining BIP and G3 according to a related embodiment. The combined excitation profile of this pulse shown in FIG. 3C results in good suppression of the sucrose signal. As illustrated in FIG. 2, the first CLUB sandwich element is located at 55 ppm and has a broadband BIP pulse with a duration of 120 μs to excite a wide range of magnetization, and is located at 81.5 ppm and has a duration of 500 μs to suppress the sucrose signal. A combination with a G3 inversion pulse is used.

[0026] In some experiments using NMR measurement techniques, it is necessary to invert or excite the magnetization on one side of the bandwidth. In various implementations, for example, as described in Hwang, T.-L.; van Zijl, P.C.M.; Garwood, M., Asymmetric adiabatic pulses for NH selection. J. Magn. Reson. 1999, 138, 173-177, two half passages derived from HS1 / 2 and tanh / tan modulation functions with different R values (R = pulse length (seconds) * bandwidth (Hz)) and pulse length (Tp) are contained in an asymmetric adiabatic full passage to narrow the transition bandwidth while achieving inversion or excitation of the bandwidth on one side of the spectrum.

[0027] Figures 3D, 3E, and 3F show 13 while suppressing the C sucrose signal 13 Three exemplary asymmetric adiabatic pulses 300d, 300e, and 300f optimized by different pulse lengths to invert the C methyl signal are shown respectively. In each of Figures 3D, 3E, and 3F, T x is the transmitter offset, and the profile was generated by incrementing the offset at 1 ppm intervals.

[0028] Figure 3D shows a pulse profile 300d shown as (1) [HS1 / 2, R = 10, 0.9Tp; tanh / tan, R = 140, 0.1Tp] with a transmitter offset of 43 ppm and a pulse length of 1500 μs, as described, for example, in Hwang, T.-L.; van Zijl, P.C.M.; Garwood, M., Asymmetric adiabatic pulses for NH selection. J. Magn. Reson. 1999, 138, 173-177. As a result, while suppressing the carbon signal of sucrose, the excitation band can cover the methyl region. The transition bandwidth of [HS1 / 2, R = 10, 0.9Tp; tanh / tan, R = 140, 0.1Tp] with a pulse length of 1500 μs is approximately 700 Hz (Figure 3D). Note that the entire pulse profile can be shifted in the vicinity according to the position of the transmitter offset of the pulse. In other words, when the transmitter offset of the pulse is located at 21 ppm, the excitation band moves to a lower ppm range accordingly, but this excitation band is C β covers the methyl region while still suppressing the carbon signal.

[0029] Figure 3E shows a pulse profile 300e shown as (2) [HS1 / 2, R = 10, 0.9Tp; tanh / tan, R = 70, 0.1Tp] with a transmitter offset of 30 ppm and a pulse length of 750 μs. The excitation band covers the methyl region of the therapeutic molecule while suppressing the carbon signal of sucrose.

[0030] Figure 3F shows a pulse profile 300f shown as (3) [HS1 / 2, R = 10, 0.9Tp; tanh / tan, R = 50, 0.1Tp] with a transmitter offset of 2 ppm and a pulse length of 375 μs. Similarly, the excitation band can cover the methyl region of the therapeutic molecule while suppressing the carbon signal of sucrose. In Figure 3F, the transition bandwidth of [HS1 / 2, R = 10, 0.9Tp; tanh / tan, R = 50, 0.1Tp] with a pulse length of 375 μs is in a very wide range, but the T 2 and T 1ρ relaxation of the mAb magnetization is extremely short, so the intensity loss of the methyl peak is reduced at a shorter pulse length.

[0031] Figure 4 is the result of Fourier-transforming the free induction decay data in the time domain into frequency domain data, and this is a graph 400 of the spectrum visualizing the NMR peaks appearing at different ppm. The X-axis is represented as ppm and is independent of the spectrometer frequency, whereby spectra can be compared at different magnetic field strengths. As shown in Figure 4, the graph 400 shows, according to the related embodiments, 1 H- 13 a comparison of the signal intensities of sucrose, acetate, and methyl peaks based on the H- 1 H- 13 C sensitivity-enhanced HSQC experiment scheme. The intensities of different components in the H-

[0032] FIG. 5 is a graph 500 showing a spectrum that can visualize NMR peaks appearing at different ppm by Fourier-transforming free induction decay data in the time domain into frequency domain data. The X-axis is represented as ppm and is independent of the spectrometer frequency, thereby allowing comparison of spectra at different magnetic field strengths. As shown in FIG. 5, the graph 500, according to some embodiments, shows a comparison of the signal intensities of sucrose and methyl peaks based on the 1 H- 13 C sensitivity enhanced HSQC experiment scheme. In particular, the signal profiles shown in FIG. 5 are from the signal intensities of different components measured by an 1 H- 13 C HSQC experiment using newly proposed refocusing pulses (i.e., Reburp, BIP+G3, and asymmetric adiabatic pulses) during the encoding period of the echo / antiecho scheme. In various embodiments, a water suppression technique (WET) scheme is applied to suppress the acetate signal. In various embodiments, a digital filter is applied to further remove the water signal.

[0033] Also, FIG. 5 shows that the intensity of the sucrose signal is on the same order as the intensity of the methyl peak. In the 2D spectrum, these sucrose signals behave like the noise of T 1 and do not interfere with the methyl peak analysis (as shown in FIGS. 6B and 6C). These spectra also show that the intensity of the methyl peak varies slightly for pulses having different pulse lengths. For example, the pulse profile of [HS1 / 2, R = 10, 0.9Tp; tanh / tan, R = 140, 0.1Tp] with a pulse length of 1500 μs located at 21 ppm does not excite the C β signal, and as shown in FIG. 5, the corresponding H β peak near 3 ppm disappears.

[0034] In various embodiments, the T of the signal of the small peptide2 and T 1ρ Relaxation is much slower than that of large mAbs. Conversely, the T of mAbs 2 and T 1ρ Relaxation and / or intensity loss due to diffusion effects can be significant with a slight difference in pulse length. As a result, any slight difference in pulse length can significantly affect the intensity of the methyl peak of mAbs. According to related embodiments of the disclosed NMR method, the pulse sequence can be improved by shortening the gradient pulse from 1000 μs to 250 μs during the echo / antiecho period. This technique is experimented with using Sample 3. Due to the different polarities of the gradients in the CLUB sandwich, eddy currents can be nullified, and thus gradient recovery can be further reduced from the conventional 200 μs to 50 μs. These optimized values are obtained by integrating the methyl peak areas from -0.5 to 2 ppm for current and new 1 H- 13 The relative integral values from different experiments when applied to the C HSQC experiment are compared in Table 1 below.

[0035]

Table 1

[0036] The data in Table 1 show that the original hard refocusing experiment with a gradient of 1 ms (1000 μs) length has a minimum relative intensity of 0.73. After shortening the gradient pulse length to approximately 250 μs, the relative methyl intensity increased significantly to 1.

[0037] Figures 6A - 6C show plots 600a, 600b, and 600c of different C 2D methyl fingerprints for comparing the effectiveness of a particular NMR enhancement method. Figure 6A shows 3%D 13 C 2D methyl fingerprints of plots 600a, 600b, and 600c respectively. Figure 6A shows 3%D 2The experimental results using the conventional NMR method (i.e., the NIST protocol) are shown for a sample containing mAb1, 50 mg / ml, 9% sucrose, 10 mM acetate, 0.01% polysorbate (PS) 80 mixed with O at pH = 5.2. The sucrose signal folded into the methyl region, and the strip of the acetate signal appeared near 2 ppm. These artifacts interfered with the methyl peak analysis. In contrast, Figure 6B represents a clear methyl region without interference from the sucrose and acetate signals. This result was obtained by using [HS1 / 2, R = 10, 0.9Tp; tanh / tan, R = 50, 0.1Tp] with a transmitter offset of 2 ppm and a pulse length of 375 μs as a refocusing element, and 1 using the WET sequence to suppress the H acetate signal. Figure 6C shows that the C β region can be further suppressed by using [HS1 / 2, R = 10, 0.9Tp; tanh / tan, R = 140, 0.1Tp] with a transmitter offset of 21 ppm and a pulse length of 1500 μs.

[0038] Therapeutic protein "Therapeutic protein" means any protein molecule that exhibits therapeutic biological activity. The therapeutic protein molecule may be, for example, a full-length protein. In other embodiments, the therapeutic protein is an active fragment of the full-length protein. The therapeutic protein may be made and purified from its natural source. Alternatively, the term "recombinant therapeutic protein" includes any therapeutic protein obtained by recombinant DNA technology.

[0039] Proteins that bind to one or more of the following can be used in the disclosed methods. These include CD proteins including CD3, CD4, CD8, CD19, CD20, CD22, CD30 and CD34, including those that interfere with receptor binding. HER receptor family proteins including HER2, HER3, HER4 and EGF receptor. Cell adhesion molecules such as LFA-I, MoI, pl50, 95, VLA-4, ICAM-I, VCAM, and α v / β 3 integrin. Growth factors such as vascular endothelial growth factor ("VEGF"), growth hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, parathyroid hormone, müllerian inhibiting substance, human macrophage inflammatory protein (MIP-I-α), erythropoietin (EPO), nerve growth factors such as NGF-β, platelet-derived growth factor (PDGF), fibroblast growth factors including, for example, aFGF and bFGF, epidermal growth factor (EGF), in particular, transforming growth factor (TGF) including TGF-α and TGF-β, for example TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5, insulin-like growth factors I and II (IGF-I and IGF-II), des(l-3)-IGF-I (brain IGF-I), and osteogenic factors. Insulin, insulin A chain, insulin B chain, proinsulin, and insulin-related proteins including insulin-like growth factor binding proteins. In particular, coagulation proteins and coagulation-related proteins such as factor VIII, tissue factor, von Willebrand factor, protein C, α-1-antitrypsin, plasminogen activators such as urokinase-type plasminogen activator and tissue plasminogen activator ("t-PA"), bombazine, thrombin and thrombopoietin; other blood proteins and serum proteins including but not limited to albumin, IgE and blood group antigens. Colony stimulating factors and their receptors such as the following, in particular, M-CSF, GM-CSF, and G-CSF, and their receptors such as CSF-1 receptor (c-fms).Receptor proteins and receptor-related proteins, including, for example, flk2 / flt3 receptor, obesity (OB) receptor, LDL receptor, growth hormone receptor, thrombopoietin receptor (“TPO-R”, “c-mpl”), glucagon receptor, interleukin receptor, interferon receptor, T cell receptor, stem cell factor receptors such as c-Kit and other receptors. Receptor ligands, including, for example, OX40L which is a ligand of OX40 receptor. Neurotrophic factors, including bone-derived neurotrophic factor (BDNF), and neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6). Relaxin A chain, relaxin B chain, and prorelaxin; Interferons and interferon receptors, including, for example, interferon-α, -β, and -γ, and their receptors. Interleukins and interleukin receptors, including IL-1 to IL-33 and IL-1 to IL-33 receptors, for example, particularly including IL-8 receptor. Virus antigens, including AIDS envelope virus antigen. Lipoproteins, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactant, tumor necrosis factor α and β, enkephalinase, RANTES (regulated on activation normally T-cell expressed and secreted), mouse gonadotropin-related peptide, DNase, inhibin and activin. Integrins, protein A or D, rheumatoid factors, immunotoxins, bone morphogenetic proteins (BMP), superoxide dismutase, surface membrane proteins, decay-accelerating factor (DAF), AIDS envelope, transport proteins, homing receptors, addressins, regulatory proteins, immunoadhesins, antibodies. Myostatin, TALL-I, amyloid proteins, for example, but not limited to, amyloid β protein, thymic stromal lymphopoietin (“TSLP”), RANK ligand (“OPGL”), TALL proteins including c-kit, TNF receptors including TNF receptor type 1, TRAIL-R2, angiopoietin, and any bioactive fragment or analog or variant of the foregoing.

