Methods directed to crystalline biomolecules

Abstract

To provide methods of preparing a composition comprising crystalline biomolecules, for example, crystalline antibodies.SOLUTION: In exemplary embodiments, the method comprises forming a fluidized bed of crystalline biomolecules using, for example, counter-flow centrifugation to exchange buffers and / or to concentrate the crystalline biomolecules in a solution. Also provided are methods of detecting crystalline biomolecules and / or amorphous biomolecules in a sample. In exemplary embodiments, a method of preparing a composition comprising crystalline biomolecules comprises forming a fluidized bed of crystalline biomolecules in a rotating chamber comprising an inlet and an outlet.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 471,358, filed on March 14, 2017, and U.S. Provisional Patent Application No. 62 / 476,359, filed on March 24, 2017, the contents of each of which are incorporated herein by reference.

Background Art

[0002] Antibodies form a cornerstone of powerful therapeutic agents, characterized by limited side effects due to their ability to specifically target distinct antigens on cells, bacteria, viruses, or toxins. In 1986, the first therapeutic monoclonal antibody, Orthoclone OKT3, was introduced to the market. Since then, this class of biologic agents has increased significantly. By the end of 2014, 47 monoclonal antibody products had been approved in the United States or Europe for the treatment of various diseases, including cancer and inflammation, cardiovascular, respiratory, and infectious diseases. Considering the current approval rate of approximately four products per year, it is estimated that approximately 70 monoclonal antibody products will be commercially available by 2020. See (Non - Patent Document 1).

[0003] Although it has been reported that the antibody market in the United States is expected to increase to over $10 billion, there are limitations in the production of such therapeutic agents. One drawback of therapeutic antibodies is the cost of downstream processing to achieve the required high - purity levels. While purity levels exceeding 90% can be achieved via protein A chromatography, the cost of the adsorption media is a major drawback. Another limiting factor for therapeutic antibodies is the sensitivity of the antibody structure to chemical and physical denaturation encountered during delivery and storage. Although researchers have developed approaches to improve the stability of antibody formulations, some of these methods lead to loss of protein activity and / or increased costs due to additional expenditure on protein - stabilizing carriers or formulations.

[0004] Protein crystallization is, in principle, recognized as an effective and scalable method of protein purification. Crystalline proteins are more stable than their protein solution counterparts and thus have a longer shelf life. Protein purification through crystallization has been shown to be feasible with test protein products, including ovalbumin and lipase. Insulin is the only therapeutic protein that is crystallized on an industrial scale. The crystallization of antibodies is not yet routine due to the complexity of their phase behavior. Precipitation, phase separation, and the formation of gel-like phases can occur, "kinetically trapping systems far from equilibrium and as a result reducing the yield of crystalline protein or completely inhibiting crystal formation". See (Non-Patent Document 2). Conventional purification techniques such as tangential flow and alternating flow filtration are not suitable for protein crystallization purification due to membrane fouling (i.e., clogging of the pores of the filter medium with crystals). Also, the high pressures required to maintain flow through the filter can lead to excessive shear, breakage, and compression of the crystals. These problems are exacerbated when the protein is an antibody, as such protein crystals are sticky and brittle.

[0005] Therefore, there is a need in the art for an effective method of preparing a composition comprising crystalline antibodies.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Means for Solving the Problems

[0007] Methods for preparing compositions comprising crystalline biomolecules, such as antigen-binding biomolecules, including but not limited to antibodies or immunoglobulins and antigen-binding fragments thereof, are disclosed herein. In representative embodiments, the method involves using counterflow gradient centrifugation (CFGC) to exchange a solution (e.g., buffer) in which the crystalline biomolecule is present and / or to concentrate a solution (e.g., buffer) containing the crystalline biomolecule. The disclosed method is advantageous because this step is performed under low shear, closed-system, and aseptic processing conditions. Multiple steps are performed in the same apparatus, thereby preventing loss of product during transfer to different apparatuses. Since a low-shear environment is maintained throughout the methods described herein, the crystalline biomolecules remain separated from each other, thereby minimizing aggregate formation. The disclosed method leads to the acquisition of a uniform crystal slurry that can be readily fed into downstream processing operations such as filling.

[0008] In representative embodiments, a method for preparing a composition comprising a crystalline biomolecule involves forming a fluidized bed of the crystalline biomolecule in a rotating chamber that includes an inlet and an outlet. Without being bound by a particular theory, the chamber is rotated about a substantially horizontal axis to generate a centrifugal force (F centrifugal ) in the chamber, and a first flow of a first solution is flowed through the inlet at a first flow rate (FR1) having a force (F centrifugal ) that cancels F centrifugal in a direction opposite to the direction of F FR1 . It is believed that a fluidized bed is generated by recovering the first solution from the chamber while substantially maintaining the formation of the fluidized bed of the crystalline biomolecule.

[0009] In representative embodiments, the method also involves replacing the first flow of the first solution with a second flow of a second solution while substantially maintaining the formation of the fluidized bed of the crystalline biomolecule. In representative aspects, the method involves flowing a second flow of a second solution to replace the first flow of the first solution while substantially maintaining the formation of the fluidized bed of the crystalline biomolecule. In representative aspects, the method involves the force of the second flow being Fcentrifugal To counteract, F centrifugal in a direction opposite to the direction of, F FR1 flow a second flow of a second solution through an inlet at a flow rate (FR) equivalent to FR1 having a force (F) equal to F, while substantially maintaining the formation of a fluidized bed of crystalline biomolecules, and recovering the second solution from the chamber.

[0010] In an exemplary embodiment, the method alternatively or further comprises concentrating crystalline biomolecules within a region of the chamber by changing FR1 to a second flow rate (FR2) having a force (F centrifugal less than the force (F FR2 ) or by increasing the rotational speed of the chamber to increase F to a level greater than F FR1 centrifugal .

[0011] In an exemplary aspect, the method comprises removing crystalline biomolecules from the chamber by flowing a second flow into the chamber through an outlet in a direction parallel to, for example, the direction of F centrifugal .

[0012] Also disclosed herein is a method for detecting crystalline and / or amorphous biomolecules in a sample. In an exemplary embodiment, the method comprises obtaining a plurality of 1 1H NMR Carr - Purcell - Meiboom - Gill (CPMG) spectra of the sample and obtaining 13 13C NMR cross - polarization (CP) spectra of the sample. In certain embodiments, for example, the following are provided: (Item 1) A method of preparing a composition comprising crystalline biomolecules, comprising: a) forming a fluidized bed of crystalline biomolecules in a rotating chamber comprising an inlet and an outlet, rotating the chamber about a substantially horizontal axis to generate a centrifugal force (F centrifugal ) in the chamber such that the fluidized bed is generated, in a direction opposite to the direction of said F centrifugal and in a direction opposite to the direction of F​centrifugal with a force (F FR1 ) that cancels it out, flowing a first flow of a first solution through the inlet, and recovering the first solution from the chamber while substantially maintaining the formation of a fluidized bed of crystallized biomolecules; b) Step (i), (ii) or (iii): i. flowing a second flow of a second solution to replace the first flow of the first solution while substantially maintaining the formation of a fluidized bed of crystallized biomolecules; ii. concentrating the crystallized biomolecules within the region of the chamber by changing FR1 to a second flow rate (FR2) having a force (F centrifugal ) less than, or increasing the rotational speed of the chamber to increase F FR2 to a level greater than; FR1 centrifugal iii. performing a combination of step (i) and step (ii); and; c) flowing a second flow through the outlet into the chamber in a direction parallel to the direction of F centrifugal to remove the crystalline biomolecules from the chamber, A method comprising. (Item 2) The method according to item 1, wherein F centrifugal is 1000 g, at least during step (i). (Item 3) The method according to item 1 or 2, wherein the supply volume of the second flow of the second solution is from about 50 to about 500 mL during step (i). (Item 4) The method according to item 3, wherein the supply volume of the second flow of the second solution is from about 100 to about 300 mL. (Item 5) The method according to any one of items 1 to 4, wherein the number of volumes of the first solution per chamber is 2 or more during step (i). (Item 6) ​​The method according to any one of items 1 to 5, wherein FR1 is about 50 mL / min to about 150 mL / min during step (i). (Item 7) The method according to item 6, wherein FR1 is about 70 mL / min to about 120 mL / min. (Item 8) The method according to any one of items 1 to 7, wherein the first solution is a hypertonic crystallization buffer. (Item 9) The method according to any one of items 1 to 8, wherein the second solution is an isotonic formulation buffer. (Item 10) The F centrifugal is 1000 g during step (ii), the method according to any one of items 1 to 9. (Item 11) The method according to any one of items 1 to 10, wherein the supply volume of the second flow of the second solution is about 100 to about 500 mL during step (ii). (Item 12) The method according to item 11, wherein the supply volume of the second flow of the second solution is about 150 to about 350 mL. (Item 13) The method according to any one of items 1 to 12, wherein the number of volumes of the first solution per chamber is 1 or 2 during step (ii). (Item 14) The method according to any one of items 1 to 13, wherein FR2 is about 10 mL / min to about 50 mL / min during step (ii). (Item 15) The method according to item 14, wherein FR2 is about 20 mL / min to about 40 mL / min. (Item 16) The method according to any one of items 1 to 15, wherein the concentration of the crystalline biomolecule increases by at least 2 times. (Item 17) The method according to item 16, wherein the concentration of the crystalline biomolecule increases by at least 3 times. (Item 18) The method according to item 17, wherein the concentration of the crystalline biomolecule increases by at least 4 times. (Item 19) Reduce F to levels of about 5 g and about 20 g centrifugal The method according to any one of items 1 to 18, comprising reducing (Item 20) Reduce F to below 10 g centrifugal The method according to item 19, reducing FR1 is adjusted by a first pump, and the first pump is set to about 10 mL / min to about 100 mL / min after step (b), the method according to any one of items 1 to 20. (Item 22) The method according to item 21, wherein the first pump is set to about 15 mL / min to about 65 mL / min. (Item 23) The method according to any one of items 1 to 22, wherein the rotary chamber is connected to a chamber peristaltic pump. (Item 24) The chamber peristaltic pump is set to feed in the same direction as the first pump and F centrifugal The method according to item 23. (Item 25) The method according to item 24, wherein the chamber peristaltic pump is set to about 50 mL / min to about 100 mL / min after step (b). (Item 26) The method according to item 25, wherein the chamber peristaltic pump is set to about 75 mL / min. (Item 27) The method according to item 26, wherein the chamber peristaltic pump is set to about 75 mL / min, the first pump is set to about 50 mL / min, and F is reduced to below 10 g. centrifugal The method according to item 26. (Item 28) The method according to any one of items 1 to 27, wherein the method is performed in an apparatus comprising a plurality of rotary chambers. (Item 29) The method according to item 28, wherein the apparatus comprises at least 2, at least 4 or at least 6 rotary chambers. (Item 30) The method according to item 29, wherein the device includes four or six chambers. (Item 31) The method according to item 30, wherein steps (a) to (c) are performed in a plurality of chambers. (Item 32) The method according to item 31, wherein the crystalline biomolecule is removed from the chamber of the device at a time separate from when the crystalline biomolecule is removed from another chamber of the device. (Item 33) The method according to any one of items 1 to 32, wherein the crystalline biomolecule is a protein or contains one or more polypeptide chains. (Item 34) The method according to item 33, wherein the crystalline protein is an immunoglobulin or an antigen-binding fragment thereof. (Item 35) A method for detecting a crystalline biomolecule and / or an amorphous biomolecule in a sample, comprising: a) obtaining a plurality of 1 H NMR Carr-Purcell-Meiboom-Gill (CPMG) spectra of the sample; and b) obtaining a 13 C NMR cross-polarization (CP) spectrum of the sample. A method comprising the above. (Item 36) The method according to item 35, comprising operating an H resonance frequency of about 250 to about 1000 MHz during step (a) of the method. 1 A method according to item 35, comprising maintaining the temperature at about 250 to about 350 K. (Item 38) (Item 37) The method according to item 35 or 36, comprising operating a magic angle spinning (MAS) probe. (Item 38) The method according to any one of items 35 to 37, comprising operating a magic angle spinning (MAS) probe. (Item 39) The method according to item 38, wherein the MAS probe includes at least two RF channels. (Item 40) The MAS probe is 1 tuned to 13 H and (Item 41) The method according to any one of Items 38 to 40, wherein the MAS probe is operated at a rotational frequency of about 2 kHz to about 8 kHz. (Item 42) The method according to any one of Items 35 to 41, including using a 90° pulse. (Item 43) About 2.5 μs 1 The method according to Item 42, including using an H 90° pulse. (Item 44) The 1 The method according to any one of Items 35 to 43, wherein the H CPMG spectrum is obtained with about 5 to about 100 π pulses having a length of about 2 μs to about 20 μs. (Item 45) The method according to Item 44, wherein each of the 20 π pulses is separated by about 10 μs to about 1 ms. (Item 46) The method according to Item 45, wherein the total time of the CPMG pulses is about 500 μs to about 50 ms. (Item 47) Rotating at a frequency up to 14 kHz while obtaining a plurality of 1 The method according to any one of Items 35 to 46, including obtaining an H CPMG spectrum. (Item 48) During the measurement, the 13 The method according to any one of Items 35 to 47, wherein the contact time of the C CP is about 100 μs to about 10 ms. (Item 49) The measurement according to Item 48, including an RF spin-lock pulse at about 20 kHz to about 100 kHz 13 on C. (Item 50) 1 The method according to Item 49, wherein the lamp pulse on H coincides. (Item 51) The method according to any one of Items 35 to 50, including quantifying the content of the biomolecule in the sample. (Item 52) The method according to any one of Items 35 to 51, wherein the crystalline biomolecule exhibits spectroscopic characteristics different from those of the amorphous biomolecule. (Item 53) The method according to any one of items 35 to 52, wherein the crystalline biomolecule exhibits spectroscopic characteristics of higher molecular mobility than the amorphous biomolecule. (Item 54) The method according to any one of items 35 to 53, wherein the crystalline biomolecule is bi-refringant. (Item 55) The method according to any one of items 35 to 54, wherein the crystalline biomolecule does not diffract. (Item 56) The method according to any one of items 35 to 55, wherein the biomolecule contains a protein or contains one or more polypeptide chains. (Item 57) The method according to item 56, wherein the protein is an antibody or a fragment thereof. (Item 58) The method according to any one of items 35 to 57, wherein the biomolecule in the sample contains one or more glycans.

Brief Description of the Drawings

[0013]

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[0014] Methods for crystalline biomolecules are disclosed herein. A method for preparing a composition comprising a crystalline biomolecule is provided. In a representative embodiment, the method includes forming a fluidized bed of the crystalline biomolecule in a rotating chamber including an inlet and an outlet, the fluidized bed rotating the chamber about a substantially horizontal axis to generate a centrifugal force (F centrifugal ) in the chamber, flowing a first stream of a first solution through the inlet in a direction opposite to the direction of F centrifugal and at a first flow rate (FR1) having a force (F centrifugal ) that cancels F FR1 , and recovering the first solution from the chamber while substantially maintaining the formation of the fluidized bed of the crystalline biomolecule.

[0015] In a representative embodiment, the method includes a buffer exchange step, a concentration step, or both a buffer exchange step and a concentration step. Further description of each step is provided below.