[0040] Other therapeutic proteins include Activase® (alteplase); alirocumab, Aranesp® (darbepoetin-alpha), Epogen® (epoetin alpha, or erythropoietin); Avonex® (interferon beta-Ia); Bexxar® (tositumomab); Betaseron® (interferon-beta); bococizumab (anti-PCSK9 monoclonal antibody called L1L3, see U.S. Patent No. 8,080,243); Campath® (alemtuzumab); Dynepo® (epoetin delta); Velcade® (bortezomib); MLN0002 (anti-α4β7 Ab); MLN1202 (anti-CCR2 chemokine receptor Ab); Enbrel® (etanercept); Eprex® (epoetin alpha); Erbitux® (cetuximab); evolocumab; Genotropin® (somatropin); Herceptin® (Trastuzumab); Humatrope (registered trademark) (somatropin [rDNA origin] for injection); Humira (registered trademark) (adalimumab); Infergen (registered trademark) (interferon alfa-con-1); Natrecor (registered trademark) (nesiritide); Kineret (registered trademark) (anakinra); Leukine (registered trademark) (sargramostim); LymphoCide (registered trademark) (epratuzumab); Benlysta (trademark) (belimumab); Metalyse (registered trademark) (tenecteplase); Mircera (registered trademark) (methoxypolyethylene glycol-epoetin beta); Mylotarg (registered trademark) (gemtuzumab ozogamicin); Raptiva (registered trademark) (efalizumab); Cimzia (registered trademark) (certolizumab pegol); Soliris (trademark) (eculizumab); pexelizumab (anti-C5 complement); MEDI-524 (Numax (registered trademark)); Lucentis (registered trademark) (ranibizumab); edrecolomab (Panorex (registered trademark)); Trabio (registered trademark) (lerdelimumab); TheraCim hR3 (nimotuzumab); Omnitarg (pertuzumab, 2C4); Osidem (registered trademark) (IDM-I); OvaRex (registered trademark) (B43.13); Nuvion (registered trademark) (visilizumab); canzumab mertansine (huC242-DMl); NeoRecormon (registered trademark) (epoetin beta); Neumega (registered trademark) (oprelvekin); Neulasta (registered trademark) (pegfilgrastim, pegylated G-CSF, pegylated hu-Met-G-CSF); Neupogen (registered trademark) (filgrastim);Orthoclone OKT3 (registered trademark) (muromonab-CD3), Procrit (registered trademark) (epoetin alfa), Remicade (registered trademark) (infliximab), Reopro (registered trademark) (abciximab), Actemra (registered trademark) (anti-IL6 receptor Ab), Avastin (registered trademark) (bevacizumab), HuMax-CD4 (zanolimumab), Rituxan (registered trademark) (rituximab), Tarceva (registered trademark) (erlotinib), Roferon-A (registered trademark) (interferon alfa-2a), Simulect (registered trademark) (basiliximab), Stelara (trademark) (ustekinumab), Prexige (registered trademark) (lumiracoxib), Synagis (registered trademark) (palivizumab); 146B7-CHO (anti-IL15 antibody, see US Patent No. 7,153,507), Tysabri (registered trademark) (natalizumab); Valortim (registered trademark) (MDX-1303, anti-Bacillus anthracis protective antigen Ab); ABthrax (trademark); Vectibix (registered trademark) (panitumumab); Xolair (registered trademark) (omalizumab), ETI211 (anti-MRSA Ab), IL-I Trap (extracellular domains of both the Fc portion of human IgG1 and the IL-I receptor component (type I receptor and receptor accessory protein)), VEGF Trap (Ig domain of VEGFR1 fused to the Fc of IgG1), Zenapax (registered trademark) (daclizumab); Zenapax (registered trademark) (daclizumab), Zevalin (registered trademark) (ibritumomab tiuxetan), Atacicept (TACI-Ig), anti-α4β7 Ab (vedolizumab); galiximab (anti-CD80 monoclonal antibody), anti-CD23 Ab (lumiliximab); BR2-Fc (huBR3 / huFc fusion protein, soluble BAFF antagonist); Simponi (trademark) (golimumab); mapatumumab (human anti-TRAIL receptor-1 Ab); ocrelizumab (anti-CD20 human Ab); HuMax-EGFR (zalutumumab); M200 (volociximab, anti-α5β1 integrin Ab); MDX-010 (ipilimumab, anti-CTLA-4 Ab and VEGFR1 (IMC-18F1); anti-BR3 Ab;Anti-C. difficile toxin A and toxin B C Abs MDX-066 (CDA-I) and MDX-1388); anti-CD22 dsFv-PE38 conjugate (CAT-3888 and CAT-8015); anti-CD25 Ab (HuMax-TAC); anti-TSLP antibody; anti-TSLP receptor antibody (see U.S. Patent No. 8,101,182); anti-TSLP antibody called A5 (see U.S. Patent No. 7,982,016); (see anti-CD3 Ab (NI-0401); adecatumumab (MT201, anti-EpCAM-CD326 Ab); MDX-060, SGN-30, SGN-35 (anti-CD30 Ab); MDX-1333 (anti-IFNAR); HuMax CD38 (anti-CD38 Ab); anti-CD40L Ab; anti-Cripto Ab; anti-CTGF idiopathic pulmonary fibrosis phase I Fibrogen (FG-3019); anti-CTLA4 Ab; anti-eotaxin Ab (CAT-213); anti-FGF8 Ab; anti-ganglioside GD2 Ab; anti-sclerostin antibody (see U.S. Patent No. 8,715,663 or U.S. Patent No. 7,592,429), anti-sclerostin antibody called Ab-5 (see U.S. Patent No. 8,715,663 or U.S. Patent No. 7,592,429); anti-ganglioside GM2 Ab; anti-GDF-8 human Ab (MYO-029); anti-GM-CSF receptor Ab (CAM-3001); anti-HepC Ab (HuMax HepC); MEDI-545, MDX-1103 (anti-IFNα Ab); anti-IGFIR Ab; anti-IGF-IR Ab (HuMax-Inflam); anti-IL12 / IL23p40 Ab (briakinumab); anti-IL-23p19 Ab (LY2525623); anti-IL13 Ab (CAT-354); anti-IL-17 Ab (AIN457); anti-IL2Ra Ab (HuMax-TAC); anti-IL5 receptor Ab; anti-integrin receptor Ab (MDX-Ol8, CNTO 95); anti-IPIO ulcerative colitis Ab (MDX-1100); anti-LLY antibody; BMS-66513; anti-mannose receptor / hCGβ Ab (MDX-1307); anti-mesothelin dsFv-PE38 conjugate (CAT-5001); anti-PDlAb (MDX-1 106 (ONO-4538));Anti-PDGFRα antibody (IMC-3G3); anti-TGFβ Ab (GC-1008); anti-TRAIL receptor-2 human Ab (HGS-ETR2); anti-TWEAK Ab; anti-VEGFR / Flt-1 Ab; anti-ZP3 Ab (HuMax-ZP3); NVS antibody #1; NVS antibody #2; and an amyloid β monoclonal antibody containing the sequences of SEQ ID NO: 8 and SEQ ID NO: 6 (see U.S. Patent No. 7,906,625) are mentioned.;

[0041] Examples of antibodies that can be used in the disclosed method include the antibodies shown in Table A. Other examples of suitable antibodies include infliximab, bevacizumab, ranibizumab, cetuximab, palivizumab, abagovomab, abciximab, actoxumab, adalimumab, afelimomab, afutuzumab, alacizumab, alacizumab pegol, ald518, alemtuzumab, allorokumab, altumomab, amatuximab, anatumomab mafenatox, anrukinzumab, apolizumab, arcitumomab, aselizumab, altinumab, atorizumab, atorlimomab, tocilizumab, bapineuzumab, basiliximab, bavituximab, bectumomab, belimumab, benralizumab, beltilimumab, besilesomab, bevacizumab, bezlotoxumab, bispecific monoclonal antibody, bivatuzumab, bivatuzumab mertansine, blinatumomab, brodalumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, canertinib mertansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, cc49, cedelizumab, certolizumab pegol, cetuximab, cixutumumab bogatox, cixutumumab, clazakizumab, clenoliximab, crizanlizumab tetraxetan, conatumumab, crenezumab, cr6261, dacetuzumab, daclizumab, darolutamide, daratumumab, demcizumab, denosumab, detumomab, dolutamide altotox, dorzigatox, durigatox, dupilumab, eculizumab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, erlotinib, elsilimomab, enavatuzumab, enlimomab pegol, enokizumab, enoticumab, ensituximab, epitumomab cituxetan, epratuzumab, erlizumab, ertumaxomab, etaracizumab, etorlimomab, exibizumab, fanolesomab, faralimumab, farletuzumab, facizumab, fbta05, felvizumab, fezakinumab, ficlatuzumab, figitumumab, flanvotumab, fontolizumab, foralumab, foravilumab, fresolimumab, fulranumab, futuximab, galiximab, ganitumab, ganetespib, gavrilimomab, gemtuzumab ozogamicin, gebokizumab, gilencitinib,Glembatumumab vedotin, golimumab, gomiliximab, gs6624, ibalizumab, ibritumomab tiuxetan, icrucumab, igovomab, imciromab, imigatuzumab, inclacumab, indatuximab ravtansine, infliximab, intezumumab, inolimomab, inotuzumab ozogamicin, ipilimumab, iratumumab, itritumumab, ixekizumab, certolizumab, labetuzumab, lebrikizumab, remalizumab, rerdelimumab, rexicizumab, rivibizumab, rigelizumab, lintuzumab, lirilumab, lorvotuzumab mertansine, lucatumumab, lumiliximab, mapatumumab, maslimomab, mabrilizumab, matuzumab, mepolizumab, metelimumab, miratumumab, minleptomonab, mitumomab, mogamulizumab, morolimumab, motavizumab, moxetumomab pasudotox, muromonab-cd3, nacotromab tafenotox, namilumab, naputromab estafenotox, nalnatumumab, natalizumab, nebacumab, nesitumumab, nerelimumab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, ocaratuzumab, ocrelizumab, odevixibat, ofatumumab, olaratumab, orocizumab, omalizumab, onartuzumab, oportuzumab monatox, oregovomab, orticumab, oteracizumab, oxelumab, ozanezumab, ozoralizumab, pazibaxizumab, parvizumab, panitumumab, panobacumab, palsatumumab, pascolizumab, pateclizumab, patritumab, pemtumomab, perakizumab, pertuzumab, pegylated infliximab, pidilizumab, pintumomab, plicamycin, ponesimod, priliximab, pritumumab, PRO 140, quilizumab, racotumomab, radretumab, raffibercept, ramucirumab, ranibizumab, raxibacumab, regorafenib, reslizumab, rilotumumab, rituximab, robatumumab, roledumab, romosozumab, rontalizumab, rovelizumab, rupatuzumab, samalizumab, sartalizumab, satumomab pentetide, secukinumab, sevirumab, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, sipuleucel-T, silucumab, sorafenib, solitomab, sonelixizumab, sonitumumab, stimalizumab, streslimab, subizumab,Tabalumab, Tocilizumab Tetraxetan, Tositumomab, Talizumab, Tanecumab, Tavilimumab Paptox, Tefibazumab, Telimomab Aritox, Tenatumomab, Teneliximab, Tepilizumab, Teplizumab, TGN1412, Tremelimumab, Chirilimumab, Chilukizumab, Chigatsuzumab, TNX-650, Tositumomab, Tralizumab, Tosatumomab, Tralokinumab, Trastuzumab, TRBS07, Tregrastuzumab, Tremelimumab, Tsukotsuzumab Celmolokin, Tubulumab, Ublituximab, Urelumab, Ultuxizumab, Ustekinumab, Bapaliximab, Batelizumab, Vedolizumab, Belzutuzumab, Bepalimumab, Besremi, Bispecific Anti-CD3xCD20 T Cell Engager, Bortezomib, Bortezomib Mafodotin, Bortezomib, Zalutumumab, Zanomeline, Zalizumab, Girentuximab, and Zolimomab Aritox.