[0016] Buffer Exchange Step In a representative embodiment, the method includes a buffer exchange step, and in a representative aspect, such a step is performed while maintaining a fluidized bed of the crystalline biomolecule. In a representative aspect, the method includes replacing a first flow of a first solution with a second flow of a second solution while substantially maintaining the formation of the fluidized bed of the crystalline biomolecule. In a representative aspect, the method includes flowing a second flow of a second solution to replace the first flow of the first solution while substantially maintaining the formation of the fluidized bed of the crystalline biomolecule.

[0017] In a representative aspect, the method is such that the force of the second flow is F centrifugal to counteract, in a direction opposite to the direction of F centrifugal and at a flow rate (FR) equivalent to FR1 having a force (F) equal to F FR1 flow the second flow or the second solution through the inlet and recover the second solution from the chamber while substantially maintaining the formation of the fluidized bed of the crystalline biomolecule. In a representative aspect, F centrifugal is suitable for maintaining the crystalline biomolecule in the fluidized bed. In certain aspects, F centrifugal is in the range of about 500 g to about 3000 g. In certain aspects, F centrifugal is in the range of about 750 g to about 1250 g. In certain aspects, F centrifugal is about 1000 g (±100 g) during this step.

[0018] The supply volume of the second flow of the second solution is sufficient to maintain the crystalline biomolecule in the fluidized bed. In a representative aspect, the supply volume of the second flow of the second solution is about 50 - 500 mL. In a representative aspect, the supply volume of the second flow of the second solution is about 100 - 300 mL. In a representative aspect, the number of volumes of the first solution per chamber is 2 or more. In a representative aspect, FR1 is sufficient to maintain the crystalline molecule in the fluidized bed. In a representative aspect, FR1 is about 50 mL / min to about 150 mL / min. In a representative aspect, FR1 is about 70 mL / min to about 120 mL / min.

[0019] Advantageously, the method is not limited to a particular type of buffer, provided that the buffer does not negatively affect the integrity of the crystalline biomolecule, e.g., the buffer does not negatively affect the size, shape, and / or quality of the product of the crystalline biomolecule. In this regard, each of the first solution and the second solution can be either a buffer or any type of solution characterized by any pH. In a representative embodiment, the pH of the solution is a physiological pH, e.g., 6.5 - 7.5. In a representative embodiment, the pH of the solution can be at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, or at least 10.5, and pH 11 or less. In a representative embodiment, one or both of the first solution and the second solution independently contain a buffer. In a representative embodiment, the buffer is selected from the group consisting of phosphate buffer (e.g., PBS), triethanolamine, Tris, Bicine, TAPS, Tricine, HEPES, TES, MOPS, PIPES, cacodylic acid, MES, acetic acid, citric acid, succinic acid, histidine, or other pharmaceutically acceptable buffers.

[0020] In a representative embodiment, the first solution is a high-osmotic protein crystallization buffer. In a representative embodiment, the second solution is a formulated buffer that is safe for administration to a mammal, e.g., a human. In a representative embodiment, the second solution has a physiological pH and is sterile.

[0021] Concentration step In a further or alternative embodiment, the method includes concentrating the crystalline biomolecule within the region of the chamber. In a representative embodiment, the method includes concentrating the crystalline biomolecule after a buffer exchange step, such as any of the steps described herein. In a representative embodiment, the concentration step is to change to a second flow rate (FR2) having a force (F centrifugal weaker than FR1 (F FR2 ), or (ii) to a level higher than F FR1 for F centrifugalIt can be done by increasing the rotation speed of the chamber to raise it or (iii) doing either (i) and (ii) or both. In a representative embodiment, F centrifugal is 1000 g at least during this stage. In a representative embodiment, the supply volume of the second flow of the second solution is about 100 - 500 mL. In a representative embodiment, the supply volume of the second flow of the second solution is about 150 mL to about 350 mL, such as about 150 mL, about 200 mL, about 250 mL, about 300 mL or about 350 mL. In a representative embodiment, the number of volumes of the first solution per chamber is 1. In a representative embodiment, FR2 is about 10 mL / min to about 50 mL / min. In a representative embodiment, FR2 is less than about 60 mL / min. In a representative embodiment, FR2 is about 10 mL / min to about 50 mL / min. In a representative embodiment, FR2 is about 20 mL / min to about 40 mL / min. In a representative embodiment, FR2 is about 20 mL / min, about 25 mL / min, about 30 mL / min, about 35 mL / min or about 40 mL / min.

[0022] In a representative embodiment, after the concentration stage, the concentration of the crystalline biomolecule increases by at least 2 times, at least 3 times or at least 4 times.

[0023] Recovery stage In a representative embodiment, the method includes removing the crystalline biomolecule from the chamber. In a representative embodiment, the method includes removing the crystalline biomolecule from the chamber after concentrating the crystalline biomolecule within the region of the chamber. In a representative embodiment, the method includes slowly removing the concentrated suspension of the crystalline biomolecule from the chamber without destroying the packed crystal suspension and / or without causing cavity formation within the packed crystal suspension. Cavity formation within the packed crystal suspension is undesirable as it destroys the concentration profile and creates a non-uniform concentration gradient across the concentrated crystal suspension.

[0024] In a representative embodiment, the method is for removing the crystalline biomolecule from the chamber by F centrifugalincluding flowing a second flow through an outlet in a direction parallel to the direction of

[0025] In a representative embodiment, the method reduces F to a level of about 5 g to about 20 g, or to a level of about 5 g to about 15 g centrifugal including. In a representative embodiment, F centrifugal is reduced to less than about 10 g, for example, to about 8 g.

[0026] In a representative embodiment, FR1 is adjusted by a first pump and the first pump is set to about 10 mL / min to about 100 mL / min after a buffer exchange and / or concentration step. In a representative embodiment, FR1 is adjusted by a first pump and the first pump is set to less than about 75 mL / min. In a representative embodiment, FR1 is adjusted by a first pump and the first pump is set to about 15 mL / min to about 65 mL / min after a buffer exchange and / or concentration step. In a representative embodiment, the first pump is set to about 45 mL / min to about 65 mL / min, optionally to about 50 mL / min.

[0027] In a representative embodiment, the rotary chamber is connected to a chamber peristaltic pump. In a representative example, the chamber peristaltic pump feeds in the same direction as the first pump and F centrifugal . Without being bound by a particular theory, the first pump acts as a "pulling" mechanism that sucks material from the chamber, while the chamber peristaltic pump acts as a "pushing" mechanism to ensure no cavity formation of the biomolecules. In a representative embodiment, the chamber peristaltic pump is set to about 50 mL / min to about 150 mL / min after a buffer exchange and / or concentration step. In a representative embodiment, the chamber peristaltic pump is set to less than 100 mL / min. In some embodiments, the chamber peristaltic pump is set to about 60 mL / min to about 90 mL / min. In a representative example, the chamber peristaltic pump is set to about 75 mL / min, the first pump is set to about 50 mL / min, and F centrifugal is reduced to less than 10 g, optionally to 8 g.

[0028] CFGC device In representative embodiments, the disclosed method is performed in a counterflow gradient centrifugation (CFGC) system. In representative embodiments, the device includes a plurality of rotating chambers. In representative embodiments, the device includes at least 2, at least 4, or at least 6 rotating chambers. In representative embodiments, the device includes 4 or 6 chambers. In representative embodiments, the method is performed in a device that includes a plurality of rotating chambers, and each chamber may be operated simultaneously with, or independently of, the other chambers of the device. In representative embodiments, the steps of the disclosed method are performed in a plurality of chambers. In representative embodiments, the steps of the disclosed method are performed in 2, 3, or 4 chambers. In representative embodiments, the steps of the disclosed method are performed in 5 or 6 chambers. In representative embodiments, the steps of the disclosed method are performed in more than about 6, more than about 10, more than about 20 chambers.

[0029] In representative embodiments, the device is a kSep® system. In representative embodiments, the device is a kSep® 400, which has 4 individual chambers, each with a limit capacity of 100 mL. In representative embodiments, the device is a kSep® 6000S, which has 6 individual chambers, each with a limit capacity of 1000 mL. The maximum flow rate of the kSep® 6000S is 720 L / hr.

[0030] In a representative embodiment, when the disclosed method is performed in a plurality of chambers of the apparatus, the crystalline biomolecules are removed from the chambers one by one at a time. For example, the crystalline biomolecules are sequentially removed from each chamber. Thus, the removal of the crystalline biomolecules from one chamber of this apparatus is performed at a different time than when the crystalline biomolecules are removed from another chamber of this apparatus. Without being bound by a particular theory, it is believed that removing the crystalline biomolecules from one chamber at a time reduces the opportunity for cavity formation of the crystalline biomolecules.

[0031] Representative embodiment In a representative aspect, a method of preparing a composition comprising a crystalline biomolecule comprises: (a) forming a fluidized bed of the crystalline biomolecule in a rotating chamber comprising an inlet and an outlet, rotating the chamber about a substantially horizontal axis to generate a centrifugal force (F centrifugal ) therein, flowing a first stream of a first solution through the inlet at a first flow rate (FR1) having a direction opposite to the direction of F centrifugal and a force (F centrifugal ) that cancels F FR1 , and recovering the first solution from the chamber while substantially maintaining the formation of the fluidized bed of the crystalline biomolecules, whereby the fluidized bed is generated; (b) performing step (i), step (ii) or step (iii), wherein step (i) is flowing a second stream of a second solution to replace the first stream of the first solution while substantially maintaining the formation of the fluidized bed of the crystalline biomolecules, step (ii) is changing FR1 to a second flow rate (FR2) having a force (F centrifugal ) less than F FR2 , or increasing the rotational speed of the chamber to increase F FR1 to a level higher than F centrifugal to concentrate the crystalline biomolecules within the region of the chamber, and step (iii) is a combination of step (i) and step (ii); and (c) removing the crystalline biomolecules from the chamber. In a representative aspect, F centrifugal is used to remove the crystalline biomolecules from the chamber.Removing the crystalline biomolecule from the chamber by flowing a second fluid through the outlet in a direction parallel to the direction of

[0032] Further steps The methods disclosed herein may include further steps. For example, the method may include one or more upstream or downstream steps involved in producing, purifying, and formulating a recombinant protein. In representative embodiments, the method includes steps for creating a host cell that expresses a recombinant protein (e.g., a recombinant antibody). The host cell can be a prokaryotic host cell, such as E. coli or Bacillus subtilis, or the host cell can be eukaryotic, such as a yeast cell, a filamentous fungal cell, a protozoan cell, an insect cell, or a mammalian cell (e.g., a CHO cell). Such host cells are described in the art. See, e.g., Frenzel et al., Front Immunol 4:217 (2013). For example, in some instances, the method includes introducing into the host cell a vector comprising a nucleic acid comprising a nucleotide sequence encoding the recombinant protein or a polypeptide chain thereof.

[0033] In representative embodiments, the method includes steps for culturing a host cell that expresses a recombinant protein (e.g., a recombinant antibody). Such steps are known in the art. See, e.g., Li et al., MAbs 2(5):466 - 477 (2010).

[0034] In representative embodiments, the method includes steps for purifying a recombinant protein (e.g., a recombinant antibody) from a culture. In representative aspects, the method includes one or more chromatography steps, such as affinity chromatography (e.g., protein A affinity chromatography), ion exchange chromatography, and / or hydrophobic interaction chromatography. In representative aspects, the method includes steps for generating a crystalline biomolecule from a solution containing the recombinant protein. In representative aspects, the method includes steps for preparing a crystalline substance, including those described in International Publication No. WO 2016 / 010927, entitled "CRYSTALLINE ANTIBODY FORMULATIONS", which is incorporated herein by reference in its entirety. The method may, in some embodiments, include adjusting factors that affect the solution state or crystallization, such as the evaporation rate of solvents, organic solvents or additives, the presence of appropriate co-solutes and buffers, pH, and temperature. A comprehensive overview of the various factors that affect protein crystallization is published by McPherson (1985, Methods Enzymol 114:112-120). As a guide, the teachings of McPherson and Gilliland (1988, J Crystal Growth, 90:51-59), including a comprehensive list of polypeptides that have been crystallized, and the conditions under which they were crystallized are available. Additionally, repositories of lists of crystals and crystallization recipes, as well as coordinates of the dissolved protein structures, are available from the Protein Data Bank at Brookhaven National Laboratory. It is maintained by the Data Bank (www.rcsb.org / pdb / ; Bernstein et al., 1977, J Mol Biol 112:535-542). Generally, crystals are generated by combining a polypeptide (i.e., an antibody) to be crystallized with an appropriate crystallization reagent, which is generally an aqueous solvent containing an appropriate aqueous solvent or salt or an appropriate crystallizing agent such as an organic solvent or additive (collectively referred to as a "crystallization reagent"). The solvent can be combined with the polypeptide and stirred at a temperature experimentally determined to be appropriate for inducing crystallization and acceptable for maintaining polypeptide activity and stability. Laboratory-scale methods for crystallization include hanging drop vapor diffusion, sitting drop vapor diffusion, microdialysis, microbatch, under oil, in gel, and sandwich drop methods. The solvent can optionally include co-crystallization additives such as precipitants, fatty acids, reducing agents, glycerol, sulfobetaines, surfactants, polyols, divalent cations, cofactors or chaotropes and amino acids as well as buffer species for adjusting the pH. "Co-crystallization additives" include compounds that promote crystallization of the polypeptide and / or compounds that stabilize the protein and protect it from denaturation. Examples of co-solutes include ammonium acetate, ammonium chloride, ammonium fluoride, ammonium formate, ammonium nitrate, ammonium phosphate, ammonium sulfate, cadmium chloride, cadmium sulfate, calcium acetate, calcium chloride, cesium chloride, cobalt chloride, CH 3 (CH 2 ) 15 N(CH 3 ) 3Br.-(CTAB), diammonium citrate, diammonium hydrogen phosphate, diammonium phosphate, diammonium tartrate, dipotassium phosphate, disodium phosphate, disodium tartrate, DL-malic acid, ferric chloride, L-proline, lithium acetate, lithium chloride, lithium nitrate, lithium sulfate, magnesium acetate, magnesium chloride, magnesium formate, magnesium nitrate, magnesium sulfate, nickel chloride, potassium acetate, potassium bromide, potassium chloride, potassium citrate, potassium fluoride, potassium formate, potassium nitrate, potassium phosphate, potassium sodium tartrate, potassium sulfate, potassium thiocyanate, sodium acetate, sodium bromide, sodium chloride, sodium citrate, sodium fluoride, sodium formate, sodium malonate, sodium nitrate, sodium phosphate, sodium sulfate, sodium thiocyanate, succinic acid, tacsimate, triammonium citrate, trilithium citrate, trimethylamine N-oxide, tripotassium citrate, trisodium citrate, zinc acetate, zinc sulfate and other compounds that function to supply a cosolute. "Crystallization" includes compounds that maintain the pH of the solution within a desired range to promote crystallization of the polypeptide.Examples include ACES (N-(2-acetamido)-2-aminoethanesulfonic acid), BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), Bicine (N,N-bis(2-hydroxyethyl)glycine), Bis-Tris (2,2-bis-(hydroxymethyl)-2,2’,2’’-nitrilotriethanol), boric acid, CAPS (3-[cyclohexylamino]-1-propanesulfonic acid), citric acid, EPPS (HEPPS, 4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid), Gly-Gly (NH.sub.2CH.sub.2CONHCH.sub.2COOH, glycyl-glycine), HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), imidazole, MES (2-morpholinoethanesulfonic acid), MOPS (3-(N-morpholino)-propanesulfonic acid), PIPES (piperazine-1,4-bis(2-ethanesulfonic acid)), potassium chloride, sodium acetate, sodium bicarbonate, monosodium phosphate (sodium dihydrogen phosphate), disodium phosphate, TAPS (N-[tris-(hydroxymethyl)methyl]-3-aminopropanesulfonic acid), TAPSO (N-[tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid), TES (N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid), tricine (N-[tris(hydroxymethyl)methyl]glycine), Tris-HCl, TRIZMA (2-amino-2-(hydroxymethyl)-1,3-propanediol), and other compounds that function to maintain the solution at a specified pH or near it.