[0042] The most preferred antibodies for use in the disclosed methods are adalimumab, bevacizumab, blinatumomab, cetuximab, conatumumab, denosumab, eculizumab, elotuzumab, evolocumab, infliximab, natalizumab, panitumumab, rilotumumab, rituximab, romosozumab, and trastuzumab, and antibodies selected from Table A.

[0043] [Table A-1]

[0044] [Table A-2]

[0045] Mutant A mutein is a protein having at least an amino acid mutation due to a mutation of a nucleic acid sequence such as substitution, deletion or insertion. Exemplary muteins include amino acid sequences having at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90% sequence homology to the wild-type amino acid sequence, or having more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99%) sequence homology. In addition, the mutein may be a fusion protein as described above. In an exemplary embodiment, the mutein comprises an amino acid sequence containing at least one amino acid substitution relative to the wild-type amino acid sequence, and the amino acid substitution is a conservative amino acid substitution. As used herein, the term "conservative amino acid substitution" means substituting one amino acid with another amino acid having similar properties, e.g., size, charge, hydrophobicity, hydrophilicity, and / or aromaticity, and includes exchanges within the range of one of the following five groups: I. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, Gly; II. Polar negatively charged residues, and their amides and esters: Asp, Asn, Glu, Gln, cysteic acid and homocysteic acid; III. Polar positively charged residues: His, Arg, Lys; ornithine (Orn) IV. Large aliphatic, nonpolar residues: Met, Leu, Ile, Val, Cys, norleucine (Nle), homocysteine V. Large aromatic residues: Phe, Tyr, Trp, acetylphenylalanine.

[0046] In an exemplary embodiment, the mutein comprises an amino acid sequence having at least one amino acid substitution relative to the wild-type amino acid sequence, and the amino acid substitution is a non-conservative amino acid substitution. As used herein, the term "non-conservative amino acid substitution" is defined herein as substituting one amino acid with another amino acid having different properties, such as size, charge, hydrophobicity, hydrophilicity, and / or aromaticity, and includes exchanges outside the scope of the above five groups.

[0047] In an exemplary embodiment, the mutein comprises an amino acid sequence having at least one amino acid substitution relative to the wild-type amino acid sequence, and the substituted amino acid is a naturally occurring amino acid. "Naturally occurring amino acid" or "standard amino acid" or "canonical amino acid" refers to one of the 20 α-amino acids (Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, Gln, Cys, Gly, Pro, Arg, His, Lys, Asp, Glu) directly encoded by codons of the universal genetic code and found in eukaryotes. In an exemplary embodiment, the mutein comprises an amino acid sequence having at least one amino acid substitution relative to the wild-type amino acid sequence, and the substituted amino acid is a non-standard amino acid or an amino acid that was not incorporated into the protein during translation. Non-standard amino acids include, but are not limited to, selenocysteine, pyrrolidine, ornithine, norleucine, β-amino acids (e.g., β-alanine, β-aminoisobutyric acid, β-phenylalanine, β-homophenylalanine, β-glutamic acid, β-glutamine, β-homotryptophan, β-leucine, β-lysine), homoamino acids (e.g., homophenylalanine, homoserine, homoarginine, monocysteine, homocystine), N-methyl amino acids (e.g., L-ablaine, N-methyl-alanine, N-methyl-isoleucine, N-methyl-leucine), 2-aminocaprylic acid, 7-aminocephalosporanic acid, 4-aminobenzoic acid, α-aminocyclohexanepropionic acid, amino-(4-hydroxyphenyl)acetic acid, 4-amino-nicotinic acid, 3-aminophenylacetic acid, and the like.

[0048] BiTE® molecule A bispecific T cell engager (BiTE) molecule is a bispecific antibody construct or bispecific fusion protein that contains two antibody-binding domains (or target regions) linked together. One arm of the molecule is modified to bind to a protein found on the surface of cytotoxic T cells, and the other arm is designed to bind to a specific protein found mainly on tumor cells. When the BiTE molecule binds to both targets, it forms a bridge between the cytotoxic T cells and the tumor cells, thereby enabling the T cells to recognize the tumor cells and eliminate them by injecting harmful molecules. For example, different BiTE antibody constructs can be generated by modifying the tumor-binding arm of the molecule to target different types of cancer.

[0049] The term "binding domain" with respect to a BiTE molecule means a domain that (specifically) binds to / interacts with / recognizes a given target epitope or a given target site on a target molecule (antigen). The structure and function of the first binding domain (recognizing the antigen of the target cell), and preferably also the structure and / or function of the second binding domain (recognizing the cytotoxic T cell), are based on the structure and / or function of an antibody, such as a full-length or complete immunoglobulin molecule.

[0050] "Epitope" means a site on an antigen to which a binding domain, such as an antibody or an immunoglobulin or a derivative or fragment of an antibody or an immunoglobulin, specifically binds. An "epitope" is antigenic and thus the term "epitope" may also be referred to herein as an "antigenic structure" or "antigenic determinant". Thus, the binding domain is an "antigen interaction site". The binding / interaction is also understood to define "specific recognition".

[0051] For example, a BiTE molecule comprises a first binding domain characterized by the presence of three light chain "complementary determining regions" (CDRs) (i.e., CDR1, CDR2, and CDR3 of the VL region), as well as three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VH region). Also, the second binding domain preferably comprises the minimal structural requirements of an antibody that enable target binding. More preferably, the second binding domain comprises at least three light chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VL region), and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VH region). The first and / or second binding domain is envisioned to be made or obtained by phage display or library screening methods, in addition to grafting CDR sequences from existing (monoclonal) antibodies onto a scaffold.

[0052] A binding domain can typically include the antibody light chain variable region (VL) and the antibody heavy chain variable region (VH), but it is not necessary to include both. The Fd fragment, for example, has two VH regions and often retains the antigen-binding function of a part of the intact antigen-binding domain. Examples of (modified) antigen-binding antibody fragments include: (1) the Fab fragment, a monovalent fragment having the VL, VH, CL, and CH1 domains; (2) the F(ab’)2 fragment, a bivalent fragment having two Fab fragments linked by a disulfide bridge in the hinge region; (3) the Fd fragment having two VH and CH1 domains; (4) the Fv fragment having the VL and VH domains of a single arm of the antibody; (5) the dAb fragment having a VH domain (Ward et al., (1989) Nature 341:544-546); (6) the isolated complementary determining region (CDR); and (7) the single-chain Fv (scFv), with the latter (e.g., from an scFv-library) being preferred.

[0053] The terms "bind (specifically)", "recognize (specifically)", "direct (specifically)" and "react (specifically)" with respect to BiTE molecules mean that the binding domain interacts or specifically interacts with one or more, preferably at least two, more preferably at least three and most preferably at least four amino acids of an epitope located on a target protein or antigen.

[0054] The term "variable" refers to a part of an antibody or immunoglobulin domain (i.e., the "variable domain") that exhibits variability within the sequence and is involved in determining the specificity and binding affinity of a particular antibody. A pair of variable heavy (VH) and variable light (VL) chains together form a single antigen-binding site. The CH domain closest to VH is designated CH1. Each light (L) chain is linked to the heavy (H) chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype.

[0055] The variability is not uniformly distributed throughout the variable domains of the antibody but is concentrated in each of the subdomains of the heavy and light chain variable regions. These subdomains are called "hypervariable regions" or "complementary determining regions" (CDRs). The more conserved (i.e., non-hypervariable) parts of the variable domains are called "framework" regions (FRMs), which provide a scaffold for the six CDRs in three-dimensional space and form the antigen-binding surface. The variable domains of the naturally occurring heavy and light chains each contain four FRM regions (FR1, FR2, FR3, and FR4), mostly in a β-sheet structure, which are connected by three hypervariable regions that form loop connections and sometimes part of the β-sheet structure. The hypervariable regions of each chain are held together in proximity by the FRMs and contribute to the formation of the antigen-binding site together with the hypervariable regions of the other chain (see Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service N.I.H., Bethesda, MD). The constant domains do not directly participate in antigen binding but exhibit various effector functions such as antibody-dependent cell-mediated cytotoxicity and complement activation.

[0056] CDR3 of the light chain and, particularly, CDR3 of the heavy chain can be the most important determinants in antigen binding within the light and heavy chain variable regions. In some antibody constructs, the heavy chain CDR3 appears to be the major contact area between the antigen and the antibody. An in vitro selection scheme that varies only CDR3 can be used to change the binding properties of the antibody or to determine which residues contribute to antigen binding. Thus, CDR3 is typically the largest source of molecular diversity within the antibody-binding site. For example, H3 can be as short as about two amino acid residues or more than 26 amino acids.

[0057] The sequences of antibody genes after construction and somatic mutation are extremely diverse, and these diversified genes are estimated to encode 10^10 different antibody molecules (Immunoglobulin Genes, 2nd ed., eds. Jonio et al., Academic Press, San Diego, CA, 1995). Thus, the immune system provides an immunoglobulin repertoire. The term "repertoire" means at least one nucleotide sequence that is wholly or partly derived from at least one sequence encoding at least one immunoglobulin. This sequence can be generated by in vivo rearrangement of the V, D, and J segments of the heavy chain, and the V and J segments of the light chain. Alternatively, this sequence can be generated from cells in response to, for example, in vitro stimulation that causes rearrangement. Alternatively, some or all of this sequence may be obtained by DNA splicing, nucleotide synthesis, mutagenesis, and other methods (see, for example, U.S. Patent No. 5,565,332). The repertoire can contain only one sequence or multiple sequences including those within a genetically diverse collection.

[0058] As used herein, the term "bispecific" means an antibody construct that is "at least bispecific", i.e., it contains at least a first binding domain and a second binding domain, where the first binding domain binds to one antigen or target and the second binding domain binds to another antigen or target. Thus, the antibody construct within the BiTE molecule has specificity for at least two different antigens or targets. The term "bispecific antibody construct" of the present invention also encompasses multispecific antibody constructs, such as a trispecific antibody construct containing three binding domains or a construct having four or more (e.g., four, five,...) specificities.

[0059] At least two binding domains and variable domains of the antibody construct within the BiTE molecule may or may not include a peptide linker (spacer peptide). According to the present invention, the term "peptide linker" defines an amino acid sequence that links the amino acid sequences of one (variable and / or binding) domain and the other (variable and / or binding) domain of the antibody construct of the present invention to each other. A highly important technical feature of such a peptide linker is that the peptide linker does not contain any polymerization activity. Suitable peptide linkers include those described in U.S. Patent Nos. 4,751,180 and 4,935,233 or International Publication No. WO 88 / 09344 pamphlet.

[0060] When a linker is used, this linker preferably consists of a length and sequence sufficient for each of the first and second domains to reliably retain their different binding specificities independently of each other. In the case of a peptide linker connecting at least two binding domains (or two variable domains) of an antibody construct within a BiTE molecule, those peptide linkers preferably contain only a few amino acid residues, for example those containing 12 or fewer amino acid residues. Thus, peptide linkers of 12, 11, 10, 9, 8, 7, 6 or 5 amino acid residues are preferred. The putative peptide linker of less than 5 amino acids contains 4, 3, 2 or 1 amino acid, and here, a Gly-rich linker is preferred. The particularly preferred "single" amino acid in relation to the "peptide linker" is Gly. Thus, the above peptide linker can consist of the single amino acid Gly. Another preferred embodiment of the peptide linker is characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e., Gly4Ser or its polymer, i.e., (Gly4Ser)x, where x is an integer of 1 or more. The characteristics of the peptide linker, including not promoting secondary structure, are known in the art and are described, for example, in Dall’Acqua et al. (Biochem. (1998) 37, 9266-9273), Cheadle et al. (Mol Immunol (1992) 29, 21-30), and Raag and Whitlow (FASEB (1995) 9(1), 73-80). A peptide linker that does not promote any secondary structure is preferred. The mutual linkage of the domains can be provided, for example, by the genetic manipulation described in the examples. Methods for preparing and expressing bispecific single-chain constructs that are fused and operably linked in mammalian cells or bacteria are well known in the art (for example, WO 99 / 54440 pamphlet or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).

[0061] The BiTE molecules of the present disclosure may include antibody constructs of a configuration selected from the group consisting of (scFv)2, scFv-single domain mAb, diabodies and oligomers of any of the above configurations.

[0062] According to a particularly preferred embodiment and as described in the accompanying examples, the antibody construct within the BiTE molecule is a "bispecific single-chain antibody construct", more preferably a bispecific "single-chain Fv" (scFv). The two domains of the Fv fragment, VL and VH, are encoded by separate genes, but these can be joined by a synthetic linker that enables them to be made as a single protein chain paired such that the VL and VH regions form a monovalent molecule using recombinant methods. See, for example, Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883. These antibody fragments are obtained using prior art known to those skilled in the art, and the fragments are evaluated for function in the same manner as full or whole antibodies. Thus, a single-chain variable fragment (scFv) is a fusion protein of the variable region of the heavy chain (VH) and the variable region of the light chain (VL) of an immunoglobulin, connected by a short linker peptide usually of about 10 to about 25 amino acids, preferably about 15 to 20 amino acids. The linker usually contains glycine for flexibility and is rich in serine or threonine for solubility, and can either connect the N-terminus of VH to the C-terminus of VL or vice versa. This protein retains the specificity of the original immunoglobulin despite removal of the constant regions and introduction of a linker.

[0063] Bispecific single-chain molecules are known in the art and are described in WO 99 / 54440, Mack, J. Immunol. (1997), 158, 3965-3970, Mack, PNAS (1995), 92, 7021-7025, Kufer, Cancer Immunol. Immunother. (1997), 45, 193-197, Loeffler, Blood (2000), 95, 6, 2098-2103, Bruehl, Immunol. (2001), 166, 2420-2426, Kipriyanov, J. Mol. Biol. (1999), 293, 41-56. Techniques described for the production of single-chain antibodies (see in particular US Pat. No. 4,946,778, Kontermann and Duebel (2010), supra and Little (2009), supra) can be adapted to produce single-chain antibody constructs that specifically recognize a selected target.

[0064] A bivalent (also called divalent) or bispecific single-chain variable fragment (bi-scFv or di-scFv having a (scFv)2 construct) can be modified by linking two scFv molecules. When these two scFv molecules have the same binding specificity, the resulting (scFv)2 molecule is preferably called bivalent (i.e., it has a valence of 2 for the same target epitope). When these two scFv molecules have different binding specificities, the resulting (scFv)2 molecule is preferably called bispecific. This linking can be done by creating a single peptide chain having two VH regions and two VL regions to generate a tandem scFv (see, e.g., Kufer P. et al., (2004) Trends in Biotechnology 22(5):238-244). Another possibility is to create scFv molecules using a linker peptide that is too short (e.g., about 5 amino acids) to fold the two variable regions together and dimerize this scFv. This type is known as a diabody (see, e.g., Hollinger, Philipp et al., (July 1993) Proceedings of the National Academy of Sciences of the United States of America 90(14):6444-8).

[0065] A single-domain antibody contains only one (monomeric) antibody variable domain that can selectively bind to a specific antigen independently of other V regions or domains. The first single-domain antibodies were modified from heavy-chain antibodies found in camels, which are called VHH fragments. Cartilaginous fish also have heavy-chain antibodies (IgNAR) from which single-domain antibodies called VNAR fragments can be obtained. An alternative approach is, for example, to split the dimeric variable domains from common immunoglobulins derived from humans or rodents into monomers, thereby obtaining VH or VL as single-domain Abs. Most research on single-domain antibodies is currently based on heavy-chain variable domains, but nanobodies derived from light chains have also been shown to specifically bind to target epitopes. Examples of single-domain antibodies are called sdAbs, nanobodies, or single variable domain antibodies.

[0066] Accordingly, (single-domain mAb)2 is a monoclonal antibody construct composed of (at least) two single-domain monoclonal antibodies individually selected from the group consisting of VH, VL, VHH, and VNAR. The linker is preferably in the form of a peptide linker. Similarly, "scFv single-domain mAb" is a monoclonal antibody construct composed of at least one single-domain antibody described above and one scFv molecule described above. Again, in this case, the linker is preferably in the form of a peptide linker.

[0067] Exemplary BiTE molecules include anti-CD33 and anti-CD3 BiTE molecules, anti-BCMA and anti-CD3 BiTE molecules, anti-FLT3 and anti-CD3 BiTE, anti-CD19 and anti-CD3 BiTE, anti-EGFRvIII and anti-CD3 BiTE molecules, anti-DLL3 and anti-CD3 BiTE, BLINCYTO (blinatumomab), and solitomab.

[0068] Pharmaceutical composition formulations and components Permissible pharmaceutical ingredients are preferably non-toxic to patients at the dosages and concentrations used. The pharmaceutical composition may include, for example, agents for modifying, maintaining or protecting the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or permeability of the composition.

[0069] In general, excipients can be classified based on the mechanisms by which they stabilize proteins against various chemical and physical stresses. Some excipients mitigate the effects of specific stresses or modulate the specific susceptibilities of particular polypeptides. Other excipients have more general effects on the physical and covalent stability of proteins. Table B shows common excipients for liquid and lyophilized protein formulations (see also Kamerzell TJ, Esfandiary R, Joshi SB, Middaugh CR, Volkin DB. 2011. Protein-excipient interactions: mechanisms and biophysical characterization applied to protein formulation development. Adv Drug Deliv Rev 63:1118-59).

[0070]

Table B-1

[0071]

Table B-2

[0072] As described above, it has been surprisingly found that by changing the pulse profile, a significant impact can be exerted on the signal-to-noise ratios in various NMR regions. For example, using a specific pulse profile with an inversion pulse, while suppressing the 13 C excipient signal derived from sucrose, the 13The C-methyl signals can be excited. These signals can be enhanced by shorter gradient pulses. These various factors affecting signal enhancement and signal suppression are further claimed in the following embodiments.

[0073] According to related embodiments, an exemplary method of fingerprinting specific molecules in a composition using NMR is described herein. The method includes providing a composition having at least a first molecule having a first NMR signal, a second molecule having a second NMR signal, and a third molecule having a third NMR signal. In this method, each of the signals originates from each of the respective molecules having a non-zero nuclear spin. The method includes applying a cycle of signal processing steps. This cycle includes applying a high-frequency (RF) pulse, applying a gradient pulse having a pulse length of 1000 μs or less, and applying a water suppression technique (WET). In this method, the first NMR signal, the second NMR signal, and the third NMR signal are 13 located in a region of the NMR spectrum near the defined ppm range of the C-methyl signal. The method also includes repeating the cycle at least three times to obtain an enhanced signal of the composition. The method further includes fingerprinting specific molecules based on the enhanced signal of the composition.

[0074] In this embodiment and related embodiments, the region of the NMR spectrum includes an NMR 13 C spectral window of about 5 ppm to about 150 ppm. The region of the NMR spectrum includes an NMR spectral window of about 5 ppm to about 100 ppm, about 5 ppm to about 50 ppm, or about 7 ppm to about 35 ppm. Further, for example, when oxidized met is used, the NMR spectral window can be about 7 ppm to about 40 ppm.

[0075] The RF pulse includes at least one of a Reburp pulse, a broadband inversion pulse (BIP), and a Gaussian (G3) inversion pulse, and an asymmetric adiabatic pulse. In the case of the Reburp pulse, a first NMR signal is excited by this pulse. In the case of the BIP, a wide range of NMR signals are excited by the BIP, and a second NMR signal is suppressed by the G3 inversion pulse. In the case of the asymmetric adiabatic pulse, a first NMR signal is excited while suppressing the second NMR signal by this pulse.