[0035] The choice of precipitant is one factor that affects crystallization. For example, PEG products, such as those with a molecular weight of 200 to 20,000 kD, can be used. PEG3350 is a long polymer precipitant or dehydrating agent that acts by a volume exclusion effect. Lyotropic salts, such as ammonium sulfate, promote the precipitation process, as do short-chain fatty acids such as capric acid. Polyionic species are also useful precipitants.

[0036] Antibodies for use in formulations for subcutaneous injection are precipitated, for example, preferably in the physiological pH range and in crystallization reagents that provide isotonic osmotic pressure. It is determined experimentally that additives, cosolutes, buffers, etc. and their concentrates are required to promote crystallization.

[0037] In industrial-scale processes, the precipitation leading to crystallization can best be carried out by a simple combination of polypeptide, precipitant, cosolute and optionally buffer in a batch process. As another option, the polypeptide can be crystallized by using a polypeptide precipitate as the starting material (“seed”). In this case, the polypeptide precipitate is added to the crystallization solution and left to stand until crystals form.

[0038] Alternative laboratory crystallization methods such as dialysis or vapor diffusion can also be adapted. McPherson, supra and Gilliland, supra include comprehensive lists of appropriate conditions in reviews of crystallization literature. Occasionally, when the crystallized polypeptide is to be crosslinked, incompatibility between the intended crosslinking agent and the crystallization medium may require exchanging the crystals into a more suitable solvent system.

[0039] According to some embodiments, polypeptide crystals, crystalline formulations and compositions are prepared by the following steps: First, the polypeptide is crystallized. Next, excipients or ingredients as described herein are added directly to the mother liquor. Alternatively, after removing the mother liquor, the crystals are suspended in a solution of excipient or other formulation components for a minimum of 1 hour to a maximum of 24 hours. The excipient concentration is generally about 0.01 - 30% w / w, corresponding to polypeptide crystal concentrations of 99.99 - 70% w / w, respectively. In one embodiment, the excipient concentration is about 0.1 - 10%, corresponding to crystal concentrations of 99.9 - 90% w / w, respectively. The mother liquor can be removed from the crystal slurry either by filtration, buffer exchange, or centrifugation.

[0040] Subsequently, at either room temperature or a temperature in the range of -20°C to 25°C, in any isotonic injectable vehicle that does not dissolve the crystals, the crystals are washed with a 50 - 100% solution of one or more organic solvents or additives such as, for example, ethanol, methanol, isopropanol, ethyl acetate, or polyethylene glycol (PEG). Further, water may be used to wash the crystals. The crystals are dried either by passing a stream of nitrogen, air, or an inert gas over the crystals. Finally, micronization of the crystals can be carried out if necessary. Drying of the polypeptide crystals is by means including drying with N 2 , air, or an inert gas; vacuum oven drying; freeze drying; washing with a volatile organic solvent or additive followed by evaporation of the solvent; or removal of water, an organic solvent or additive, or a liquid polymer by means including evaporation in a draft. Generally, drying is achieved when the crystals become a free-flowing powder. Drying can be carried out by passing a gas stream over the wet crystals. The gas can be selected from the group consisting of nitrogen, argon, helium, carbon dioxide, air, or combinations thereof. The diameter of the particles achieved can be in the range of 0.1 - 100 micrometers or in the range of 0.2 - 10 micrometers or in the range of 10 - 50 micrometers or in the range of 0.5 - 2 micrometers. For formulations intended for administration by inhalation, in one embodiment, the particles formed from the polypeptide crystals are in the range of 0.5 - 1 micrometer.

[0041] According to some embodiments, when preparing protein crystals, no protein crystal formulation or composition, enhancer, such as a surfactant, etc., is added during crystallization. According to some other embodiments, when preparing protein crystals, a protein crystal formulation or composition, enhancer, such as a surfactant, etc., is added during crystallization. After crystallization, excipients or components are added to the mother liquor at a concentration of about 1-10% w / w, or about 0.1-25% w / w, or about 0.1-50% w / w. These concentrations correspond to crystal concentrations of 99-90% w / w, 99.9-75% w / w, and 99.9-50% w / w, respectively. The excipients or components are incubated with the crystals in the mother liquor for about 0.1-3 hours, or incubated for 0.1-12 hours, or incubated for 0.1-24 hours.

[0042] In some or any of the embodiments, the component or excipient is dissolved in a solution other than the mother liquor, the protein crystal is taken out from the mother liquor, and suspended in the excipient or component solution. In some embodiments, the excipient or component solution (or suspension vehicle) is a mixture of an excipient or component or surfactant that is isotonic and injectable. In some embodiments, the excipient or component solution (or resuspension vehicle) is not a mixture of an excipient or component or surfactant that is isotonic and injectable. The component or excipient concentration and incubation time are the same as those described above.

[0043] In a representative embodiment, the method includes steps for formulating a purified biomolecule (such as an antibody). Representative steps are described in Formulation and Process Development Strategies for Manufacturing, eds. Jameel and Hershenson, John Wiley & Sons, Inc. (Hoboken, NJ), 2010.

[0044] In a representative embodiment, the method includes analyzing a sample for crystalline and amorphous forms of a biomolecule. In a representative aspect, the method includes quantitative analysis of the sample.

[0045] Biomolecule As used herein, the term "biomolecule" or "biological molecule" refers to large macromolecules that are structurally based on molecules that are present in, or can be produced or metabolized by, living organisms. Biomolecules include, but are not limited to, polypeptides, proteins, polysaccharides, lipids (e.g., glycolipids, phospholipids, sterols) and polynucleotides or nucleic acids (e.g., DNA or RNA).

[0046] The methods disclosed herein are not limited to any particular type of biomolecule, so long as the biomolecule can assume a crystalline form. In a representative embodiment, the crystalline biomolecule is a protein comprising one or more polypeptide chains. In a representative embodiment, the crystalline protein is a hormone, growth factor, cytokine, cell surface receptor or some other natural or non-natural ligand that binds to a cell surface receptor (e.g., epidermal growth factor receptor (EGFR), T cell receptor (TCR), B cell receptor (BCR), CD28, platelet-derived growth factor receptor (PDGF), nicotinic acetylcholine receptor (nAChR), etc.).

[0047] In representative examples, the biomolecule is an antibody or immunoglobulin or a fragment thereof, such as an antigen-binding antibody fragment. As used herein, the term “antibody” refers to a protein having the conventional immunoglobulin format, including heavy and light chains, and including variable and constant regions. For example, the antibody can be IgG, which is a “Y-shaped” structure of two identical pairs of polypeptide chains, each pair having one “light” (generally having a molecular weight of about 25 kDa) and one “heavy” chain (generally having a molecular weight of about 50-70 kDa). Antibodies have variable and constant regions. In the IgG format, the variable region is generally about 100-110 or more amino acids, contains three complementarity-determining regions (CDRs), is mainly involved in antigen recognition, and varies substantially among other antibodies that bind different antigens. The constant region mobilizes cells and molecules of the immune system to the antibody. The variable region is composed of the N-terminal regions of each light and heavy chain, while the constant region is composed of the respective C-terminal portions of the heavy and light chains. (Janeway et al., “Structure of the Antibody Molecule and the Immunoglobulin Genes”, Immunobiology: The Immune System in Health and Disease, 4 th ed. Elsevier Science Ltd. / Garland Publishing, (1999)).