[0076] The first NMR signal is an NMR signal related to the 13 C methyl of the therapeutic molecule, and the second NMR signal is 13 a signal related to 1 C sucrose, and the third NMR signal is 1 a signal related to at least 13 the H acetate or other

[0077] Exemplary methods for using NMR can be performed in a frequency range of about 100 MHz to about 2000 MHz, such as 1200 MHz, currently commercially available.

[0078] The Reburp pulse has a pulse length of about 500 μs to about 1000 μs. The Reburp pulse has a pulse length of about 600 μs to about 900 μs, or about 600 μs to about 800 μs.

[0079] The combination of the BIP and the G3 inversion pulse has a total pulse length of about 200 μs to about 2500 μs. The combination of the BIP and the G3 inversion pulse has a pulse length of about 200 μs to about 2000 μs, about 200 μs to about 1500 μs, about 250 μs to about 1000 μs, or about 250 μs to about 750 μs. The combination of the BIP and the G3 inversion pulse has a pulse length of about 620 μs. The BIP has a pulse length of about 120 μs, and the G3 inversion pulse has a pulse length of about 500 μs.

[0080] The asymmetric heat insulation pulse has a pulse length of about 50 μs to about 2500 μs, about 50 μs to about 2000 μs, about 50 μs to about 1500 μs, about 50 μs to about 1000 μs, or about 100 μs to about 800 μs.

[0081] The gradient pulse has a pulse length of about 1500 μs or less, or about 1000 μs or less. The gradient pulse has a pulse length of about 50 μs to about 1500 μs, about 50 μs to about 1200 μs, about 50 μs to about 1000 μs, about 50 μs to about 800 μs, about 50 μs to about 600 μs, about 50 μs to about 500 μs, about 50 μs to about 400 μs, about 50 μs to about 300 μs, about 50 μs to about 250 μs, about 50 μs to about 200 μs, about 50 μs to about 150 μs, or about 50 μs to about 100 μs.

[0082] Following the gradient pulse, there is at least one reverse gradient pulse having a pulse length of about 50 μs to about 990 μs, about 50 μs to about 900 μs, about 50 μs to about 800 μs, about 50 μs to about 700 μs, about 50 μs to about 600 μs, about 50 μs to about 500 μs, about 50 μs to about 400 μs, about 50 μs to about 300 μs, about 50 μs to about 250 μs, about 50 μs to about 200 μs, about 50 μs to about 150 μs, or about 50 μs to about 100 μs.

[0083] Following at least one reverse gradient pulse, there is another gradient pulse having a pulse length of about 50 μs to about 990 μs, about 50 μs to about 900 μs, about 50 μs to about 800 μs, about 50 μs to about 700 μs, about 50 μs to about 600 μs, about 50 μs to about 500 μs, about 50 μs to about 400 μs, about 50 μs to about 300 μs, about 50 μs to about 250 μs, about 50 μs to about 200 μs, about 50 μs to about 150 μs, or about 50 μs to about 100 μs.

[0084] Another exemplary method for fingerprinting specific molecules in a composition using NMR is described herein. The method includes providing a composition having at least a first molecule having a first NMR signal, a second molecule having a second NMR signal, and a third molecule having a third NMR signal. Each of the signals results from each of the respective molecules having a non-zero nuclear spin. The method includes applying a cycle of signal processing steps. The cycle includes applying a radio frequency (RF) pulse and applying a gradient pulse. In this method, the first NMR signal, the second NMR signal, and the third NMR signal are located in the region of an NMR spectral window of about 5 ppm to about 150 ppm. The method also includes repeating the cycle at least three times to obtain an enhanced signal of the composition. The method further includes fingerprinting specific molecules based on the enhanced signal of the composition.

[0085] The cycle further includes applying a water suppression technique (WET) sequence.

[0086] The region of the NMR spectrum includes an NMR spectral window of about 5 ppm to about 100 ppm, about 5 ppm to about 50 ppm, or about 7 ppm to about 35 ppm.

[0087] The RF pulse includes at least one of a Reburp pulse, a combination of a broadband inversion pulse (BIP) and a Gaussian (G3) inversion pulse, or an asymmetric adiabatic pulse.

[0088] In the case of the Reburp pulse, the first NMR signal is excited by this pulse. The broadband inversion pulse excites a wide range of NMR signals, and the G3 inversion pulse suppresses the second NMR signal. The asymmetric adiabatic pulse excites the first NMR signal while suppressing the second NMR signal.

[0089] The first NMR signal is of a therapeutic molecule 13The NMR signal related to C-methyl, and the second NMR signal is 13 The signal related to C-sucrose, and the third NMR signal is 1 H-acetate or other 1 H / 13 The signal is at least related to the C NMR signal.

[0090] Exemplary methods for using NMR can be performed in a frequency range of about 100 MHz to about 2000 MHz, such as 1200 MHz.

[0091] The Reburp pulse has a pulse length of about 300 μs to about 1000 μs, about 600 μs to about 900 μs, or about 600 μs to about 800 μs.

[0092] The combination of the BIP and G3 inversion pulses has a total pulse length of about 200 μs to about 2500 μs, about 200 μs to about 2000 μs, about 200 μs to about 1500 μs, about 250 μs to about 1000 μs, or about 250 μs to about 750 μs. The combination of the BIP and G3 inversion pulses has a pulse length of about 620 μs to 660 μs. The BIP has a pulse length of about 120 μs to 160 μs, and the G3 inversion pulse has a pulse length of about 500 μs.

[0093] The asymmetric adiabatic pulse has a pulse length of about 50 μs to about 2500 μs, about 50 μs to about 2000 μs, about 50 μs to about 1500 μs, about 50 μs to about 1000 μs, or about 100 μs to about 800 μs.

[0094] The gradient pulse has a pulse length of 1000 μs or less. In some implementations, the gradient pulse has a pulse length of about 50 μs to about 990 μs, about 50 μs to about 900 μs, about 50 μs to about 800 μs, about 50 μs to about 700 μs, about 50 μs to about 600 μs, about 50 μs to about 500 μs, about 50 μs to about 400 μs, about 50 μs to about 300 μs, about 50 μs to about 250 μs, about 50 μs to about 200 μs, about 50 μs to about 150 μs, or about 50 μs to about 100 μs.

[0095] In some embodiments, at least one inversion gradient pulse having a pulse length of 1000 μs or less follows the gradient pulse. After the gradient pulse, at least one inversion gradient pulse having a pulse length of about 50 μs to about 990 μs, about 50 μs to about 900 μs, about 50 μs to about 800 μs, about 50 μs to about 700 μs, about 50 μs to about 600 μs, about 50 μs to about 500 μs, about 50 μs to about 400 μs, about 50 μs to about 300 μs, about 50 μs to about 250 μs, about 50 μs to about 200 μs, about 50 μs to about 150 μs, or about 50 μs to about 100 μs follows.

[0096] After at least one inversion gradient pulse, another gradient pulse having a pulse length of 1000 μs or less follows. After at least one inversion gradient pulse, another gradient pulse having a pulse length of about 50 μs to about 990 μs, about 50 μs to about 900 μs, about 50 μs to about 800 μs, about 50 μs to about 700 μs, about 50 μs to about 600 μs, about 50 μs to about 500 μs, about 50 μs to about 400 μs, about 50 μs to about 300 μs, about 50 μs to about 250 μs, about 50 μs to about 200 μs, about 50 μs to about 150 μs, or about 50 μs to about 100 μs follows.

[0097] Another exemplary method for fingerprinting specific molecules in a composition using NMR is described herein. The method includes providing a composition having at least a first molecule having a first NMR signal, a second molecule having a second NMR signal, and a third molecule having a third NMR signal. In this method, each of the signals results from each of the respective molecules having a non-zero nuclear spin. The method includes applying a radio frequency (RF) pulse to the composition to excite the first NMR signal while suppressing the second NMR signal. The RF pulse includes at least one of a Reburp pulse, a combination of a broadband inversion pulse and a Gaussian inversion pulse, or an asymmetric adiabatic pulse. The method also includes applying a gradient pulse having a pulse length of 1000 μs or less and applying a water suppression technique (WET) sequence to suppress the third NMR signal. The method also includes repeating the cycle at least three times to acquire the signal of the enhanced composition. The method further includes fingerprinting a specific molecule based on the signal of the enhanced composition.

[0098] The first NMR signal, the second NMR signal, and the third NMR signal are 13 located in a region of the NMR spectrum near the C methyl signal.

[0099] The first NMR signal, the second NMR signal, and the third NMR signal are located in an NMR spectral window of about 5 ppm to about 150 ppm. In various implementations, the first NMR signal, the second NMR signal, and the third NMR signal are located in an NMR spectral window of about 5 ppm to about 100 ppm, about 5 ppm to about 50 ppm, or about 7 ppm to about 35 ppm.

[0100] An exemplary method for using NMR can be performed in a frequency range of about 100 MHz to about 2000 MHz, such as 1200 MHz, currently commercially available.

[0101] The Reburp pulse has a pulse length of about 300 μs to about 1000 μs, about 600 μs to about 900 μs, or about 600 μs to about 800 μs.

[0102] The combination of the BIP and G3 inversion pulses has a total pulse length of about 200 μs to about 2500 μs, about 200 μs to about 2000 μs, about 200 μs to about 1500 μs, about 250 μs to about 1000 μs, or about 250 μs to about 750 μs.

[0103] The combination of the BIP and G3 inversion pulses has a pulse length of about 620 μs to 660 μs. The BIP has a pulse length of about 120 μs to 160 μs, and the G3 inversion pulse has a pulse length of about 500 μs.

[0104] The asymmetric adiabatic pulse has a pulse length of about 50 μs to about 2500 μs, about 50 μs to about 2000 μs, about 50 μs to about 1500 μs, about 50 μs to about 1000 μs, or about 100 μs to about 800 μs.

[0105] The gradient pulse has a pulse length of about 50 μs to about 1500 μs, about 50 μs to about 1200 μs, about 50 μs to about 1000 μs, about 50 μs to about 800 μs, about 50 μs to about 600 μs, about 50 μs to about 500 μs, about 50 μs to about 400 μs, about 50 μs to about 300 μs, about 50 μs to about 250 μs, about 50 μs to about 200 μs, about 50 μs to about 150 μs, or about 50 μs to about 100 μs.

[0106] Following the gradient pulse, there is at least one inversion gradient pulse having a pulse length of about 50 μs to about 990 μs, about 50 μs to about 900 μs, about 50 μs to about 800 μs, about 50 μs to about 700 μs, about 50 μs to about 600 μs, about 50 μs to about 500 μs, about 50 μs to about 400 μs, about 50 μs to about 300 μs, about 50 μs to about 250 μs, about 50 μs to about 200 μs, about 50 μs to about 150 μs, or about 50 μs to about 100 μs.

[0107] After at least one inversion gradient pulse, another gradient pulse having a pulse length of about 50 μs to about 990 μs, about 50 μs to about 900 μs, about 50 μs to about 800 μs, about 50 μs to about 700 μs, about 50 μs to about 600 μs, about 50 μs to about 500 μs, about 50 μs to about 400 μs, about 50 μs to about 300 μs, about 50 μs to about 250 μs, about 50 μs to about 200 μs, about 50 μs to about 150 μs, or about 50 μs to about 100 μs follows.

[0108] In various implementations, applying the RF pulse, the gradient pulse, and the WET sequence constitutes a cycle of the signal processing step, and the method further includes repeating this cycle at least 3 times.

[0109] The method includes repeating the cycle less than 1024 times, less than 512 times, less than 500 times, less than 400 times, less than 300 times, less than 256 times, less than 250 times, less than 200 times, less than 150 times, less than 128 times, less than 100 times, less than 96 times, less than 80 times, less than 70 times, less than 64 times, less than 60 times, less than 50 times, less than 48 times, less than 40 times, less than 36 times, less than 30 times, less than 25 times, less than 20 times, or less than 16 times.

[0110] Other excipients are known in the art (see, for example, Powell MF, Nguyen T, Baloian L. 1998. Compendium of excipients for parenteral formulations. PDA J Pharm Sci Technol 52:238 - 311). One of ordinary skill in the art can determine what amount or range of excipients may be included in any particular formulation to obtain a biopharmaceutical composition that promotes the maintenance of the stability of the biopharmaceutical. For example, the amount and type of salt to be included in the biopharmaceutical composition can be selected based on the desired osmolality of the final solution (i.e., isotonic, hypotonic, or hypertonic) and the amount and osmolality of the other components to be included in the formulation.

[0111]

Table C

Example

[0112] Experimental results, materials and methods Example 1 To perform the measurements of Example 1, a 5 mm CPTCI cryoprobe 1 H{ 19 F}- 13 C / 15 N / D-ZGRD z-gradient equipped Bruker Avance III 600 MHz NMR spectrometer (10040043) was used to acquire NMR data at 310 K (37 °C). Data processing was performed using spectrometer software (TopSpin, Bruker BioSpin North America; Billerica, MA) and MNova software (Mestrelab Research S.L. (USA); Escondido, CA).

[0113] To evaluate the disclosed NMR method, the following samples were used.

[0114] Sample 1: 5% D 2 O mixed, 50 mM acetate, 5% sucrose, 0.01% PS80, pH = 5, having 42 amino acids, M.W. 4651.38 Da, 30 mg / ml, 6 mM peptide. Approximately 200 μl of the solution was placed in a 4 mm tube manufactured by Shigemi, Inc. for NMR analysis.

[0115] Sample 2: 3% D 2 O mixed, mAb1, 50 mg / ml, 9% sucrose, 10 mM acetate, 0.01% PS80, pH = 5.2. Approximately 600 μl of the solution was placed in a 5 mm tube manufactured by Wilmad for NMR analysis.

[0116] Sample 3: Approximately 1 mL of D 2Proline, 32.22 mg (ca. 280 mM) (Sigma-Aldrich), sucrose, 87.92 mg (Sigma-Aldrich), dissolved in 99.9% D (Sigma-Aldrich; St. Louis, MO). Approximately 600 μl of the solution was placed into a 5 mm Wilmad tube for NMR analysis.

[0117] Sample 4: D 2 0.1 mg / ml GdCl in D 3 1% water containing

[0118] Example 2 To perform the measurements of Example 2, NMR data were acquired at 310 K (37 °C) for Samples 1 and 2, and at 300 K (27 °C) for Sample 3 using a Bruker Avance III 600 MHz NMR spectrometer (S / N 10040043) equipped with a 5 mm CPTCI cryoprobe 1 H{ 19 F}- 13 C / 15 N / D-ZGRD z-gradient (S / N Z128744 / 0001). 1 H{ 19 F}- 13 C / 15 N / D-ZGRD z-gradient (S / N Z128744 / 0001).

[0119] In this example, a 2D methyl fingerprint pulse sequence was applied to suppress the excipient signals of mAb1 samples in A52Su buffer (10 mM acetate, 9% sucrose, pH: 5.2) with the addition of (1) 10 mM glutamate or (2) 200 mM proline, and of "Protein 1" (an antigen-binding protein with a standard BiTE molecular structure) in G42Su buffer (15 mM glutamate, 9% sucrose, pH: 4.2).

[0120] In addition to suppressing the signals from sucrose and acetate, three samples were prepared to test the ability of the NMR pulse sequence to suppress the signals from glutamate and proline.

[0121] Sample 1: 10 mM glutamate and 5% D 2mAb1 with added O, 50 mg / ml, 9% sucrose, 10 mM acetate.

[0122] Sample 2: 200 mM proline and 5% D 2 mAb1 with added O, 50 mg / ml, 9% sucrose, 10 mM acetate.

[0123] Sample 3: Protein 1, 10 mg / ml, 9% sucrose, 15 mM glutamate and 5% D 2 O.

[0124] Referring now to FIG. 7, an exemplary NMR signal enhancement pulse sequence 700 based on an H- 1 H 13 C sensitivity enhanced HSQC experiment scheme for suppressing the excipient signal from sucrose is shown. As shown in FIG. 7, while using the WET portion of the pulse sequence to suppress the proton signal of acetate, a newly formed pulse is used during the HSQC experiment to suppress the carbon signal from sucrose while exciting the carbon signal from the methyl region of the therapeutic protein. In this example, the pulse used in the WET portion of the sequence is redesigned to suppress signals from other excipients, exemplified by glutamate and proline. The pulses in the WET portion of the sequence can be generated using Bruker Topspin software depending on what signals from excipients need to be suppressed.

[0125] FIG. 8 shows spectra 800 from the first increment of HSQC data in a state (802) where the signals from 10 mM glutamate and 10 mM acetate in Sample 1 of Example 2 are not suppressed, and in a suppressed state (804). The WET pulse was designed to suppress signals from glutamate and acetate in particular. The peak intensity at 2.418 ppm has decreased to the baseline level. These peaks have approximately the same intensity as the peaks in the methyl region, even though the peak intensities at 2.144 ppm and 2.080 ppm have decreased by about 50%.

[0126] Figure 9A shows the 2D methyl region of HSQC spectrum 900a in a state where the signals from 10 mM glutamate and 10 mM acetate in Sample 1 of Example 2 are not suppressed. Figure 9B shows the 2D methyl region of HSQC spectrum 900b in a state where the signals from 10 mM glutamate and 10 mM acetate in Sample 1 of Example 2 are suppressed. These spectra prove that, as shown in Figure 8, when it is possible to compare the signal intensity from the excipient with the signal intensity from the methyl peak, these signals do not cause a strip along the carbon dimension, or there is no possibility of causing a problem with the phase alignment of the 2D spectrum. Artifacts resulting from strip and alignment problems may interfere with the data analysis of methyl peaks near the artifact.

[0127] Figure 10 shows spectra 1000 from the first increment of HSQC data in a state (1002) where the signal from 15 mM glutamate in Sample 3 of Example 2 is not suppressed, and in a suppressed state (1004). The peak from glutamate is efficiently suppressed by using the WET sequence.

[0128] Figure 11A shows the 2D methyl region of HSQC spectrum 1100a in a state where the signal from 15 mM glutamate in Sample 3 of Example 2 is not suppressed. Figure 11B shows the 2D methyl region of HSQC spectrum 1100b in a state where the signal from 15 mM glutamate in Sample 3 of Example 2 is suppressed. These spectra clarify that when the signal intensity from the excipient is much higher than the signal intensity from the methyl peak, these signals may cause a strip in the carbon dimension, thereby interfering with the analysis of peaks near the strip in the methyl region.

[0129] Figure 12 shows spectrum 1200 from the first increment of HSQC data in a state (1202) where the signals from 200 mM proline and 10 mM acetate in sample 2 of Example 2 are not suppressed, and in a suppressed state (1204). The intensity from 200 mM proline is much greater than the intensity from the peaks in the methyl region.

[0130] Figure 13 shows another exemplary NMR signal enhancement pulse sequence 1300 based on the double-WET scheme according to various embodiments. Using the double-WET scheme shown in Figure 13, the proline signal was suppressed to the baseline level. The double-WET scheme is more efficient than a single-WET scheme and has been shown to effectively suppress the peaks from proline without resulting in a carbon dimension strip as shown in Figures 14A and 14B. Nevertheless, the intensity of the peaks in the methyl region decreased by about 15% when the double-WET scheme was used, compared to the intensity obtained from a single-WET scheme.

[0131] Figure 14A shows the 2D methyl region of HSQC spectrum 1400a in a state where the signals from 200 mM proline and 10 mM acetate in sample 2 of Example 2 are not suppressed. Figure 14B shows the 2D methyl region of HSQC spectrum 1400b in a state where the signals from 200 mM proline and 10 mM acetate in sample 2 of Example 2 are suppressed. In the state where the peaks from proline are not suppressed, there are strips along the carbon dimension and the proton dimension as shown in Figure 14A. When the double-WET sequence is used to suppress the proline signal, the 2D spectrum in Figure 14B is suitable for the analysis of the peaks in the methyl region.

[0132] Example 3 As described herein, when applying these pulses in NMR spectrometers with different magnetic field strengths, the pulses can be adjusted in pulse length or the transmitter offset can be placed separately. The results of this example demonstrate such an application at 800 MHz. In particular, Example 3 was carried out using the following parameters: 3%D 2 NMR data at 800 MHz in mAb1, 50 mg / ml, 9% sucrose, 10 mM acetate, 0.01% polysorbate (PS) 80 with pH = 5.2, mixed with 3%D

[0133] When using the same type of probe in the experiment, the 800 MHz NMR system has higher spectral sensitivity and better spectral resolution compared to the 600 MHz NMR system. That is, for example, 1 ppm in the carbon dimension is 200 Hz and 150 Hz in the 800 MHz and 600 MHz NMR systems, respectively. Therefore, in the spectrum obtained by the 800 MHz NMR system, the peaks may be further broadened.

[0134] Figures 15A - 15E show 13 Exemplary excitation profiles of pulses with different shapes that can be applied at 800 MHz to suppress the C sucrose signal. Figure 15A shows the 13It shows the pulse profile 1500a of the C signal. Figure 15B shows the pulse profile 1500b of the Reburp profile, which is adjusted to 575 μs to maintain the same excitation profile as the 750 μs Reburp pulse at 600 MHz. Figure 15C shows the pulse profile 1500c. Since the carbon spectral width at Hz is large at 800 MHz, in order to maintain a similar excitation profile as shown in Figure 15C, instead of a 2 ppm transmitter offset at 600 MHz, the transmitter offset is placed at 16 ppm for [HS1 / 2, R = 10, 0.9Tp; tanh / tan, R = 140, 0.1Tp] with a pulse length of 375 μs at 800 MHz. Figure 15D shows the pulse profile 1500d with parameters [HS1 / 2, R = 10, 0.9Tp; tanh / tan, R = 70, 0.1Tp] of a transmitter offset of 18 ppm and a pulse length of 750 μs. Figure 15E shows the pulse profile 1500e with parameters [HS1 / 2, R = 10, 0.9Tp; tanh / tan, R = 1400, 0.1Tp] of a transmitter offset of 27 ppm and a pulse length of 1500 μs. Using these profiles 1500d and 1500e, C β suppresses the carbon signal.