[0048] The general structures and characteristics of the CDRs of antibodies are described in the art. Briefly, in the antibody backbone, the CDRs are embedded within the frameworks in the heavy and light chain variable regions, and they constitute regions that are highly involved in antigen binding and recognition. The variable regions typically contain at least three heavy or light chain CDRs (see also Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service N.I.H., Bethesda, Md.; Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:877-883) within the framework regions (referred to as framework regions 1-4, FR1, FR2, FR3, and FR4 by Kabat et al., 1991).

[0049] Antibodies can include any constant regions known in the art. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, which define the antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgM1 and IgM2. Embodiments of the present invention include all such classes or isotypes of antibodies. The light chain constant region can be, for example, a kappa or lambda light chain constant region, such as a human kappa or lambda light chain constant region. The heavy chain constant region can be, for example, an alpha, delta, epsilon, gamma, or mu heavy chain constant region, such as a human alpha, delta, epsilon, gamma, or mu heavy chain constant region. Thus, in a representative embodiment, the present antibody is an antibody of isotype IgA, IgD, IgE, IgG, or IgM, including any one of IgG1, IgG2, IgG3, or IgG4.

[0050] The antibody can be a monoclonal antibody or a polyclonal antibody. In some embodiments, the antibody comprises a sequence that is substantially similar to a natural antibody produced by a mammal, such as a mouse, rabbit, goat, horse, chicken, hamster, human, etc. In this regard, the antibody can be considered a mammalian antibody, such as a mouse antibody, rabbit antibody, goat antibody, horse antibody, chicken antibody, hamster antibody, human antibody, etc. In certain embodiments, the recombinant protein is a human antibody. In certain embodiments, the biomolecule is an antibody such as a human antibody. In certain embodiments, the biomolecule is a chimeric antibody or a humanized antibody. The term "chimeric antibody" refers to an antibody that contains domains from two or more different antibodies. A chimeric antibody can contain, for example, a constant domain from one species and a variable domain from a second species, or more generally, a stretch of amino acid sequences from at least two species. A chimeric antibody can also contain domains from two or more different antibodies within the same species. The term "humanized", when used in reference to an antibody, refers to an antibody having at least the CDR regions from a non-human origin that has been engineered to have a structure and immunological function that is more similar to a true human antibody than the antibody of the original origin. For example, humanization can involve transplanting the CDRs from a non-human antibody, such as a mouse antibody, etc., into a human antibody. Humanization can also involve the selection of amino acid substitutions to make the non-human sequences more similar to human sequences.

[0051] The antibody can be cleaved into fragments by enzymes such as papain and pepsin. Papain cleaves the antibody to produce two Fab fragments and one Fc fragment. Pepsin cleaves the antibody to produce F(ab’) 2 fragments and pFc’ fragments. In representative embodiments, the biomolecule can be an antibody fragment that retains at least one glycosylation site, such as Fab, Fc, F(ab’) 2 or pFc’.

[0052] The structure of antibodies has been utilized to effect a range expansion of alternative antibody formats that span a molecular weight range of at least about 12 to 150 kDa and a valence (n) range from monomer (n = 1) to dimer (n = 2) and trimer (n = 3) to tetramer (n = 4) and potentially higher; such alternative antibody formats are referred to herein as "antibody protein products".

[0053] Antibody protein products include those based on antibody fragments that retain full antigen-binding ability, such as scFv, Fab, and VHH / VH (described below). The smallest antigen-binding fragment that retains its full antigen-binding site is the Fv fragment (fragment, antigen-binding), which consists entirely of the variable (V) regions. To link the V regions for molecular stability to form an scFv (single-chain fragment variable) fragment, a soluble flexible amino acid peptide linker is used, or a constant (C) domain is added to the V regions to generate a Fab fragment. Both scFv and Fab are widely used fragments that can be readily produced in host cells, such as prokaryotic host cells. Other antibody protein products include disulfide bond-stabilized scFv (ds-scFv), single-chain Fab (scFab), as well as dimeric and multimeric antibody formats, such as di-, tri-, and tetrabody or minibody (miniAbs) consisting of scFv linked to an oligomerization domain. The smallest fragments are the VHH / VH of camelid heavy chain Abs as well as single-domain Abs (sdAbs). The most frequently used basic unit for generating novel antibody formats is the single-chain variable (V) domain antibody fragment (scFv), which contains V domains (VH and VL domains) from the heavy and light chains linked by a peptide linker of ~15 amino acid residues. Peptibodies or peptide-Fc fusions are yet another antibody protein product. The structure of a peptibody consists of a biologically active peptide grafted onto an Fc domain. Peptibodies are described in detail in the art. See, for example, Shimamoto et al., mAbs 4(5):586-591 (2012).

[0054] Other antibody protein products include single-chain antibodies (SCA); diabodies; triabodies; tetra-bodies; bispecific or trispecific antibodies, etc. Bispecific antibodies can be divided into five main classes: BsIgG, additional IgG, BsAb fragments, bispecific fusion proteins and BsAb conjugates. See, for example, Spiess et al., Molecular Immunology 67(2) Part A:97-106(2015).

[0055] In a representative embodiment, the biomolecule comprises any one of these antibody protein products. In a representative embodiment, the biomolecule is an scFv, Fab VHH / VH, Fv fragment, ds-scFv, scFab, dimeric antibody, multimeric antibody (e.g., diabody, triabody, tetrabody), mini Ab, peptibody VHH / VH of heavy-chain antibody of camelidae, sdAb, diabody; triabody; tetrabody; bispecific or trispecific antibody, BsIgG, additional IgG, BsAb fragment, bispecific fusion protein and BsAb conjugate, any one of them.

[0056] The biomolecule can be a monomeric form or a multimeric, oligomeric or multimeric antibody protein product. In certain embodiments where the antibody comprises two or more separate antigen-binding region fragments, the antibody is considered bispecific, trispecific or multispecific or bivalent, trivalent or multivalent depending on the number of individual epitopes recognized and bound by the antibody.

[0057] Methods for Detecting Crystalline and Amorphous Biomolecules To ensure robust and safe formulations, the crystallinity or degree of disorder is optionally monitored during various processing stages (e.g., scale-up of bulk materials, formulation development, manufacturing) and throughout the shelf life of the formulation. The amorphous forms of pharmaceutical raw materials are described in detail along with their downstream consequences in formulations and biological systems. See, for example, Shah et al., J Pharm Sci 95(8):1641(2006). Amorphous solids are typically defined in relation to crystalline solids and generally lack the long-range translational symmetry that is characteristic of crystalline structures. The amorphous phase can occur throughout the particle or in part of the particle such as the surface. Detection of the amorphous form of a solid can be very difficult if the disorder is too small to be easily detected. If the disorder is large enough, it can cause changes in product performance, such as affecting hardness after compression, enhancing dissolution rate, decreasing chemical stability, and inducing recrystallization due to moisture during storage. See Shah et al., supra 2006. There are various quantitative techniques for detecting the amorphous form of a solid (relative to a crystalline solid), such as powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), isothermal microcalorimetry (IMC), solution calorimetry (SC), infrared spectroscopy (IRR), Fourier transform (FT) Raman spectroscopy, and solid state NMR (ssNMR). However, each has one or more drawbacks.