[0135] Figures 16A and 16B show different 13 plots 1600a and 1600b of the C 2D methyl fingerprint obtained on an 800 MHz NMR spectrometer to compare the effectiveness of a specific NMR enhancement method. Figure 16A shows a distinct methyl region obtained by using, as a refocusing element, [HS1 / 2, R = 10, 0.9Tp; tanh / tan, R = 50, 0.1Tp] with a transmitter offset of 16 ppm and a pulse length of 375 μs, and 1 using a WET sequence to suppress the H acetate signal. Figure 16B shows Cβ It shows that the region can be suppressed.

[0136] Figure 17 shows a comparison of the signal intensities of 1700 of the methyl peaks in an exemplary HSQC experiment obtained using an 800 MHz NMR system with different RF pulses. 1 H- 13 Based on the H- β C sensitivity-enhanced HSQC experiment scheme, a comparison of the signal intensities of the methyl peaks at 1700 is shown in the figure. Note that the H signal around 3 ppm disappears when using a shaping pulse [HS1 / 2, R = 10, 0.9Tp; tanh / tan, R = 70, 0.1Tp] with a pulse length of 750 μs and a transmitter offset of 18 ppm, and [HS1 / 2, R = 10, 0.9Tp; tanh / tan, R = 1400, 0.1Tp] with a pulse length of 1500 μs and a transmitter offset of 27 ppm. The relative methyl intensities obtained by integrating the peak areas from -0.5 to 2 ppm in Figure 17 are shown in Table 2. To compare the values in Table 2 with those in Table 1, the intensity of the methyl peak area by using the Reburp pulse was normalized to 0.88. The relative methyl intensities obtained at 600 MHz and 800 MHz are similar.

[0137]

Table 2

[0138] This specification includes details of many specific implementations, which should not be construed as limitations on the scope of any invention or what may be claimed, but rather as descriptions of features particular to specific implementations of a particular invention. Also, some features described herein in the context of separate implementations may be implemented in combination in a single implementation. Conversely, various features described in the context of a single implementation may be implemented separately in multiple implementations or in any suitable sub-combination. Further, features are described above as functioning in certain combinations and may even be initially claimed as such, but one or more features from the claimed combination may in some cases be excluded from that combination, and the claimed combination may be directed to a sub-combination or a variant of a sub-combination.

[0139] Similarly, operations are shown in the drawings in a particular order, which should not be understood as requiring that such operations be performed in the particular order shown or in a sequential order to achieve a desired result, or that all illustrated operations be performed.

[0140] References to "or" may be construed as inclusive in that any terms described using "or" may indicate any one, more than one, and all of the terms so described. Designations such as "first", "second", "third", etc. do not necessarily mean an indication of order and are generally used merely to distinguish between items or elements of the same or similar type.

[0141] Various modifications to the implementations described in this disclosure may be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other implementations without departing from the spirit or scope of this disclosure. Accordingly, the claims are not intended to be limited to the implementations shown herein, but rather should be accorded the widest scope consistent with this disclosure and the principles and novel features disclosed herein.

[0142] All the reference documents cited within the permitted scope of rights are incorporated herein by reference.

Claims

1. A method for determining a specific molecule in a composition based on a fingerprint of the composition obtained by using nuclear magnetic resonance (NMR), comprising: providing a composition comprising at least a first molecule having a first NMR signal, a second molecule having a second NMR signal, and a third molecule having a third NMR signal, each of said signals arising from each of said respective molecules having a non-zero nuclear spin; applying a cycle of signal processing steps, said cycle comprising: applying a radio frequency (RF) pulse; applying a gradient pulse having a pulse length of 1000 μs or less, said gradient pulse being accompanied by an echo / antiecho scheme; applying a water suppression technique (WET) to suppress said third NMR signal; and The first NMR signal, the second NMR signal, and the third NMR signal are １３ located in a region of the NMR spectrum within a defined ppm range of C methyl signals, wherein the first NMR signal is a １３ signal related to C methyl of the therapeutic molecule, the second NMR signal is a １３ signal related to C sucrose, and the third NMR signal is a signal related to at least １ H acetate or other excipients. applying a cycle of signal processing steps; repeating said cycle at least three times to obtain an enhanced signal of said composition; determining said specific molecule based on said enhanced signal of said composition. A method as described above.

2. The method according to claim 1, wherein said region of the NMR spectrum comprises an NMR spectral window of about 5 ppm to about 150 ppm.

3. The method according to claim 1, wherein said region of the NMR spectrum comprises an NMR spectral window of about 5 ppm to about 100 ppm.

4. The method according to claim 1, wherein said region of the NMR spectrum comprises an NMR spectral window of about 5 ppm to about 50 ppm.

5. The method according to claim 1, wherein said region of the NMR spectrum comprises an NMR spectral window of about 7 ppm to about 35 ppm.

6. The method according to claim 1, wherein said RF pulse comprises at least one of a combination of a Reburp pulse, a broadband inversion pulse (BIP), and a Gaussian (G3) inversion pulse, or an asymmetric adiabatic pulse.

7. The method according to claim 6, wherein said Reburp pulse excites said first NMR signal.

8. The method according to claim 6, wherein said broadband inversion pulse excites each of said NMR signals, and said G3 inversion pulse suppresses said second NMR signal.

9. The method according to claim 6, wherein the asymmetric adiabatic pulse excites the first NMR signal while suppressing the second NMR signal.

10. the third NMR signal is １ from one of H acetate, or glutamate, proline, arginine, or mannitol １ H / １３ a signal that is at least related to the C NMR signal, the method according to claim 1.

11. The method according to claim 1, wherein the method for using NMR is performed in a frequency range of about 100 MHz to about 2000 MHz.

12. The method according to claim 6, wherein the RF pulse includes the Reburp pulse, and the Reburp pulse has a pulse length of about 500 μs to about 1000 μs.

13. The method according to claim 6, wherein the RF pulse includes the Reburp pulse, and the Reburp pulse has a pulse length of about 600 μs to about 900 μs.

14. The method according to claim 6, wherein the RF pulse includes the Reburp pulse, and the Reburp pulse has a pulse length of about 600 μs to about 800 μs.

15. The method according to claim 6, wherein the RF pulse includes the combination of the BIP and the G3 inversion pulse, and the combination of the BIP and the G3 inversion pulse has a pulse length of about 200 μs to about 2500 μs.

16. The method according to claim 6, wherein the RF pulse includes the combination of the BIP and the G3 inversion pulse, and the combination of the BIP and the G3 inversion pulse has a pulse length of about 200 μs to about 2000 μs.

17. The method according to claim 6, wherein the RF pulse includes the combination of the BIP and the G3 inversion pulse, and the combination of the BIP and the G3 inversion pulse has a pulse length of about 200 μs to about 1500 μs.

18. The method according to claim 6, wherein the RF pulse includes the combination of the BIP and the G3 inversion pulse, and the combination of the BIP and the G3 inversion pulse has a pulse length of about 250 μs to about 1000 μs.

19. The method according to claim 6, wherein the RF pulse includes the combination of the BIP and the G3 inversion pulse, and the combination of the BIP and the G3 inversion pulse has a pulse length of about 250 μs to about 750 μs.

20. The method according to claim 6, wherein the RF pulse includes the combination of the BIP and the G3 inversion pulse, and the combination of the BIP and the G3 inversion pulse has a pulse length of about 620 μs to 660 μs.

21. The method according to claim 20, wherein the BIP has a pulse length of about 120 μs to 160 μs, and the G3 inversion pulse has a pulse length of about 500 μs.

22. The method according to claim 6, wherein the RF pulse includes the asymmetric adiabatic pulse, and the asymmetric adiabatic pulse has a pulse length of about 50 μs to about 2500 μs.

23. The method according to claim 6, wherein the RF pulse includes the asymmetric adiabatic pulse, and the asymmetric adiabatic pulse has a pulse length of about 50 μs to about 2000 μs.

24. The method according to claim 6, wherein the RF pulse includes the asymmetric adiabatic pulse, and the asymmetric adiabatic pulse has a pulse length of about 50 μs to about 1500 μs.

25. The method according to claim 6, wherein the RF pulse includes the asymmetric adiabatic pulse, and the asymmetric adiabatic pulse has a pulse length of about 50 μs to about 1000 μs.

26. The method according to claim 6, wherein the RF pulse includes the asymmetric adiabatic pulse, and the asymmetric adiabatic pulse has a pulse length of about 100 μs to about 800 μs.

27. The method according to claim 1, wherein the gradient pulse has a pulse length range of about 50 μs to about 990 μs.

28. The method according to claim 1, wherein the gradient pulse has a pulse length range of about 50 μs to about 900 μs.

29. The method according to claim 1, wherein the gradient pulse has a pulse length range of about 50 μs to about 800 μs.

30. The method according to claim 1, wherein the gradient pulse has a pulse length range of about 50 μs to about 700 μs.

31. The method according to claim 1, wherein the gradient pulse has a pulse length range of about 50 μs to about 600 μs.

32. The method according to claim 1, wherein the gradient pulse has a pulse length range of about 50 μs to about 500 μs.

33. The method according to claim 1, wherein the gradient pulse has a pulse length range of about 50 μs to about 400 μs.

34. The method according to claim 1, wherein the gradient pulse has a pulse length range of about 50 μs to about 300 μs.

35. The method according to claim 1, wherein the gradient pulse has a pulse length range of about 50 μs to about 250 μs.

36. The method according to claim 1, wherein the gradient pulse has a pulse length range of about 50 μs to about 200 μs.

37. The method according to claim 1, wherein the gradient pulse has a pulse length range of about 50 μs to about 150 μs.

38. The method according to claim 1, wherein the gradient pulse has a pulse length range of about 50 μs to about 100 μs.

39. The method according to any one of claims 27 to 38, wherein at least one inversion gradient pulse having the same pulse length range follows the gradient pulse.

40. The method according to claim 39, wherein another gradient pulse having the same pulse length range follows the at least one inversion gradient pulse.

41. The method according to claim 1, wherein repeating the cycle at least three times includes a delay in the repetition in the range of about 10 μs to about 990 μs.

42. The method according to claim 41, wherein the delay is about 30 μs to about 900 μs, about 50 μs to about 800 μs, about 50 μs to about 700 μs, about 100 μs to about 600 μs, about 150 μs to about 500 μs, or about 200 μs to about 300 μs.