[0058] In addition to methods for preparing compositions containing crystalline biomolecules, an efficient and convenient method for detecting crystalline and / or amorphous biomolecules in a sample is provided herein.

[0059] In a representative embodiment, a method for detecting crystalline and / or amorphous biomolecules in a sample comprises performing high-resolution ssNMR (solid-state nuclear magnetic resonance) analysis on the sample. In a representative embodiment, the method comprises using proton decoupling and magic angle spinning (MAS) to obtain a high-resolution 13 13C ssNMR spectrum, with sensitivity enhancement achieved by cross-polarization (CP). In a representative aspect, a method for detecting crystalline and / or amorphous biomolecules in a sample comprises a plurality of 1Obtaining an H Carr-Purcell-Meiboom-Gill (CPMG) spectrum and the 13 C cross-polarization (CP) spectrum. In a representative embodiment, the method includes operating at an H resonance frequency of about 250 to about 1000 MHz (optionally about 500 MHz, about 700 MHz, about 800 MHz, about 900 MHz). In a representative embodiment, the method includes maintaining the temperature at about 250 to 350 K, such as about 250 K, about 275 K, about 300 K, about 325 K or about 350 K. In a representative embodiment, the method includes operating a magic angle spinning (MAS) probe. In a representative example, the method includes operating an MAS probe that includes at least two radio frequency (rf) channels. In a representative embodiment, the MAS probe is 1 tuned for H and 1 C. In certain embodiments, the MAS probe is operated at a rotation frequency of about 2 kHz to about 8 kHz or about 3 kHz to about 5 kHz, such as about 4 kHz. In some embodiments, the method includes using a 90-degree pulse, such as 13 an H 90-degree pulse of about 2.5 μs. In some embodiments, an H CPMG spectrum is obtained with about 5 to about 100 π pulses (such as about 10 to about 90, about 20 to about 80 pulses) that are about 2 μs to about 20 μs long (for example, about 5 μs, about 10 μs, about 15 μs, about 20 μs). For example, each of the π pulses can be separated by 500 μs, and optionally the total time of the CPMG pulses is 10 ms. In a representative embodiment, the method includes obtaining a plurality of 1 H CPMG spectra while rotating at a frequency of 5 kHz or less, more than 8 kHz or about 14 kHz. In some embodiments, the contact time of the 1 C CP during the measurement is about 100 μs to about 10 ms, such as about 250 μs, about 500 μs, about 750 μs, about 1 ms, about 2 ms, about 3 ms, about 4 ms, about 5 ms, about 6 ms, about 7 ms, about 8 ms, about 9 ms, about 10 ms). In a representative embodiment, the measurement is at about 20 kHz to about 100 kHz, such as about 50 kHz 1 of 13 H CPMG spectra. In some embodiments, the 13included an RF spin-lock pulse in C. In a representative embodiment, 1 match the lamp pulse in H.

[0060] In a representative embodiment in some embodiments, the method of detecting includes quantifying the content of crystalline biomolecules in a sample. In a representative example, crystalline biomolecules exhibit spectroscopic characteristics different from those of amorphous biomolecules. In a representative example, crystalline biomolecules exhibit spectroscopic characteristics of higher molecular mobility than amorphous biomolecules.

[0061] Accordingly, methods for quantifying the content of crystalline and / or amorphous biomolecules in a sample are provided herein. In a representative embodiment, the method comprises (A) a plurality of 1 H obtaining an NMR Carr-Purcell-Meiboom-Gill (CPMG) spectrum of the sample and (B) 13 obtaining a 13C NMR cross-polarization (CP) spectrum of the sample. In accordance with the teachings referring to methods for detecting crystalline and / or amorphous biomolecules in a sample, the methods for quantifying the content disclosed herein can be performed.

[0062] In representative embodiments of the methods of detection or quantification, crystalline biomolecules are bi-refringant and optionally do not diffract. In representative embodiments, crystalline biomolecules are from about 1 Å to about 2 Å and do not diffract or diffract poorly. With respect to the detection methods described herein, the biomolecule can be any of the biomolecules described herein, including, for example, polypeptides, proteins, polysaccharides, lipids (e.g., glycolipids, phospholipids, sterols), polynucleotides or nucleic acids (e.g., DNA or RNA). In representative embodiments, the biomolecule is a protein comprising one or more polypeptide chains. In a representative example, the biomolecule is an antibody or immunoglobulin or an antigen-binding antibody fragment thereof and includes any of those described herein under the section entitled "Biomolecules". In a representative example, the protein comprises one or more glycans, i.e., is a glycanated biomolecule. In representative embodiments, the biomolecule is a glycanated antibody.

[0063] The following examples are given merely to illustrate the invention and are not intended to limit its scope.

Examples

[0064] Example 1: Buffer Exchange of Crystalline Monoclonal Antibodies The kSep® system (Sartorius Stedim North America, Inc., Bohemia, NY) is a countercurrent gradient centrifugation system designed for biological processes to recover cells as a product or discard cells and recover the supernatant as a product during manufacturing. This system is described in Kelly et al., Biotechnol. Prog. 32(6):1520 - 1530 (2016) and U.S. Patent Application Publication No. 2011 / 0207222. The kSep® system differs from conventional centrifugation systems where the opposing forces of the continuous flow of fluid (e.g., media, buffer) and centrifugal force are balanced, creating a fluidized bed system of cells or particles within the device. See Dechsiri, C. (2004). Particle transport in fluidized beds: experiments and stochastic models Groningen: s.n. In a conventional centrifuge unit, a packed bed system is achieved. The kSep® system also utilizes disposable components that minimize the need for cleaning and contribute to aseptic processing through aseptic welded connections. The kSep® system is available as a laboratory or manufacturing scale system. The kSep® 400 system is a laboratory scale system with the ability to process 400 mL / cycle at a maximum flow rate of 114 L / hr. The kSep® 400 system has four individual 100 mL chambers and each chamber can be operated alone or simultaneously with other chambers. The kSep® 6000S system is a manufacturing scale system (see Figure 1) with the ability to process 6000 mL / cycle at a maximum flow rate of 720 L / hr. The kSep® 6000S system has six individual 1000 mL chambers and each chamber can be operated alone or simultaneously with other chambers.

[0065] While the kSep® system has been evaluated for its use in protein crystallization (McPherson, Methods 34(3):254-265(2004)), it has not been used for further downstream processing. Here, after protein crystallization unit operation, the kSep® system is used to replace the hypertonic crystallization buffer with an isotonic formulation buffer appropriate for patient administration (see, for example, Figure 2). The kSep® system is also used when concentrating the protein crystallization suspension to a concentration suitable for patient administration.

[0066] When the kSep® system was used, the protein crystal suspension containing crystalline full-length human monoclonal antibody was buffer-exchanged with a buffer exceeding 90% efficiency at 1 buffer exchange volume and exceeding 99% efficiency at 2 buffer exchange volumes. By varying the balance of centrifugal force and fluid flow force within the kSep® system, the monoclonal antibody protein crystal suspension was concentrated before dispensing from the kSep® unit. Based on the amount of crystals loaded in the unit and the fluid flow force relative to the centrifugal force, the protein crystal suspension can be concentrated in the range of 215 - 300 mg / mL from 63 mg / mL. Therefore, countercurrent gradient centrifugation via the kSep® system is an effective means for buffer-exchanging the crystallization buffer with the formulation buffer and concentrating the protein crystals to a concentration suitable for patient administration.

[0067] Materials and Methods Machine: The kSep® 400 unit was used for protein crystal suspension buffer exchange and concentration operations. The laboratory-scale kSep® unit provided a platform for evaluating these operations without consuming large amounts of material. To reduce the amount of material used for evaluation, the use of chambers was reduced from the standard four kSep® system chambers used at a minimum of 400 mL for two or either one of the kSep® system chambers to reduce the volume used to 100 - 300 mL. A concentration-washing-recovery, CWH, disposable tube set was used for this evaluation because the configuration provided proper channels for recovering protein crystals after buffer exchange and concentration unit operations.