43. A method for determining a specific molecule in a composition based on a fingerprint of the composition obtained by using nuclear magnetic resonance (NMR), comprising: providing a composition comprising at least a first molecule having a first NMR signal, a second molecule having a second NMR signal, and a third molecule having a third NMR signal, each of the signals arising from each of the respective molecules having a non-zero nuclear spin; applying a cycle of a signal processing step, the cycle comprising: applying a high-frequency (RF) pulse; applying a gradient pulse, the gradient pulse being applied with an echo / antiecho scheme; and The first NMR signal, the second NMR signal, and the third NMR signal are located in the region of an NMR spectral window of from about 5 ppm to about 150 ppm, and the first NMR signal is １３ an NMR signal related to C methyl, and the second NMR signal is １３ a signal related to an NMR signal related to C sucrose, and the third NMR signal is at least １ a signal related to H acetate or other excipients, and applying a cycle of a signal processing step repeating the cycle at least three times to obtain an enhanced signal of the composition; determining the specific molecule based on the enhanced signal of the composition; and further comprising applying a water suppression technique (WET) sequence to suppress the third NMR signal. Method.

44. The method according to claim 43, wherein the region of the NMR spectrum includes an NMR spectrum window of about 5 ppm to about 100 ppm, about 5 ppm to about 50 ppm, or about 7 ppm to about 35 ppm.

45. The method according to claim 43, wherein the RF pulse includes at least one of a Reburp pulse, a broadband inversion pulse (BIP), and a Gaussian (G3) inversion pulse, and an asymmetric adiabatic pulse.

46. The method according to claim 45, wherein the RF pulse includes the Reburp pulse, and the Reburp pulse excites the first NMR signal.

47. The method according to claim 45, wherein the RF pulse includes the combination of the BIP and the G3 inversion pulse, the broadband inversion pulse excites a wide range of NMR signals, and the G3 inversion pulse suppresses the second NMR signal.

48. The method according to claim 45, wherein the RF pulse includes the asymmetric adiabatic pulse, and the asymmetric adiabatic pulse excites the first NMR signal while suppressing the second NMR signal.

49. the third NMR signal being １ from at least one of H acetate, or glutamate, proline, arginine, or mannitol １３ The method according to claim 43, which is a signal at least related to the C NMR signal

50. The method according to claim 43, wherein the NMR is performed in a frequency range of about 100 MHz to about 2000 MHz.

51. The method according to claim 45, wherein the RF pulse includes the Reburp pulse, and the Reburp pulse has a pulse length of about 500 μs to about 1000 μs, about 600 μs to about 900 μs, or about 600 μs to about 800 μs.

52. The method according to claim 45, wherein the RF pulse includes the combination of the BIP and the G3 inversion pulse, and the combination of the BIP and the G3 inversion pulse has a pulse length of about 200 μs to about 2500 μs, about 200 μs to about 2000 μs, about 200 μs to about 1500 μs, about 250 μs to about 1000 μs, or about 250 μs to about 750 μs.

53. The method according to claim 45, wherein the RF pulse includes the combination of the BIP and the G3 inversion pulse, and the combination of the BIP and the G3 inversion pulse has a pulse length of about 620 μs to 660 μs.

54. The method according to claim 53, wherein the BIP has a pulse length of about 120 μs to 160 μs, and the G3 inversion pulse has a pulse length of about 500 μs.

55. The method according to claim 45, wherein the RF pulse includes the asymmetric adiabatic pulse, and the asymmetric adiabatic pulse has a pulse length of about 50 μs to about 2500 μs, about 50 μs to about 2000 μs, about 50 μs to about 1500 μs, about 50 μs to about 1000 μs, or about 100 μs to about 800 μs.

56. The method according to claim 43, wherein the gradient pulse has a pulse length of 1000 μs or less.

57. The method according to claim 43, wherein the gradient pulse has a pulse length range of about 50 μs to about 1000 μs, about 50 μs to about 900 μs, about 50 μs to about 800 μs, about 50 μs to about 700 μs, about 50 μs to about 600 μs, about 50 μs to about 500 μs, about 50 μs to about 400 μs, about 50 μs to about 300 μs, about 50 μs to about 250 μs, about 50 μs to about 200 μs, about 50 μs to about 150 μs, or about 50 μs to about 100 μs.

58. The method according to any one of claims 56 or 57, wherein at least one inversion gradient pulse having the same pulse length or the same pulse length range follows the gradient pulse.

59. The method according to claim 58, wherein another gradient pulse having the same pulse length range follows the at least one inversion gradient pulse.

60. The method according to claim 43, wherein repeating the cycle at least three times includes a delay in the repetition in the range of about 10 μs to about 990 μs, about 30 μs to about 900 μs, about 50 μs to about 800 μs, about 50 μs to about 700 μs, about 100 μs to about 600 μs, about 150 μs to about 500 μs, or about 200 μs to about 300 μs.

61. A method for determining a specific molecule in a composition based on a fingerprint of the composition obtained by using nuclear magnetic resonance (NMR), To provide a composition comprising at least a first molecule having a first NMR signal, a second molecule having a second NMR signal, and a third molecule having a third NMR signal, each of said signals arising from each of said respective molecules having a non-zero nuclear spin, wherein the first NMR signal is １３ an NMR signal related to C methyl, and the second NMR signal is １３ a signal related to an NMR signal related to C sucrose, and the third NMR signal is at least １ a signal related to H acetate or other excipients, and to provide a composition applying a radio frequency (RF) pulse to the composition to excite the first NMR signal while suppressing the second NMR signal, wherein the RF pulse includes at least one of a combination of a Reburp pulse, a broadband inversion pulse (BIP), and a Gaussian (G3) inversion pulse, and an asymmetric adiabatic pulse; applying a gradient pulse having a pulse length of 1000 μs or less, wherein the gradient pulse is accompanied by an echo / antiecho scheme. Applying a water suppression technique (WET) sequence to suppress the third NMR signal; Obtaining the signal of the enhanced composition; Determining the specific molecule based on the signal of the enhanced composition; A method comprising the above steps.

62. The first NMR signal, the second NMR signal, and the third NMR signal are １３ The method according to claim 61, which is located in a region of an NMR spectrum in the vicinity of a C methyl signal.

63. The method according to claim 61, wherein the first NMR signal, the second NMR signal, and the third NMR signal are located in an NMR spectral window of about 5 ppm to about 150 ppm.

64. The method according to claim 61, wherein the first NMR signal, the second NMR signal, and the third NMR signal are located in an NMR spectral window of about 5 ppm to about 100 ppm, about 5 ppm to about 50 ppm, or about 7 ppm to about 35 ppm.

65. The method according to claim 61, wherein the NMR is performed in a frequency range of about 100 MHz to about 2000 MHz.

66. The method according to claim 61, wherein the RF pulse includes the Reburp pulse, and the Reburp pulse has a pulse length of about 500 μs to about 1000 μs, about 600 μs to about 900 μs, or about 600 μs to about 800 μs.

67. The method according to claim 61, wherein the RF pulse includes the combination of the BIP and the G3 inversion pulse, and the combination of the BIP and the G3 inversion pulse has a pulse length of about 200 μs to about 2500 μs, about 200 μs to about 2000 μs, about 200 μs to about 1500 μs, about 250 μs to about 1000 μs, or about 250 μs to about 750 μs.

68. The method according to claim 61, wherein the RF pulse includes the combination of the BIP and the G3 inversion pulse, and the combination of the BIP and the G3 inversion pulse has a pulse length of about 620 μs to 660 μs.

69. The method according to claim 68, wherein the BIP has a pulse length of about 120 μs to 160 μs, and the G3 inversion pulse has a pulse length of about 500 μs.

70. The method according to claim 61, wherein the RF pulse includes the asymmetric adiabatic pulse, and the asymmetric adiabatic pulse has a pulse length of about 50 μs to about 2500 μs, about 50 μs to about 2000 μs, about 50 μs to about 1500 μs, about 50 μs to about 1000 μs, or about 100 μs to about 800 μs.

71. The method according to claim 61, wherein the gradient pulse has a pulse length range of about 50 μs to about 990 μs, about 50 μs to about 900 μs, about 50 μs to about 800 μs, about 50 μs to about 700 μs, about 50 μs to about 600 μs, about 50 μs to about 500 μs, about 50 μs to about 400 μs, about 50 μs to about 300 μs, about 50 μs to about 250 μs, about 50 μs to about 200 μs, about 50 μs to about 150 μs, or about 50 μs to about 100 μs.

72. The method according to claim 71, wherein at least one inversion gradient pulse having the same pulse length range follows the gradient pulse.

73. The method according to claim 72, wherein another gradient pulse having the same pulse length range follows the at least one inversion gradient pulse.

74. Applying the RF pulse, the gradient pulse, and the WET sequence constitutes a cycle of a signal processing step, and the method The method according to claim 61, further comprising repeating the cycle at least 3 times to obtain the signal of the enhanced composition.

75. The method according to claim 74, wherein repeating the cycle at least 3 times includes a delay in the repetition in the range of about 10 μs to about 990 μs, about 30 μs to about 900 μs, about 50 μs to about 800 μs, about 50 μs to about 700 μs, about 100 μs to about 600 μs, about 150 μs to about 500 μs, or about 200 μs to about 300 μs.

76. １３ wherein the first NMR signal related to C-methyl is brought about by a BiTE® molecule selected from the group consisting of an anti-CD33 and anti-CD3 BiTE molecule, an anti-BCMA and anti-CD3 BiTE molecule, an anti-FLT3 and anti-CD3 BiTE, an anti-CD19 and anti-CD3 BiTE, an anti-EGFRvIII and anti-CD3 BiTE molecule, an anti-DLL3 and anti-CD3 BiTE, BLINCYTO (blinatumomab) and solitomab; an antibody selected from the group consisting of adalimumab, bevacizumab, blinatumomab, cetuximab, conatumumab, denosumab, eculizumab, elotuzumab, evolocumab, infliximab, natalizumab, panitumumab, lirilumab, rituximab, romosozumab, and trastuzumab, and an antibody selected from Table A; and a protein selected from the group consisting of combinations thereof, the method according to any one of claims 10 or 49.

77. the third NMR signal being １ from H acetate, or one of glutamate, proline, arginine, or mannitol １ H / １３ C NMR signal, at least related to the signal, the method according to claim 61.

78. The method according to any one of claims 11, 50, or 65, wherein the method for using NMR is performed in a frequency range of about 500 MHz to about 2000 MHz.

79. The method according to any one of claims 11, 50, or 65, wherein the method for using NMR is performed in a frequency range of about 500 MHz to about 1000 MHz.

80. The method according to any one of claims 11, 50, or 65, wherein the method for using NMR is performed in a frequency range of about 900 MHz.

81. The method according to any one of claims 11, 50, or 65, wherein the method for using NMR is performed in a frequency range of about 800 MHz.

82. The method according to any one of claims 11, 50, or 65, wherein the method for using NMR is performed in a frequency range of about 700 MHz.

83. The method according to any one of claims 11, 50 or 65, wherein the method for using NMR is performed in a frequency range of about 600 MHz.

84. The method according to any one of claims 11, 50 or 65, wherein the method for using NMR is performed in a frequency range of about 500 MHz.

85. The method according to any one of claims 10, 49 or 77, wherein the third NMR signal is related to glutamate or proline.

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