[0068] Materials: For this evaluation, a fully humanized monoclonal antibody was used. At the start of crystallization, the antibody formulation contained 20 mM sodium acetate, 220 mM proline, and 0.01% polysorbate 80 at a concentration of 140 mg / mL and pH 5.0. The crystallization buffer added to this system contained 16 mM sodium phosphate and 20% PEG 3350 at pH 8.4. The final protein crystal suspension at the end of the crystallization unit operation consisted of approximately 9 mM sodium acetate, 98 mM proline, 0.004% polysorbate 80, 7 mM sodium phosphate, and 8.9% PEG 3350 at pH 6.2. The crystal concentration was 62.2 mg / mL.

[0069] Buffer Exchange Buffer: The buffer used for buffer exchange and concentration was an isotonic buffer consisting of the following components: 27 mM succinic acid, 15% PEG 3350, and 0.1% PS80 at pH 5.2.

[0070] Results and Observations Buffer Exchange Using the pre-programmed application type of concentration - washing - recovery of the kSep (registered trademark) system, a buffer exchange operation was performed (see, for example, Figure 3). The significant parameters that affected the overall buffer exchange were the washing flow rate and the number of volumes per chamber (equivalent to diavolume). The washing flow rate parameter affected both the processing time and the buffer exchange efficiency. The faster the washing flow rate, the shorter the processing time, but the lower the buffer exchange efficiency. Conversely, the slower the washing flow rate, the longer the processing time, but the higher the buffer exchange efficiency. For the number of volumes per chamber, the volume was determined by the bioreactor volume as specified in the recommended protocol of the kSep (registered trademark) system. To quantify the buffer exchange efficiency, the pH of the outlet flow was measured and the convergence of the outlet pH to the buffer exchange buffer pH was observed. Since the inlet feed substance pH started at 6.2 and the buffer exchange buffer pH was 5.2, when the outlet pH reached the pH of the buffer exchange buffer, the buffer exchange was considered complete.

[0071] The results of the buffer exchange experiments are summarized in Table 1. As the number of volumes per chamber increased, the outlet pH from Experiment 1 was 5.34 and the outlet pH from Experiment 4 was 5.22, so the buffer exchange efficiency improved. Other variable factors such as the washing flow rate and the supply volume also affected the final outlet pH, but not to the same extent as the number of volumes per chamber.

[0072]

Table 1

[0073] As shown in Figure 2, the kSep (registered trademark) system functioned better with respect to buffer exchange efficiency when compared to a conventional benchtop centrifuge. After a single spin and decant operation for benchtop centrifugation (equivalent to one volume number per chamber in the kSep (registered trademark) system), the outlet pH for the conventional centrifuge was 5.7, whereas it was approximately 5.3 for the kSep (registered trademark) system. A supply volume of 100 - 300 mL, a wash 1 flow rate of 70 - 120 mL / min, and 1 - 2 buffer exchange volumes were evaluated to determine the extent to which complete buffer exchange occurred.

[0074] Concentration After the buffer exchange operation, the protein crystal suspension was concentrated by changing the balance between the centrifugal force and the fluid flow rate. By making the centrifugal force greater than the fluid flow rate force, the protein crystals moved towards one end of the kSep (registered trademark) system chamber and were effectively concentrated within the chamber. The methods to achieve this result included increasing the centrifugal force while maintaining the fluid flow rate constant, decreasing the fluid flow rate while maintaining the centrifugal force constant, or a combination of the previous two. There were two input variables that affected the outlet concentration variable: wash flow rate / centrifugal force and supply volume. Using the wash 2 flow rate function along with the kSep (registered trademark) system's concentration wash recovery program (see, for example, Figure 3), a supply volume of 150 - 300 mL and a wash 2 flow rate of 20 - 35 mL / min were evaluated. Based on these input variables, as shown in Table 2, concentrations in the range of 168 - 303 mg / mL were achieved using kSep (registered trademark).

[0075]

Table 2

[0076] In the previous experiment, the protein crystal suspension was concentrated to an outlet concentration in the range of 122 - 229 mg / mL by changing the feed volume from 260 to 500 mL and maintaining the wash 2 flow rate at 30 mL / min. Despite observing outlet concentrations exceeding 200 mg / mL, protein crystal aggregation and deterioration of crystallinity were also observed via a polarized light microscope due to the use of different buffer exchange buffers (10 mM NaPO4 pH 6.2, 10% PEG3350, 120 mM lysine, 0.1% Ps80 buffer (lysine)). The results from the initial failed experiments are shown in Table 3.

[0077]

Table 3

[0078] Recovery After concentrating the protein crystal suspension to the desired concentration, the suspension must be dispensed (recovered) from the kSep® unit. Generally, to remove substances from the kSep® system, the unit is operated in reverse and substances are pushed out through the inlet of the kSep® chamber. To prevent large pressure differences from the inlet and outlet, while the buffer pulses in the buffer, the individual chamber peristaltic pumps push out substances in reverse to remove them. The pre-programmed recovery operation in the kSep® concentration - wash - recovery application is designed to recover the non-concentrated suspension from this unit. As a result, many initial experiments failed because either substances clogged in the disposable tubes of this unit or the filled crystal suspension was destroyed by the pulsed buffer pump or the individual chamber peristaltic pumps.

[0079] The initial experiments used a pre-programmed recovery operation at a centrifugation speed of 60 g and a recovery (i.e., chamber peristaltic pump) flow rate of 50 mL / min. As a result of using the pre-programmed operation, many failures occurred where substances could not be recovered from the chamber. After many attempts using the pre-programmed recipe ended in failure, the recovery operation was switched to manual control to manipulate the flow rate and valve position in real-time. The centrifugation speed was decreased and maintained at a constant value to reduce the number of conditions to evaluate, and the buffer pump was maintained without pulsing the buffer. The buffer pump was maintained such that the pressure from the buffer flow rate increased steadily. The individual chamber peristaltic pumps slowly draw substances from the crystals and gradually push the packed crystal suspension out of the chamber by increasing the pressure while preventing cavity formation in the packed crystal suspension. Deterioration of the packed crystal suspension negatively affects the final crystal concentration. Table 4 summarizes the experiments related to recovering the packed (concentrated) crystal suspension from the kSep® system. Table 4 reveals some of the recovery experiments and observations made during the experiments. The packed crystal suspension could not be recovered with the pre-programmed recovery automation as currently set. The recovery operation needed to be switched from relying on automated control to a manual operation where the experimenter could adjust the settings in real-time. Experiments called "a" and "b" are part of the same experiment where the conditions were adjusted after attempting recovery and failing.

[0080]

Table 4

[0081] Another factor to consider is the individual chamber pump; the individual chamber pump needs to draw material from the chamber without causing any cavitation in the filled crystal suspension. Cavitation disrupts the concentration profile and creates a non-uniform concentration gradient across the concentrated crystal suspension, as shown in Experiments 4 and 5 of Table 4. To collect the concentrated crystal suspension, one chamber was collected at a time. The possibility of cavitation increased by collecting multiple chambers at a time. The centrifugal force was maintained at 8 g, the buffer pump was set at 25 - 50 mL / min, and the individual chamber pump was set at 75 mL / min (Figure 4). The centrifugal force and the buffer pump create a propulsive force to extrude the material from the chamber, while the individual chamber pump acts as a pulling mechanism to ensure that cavitation is limited.

[0082] Example 2: Identification Washing of samples in formulation buffer: Basically, samples of the crystalline material were prepared by the batch method as described in the pamphlet of International Publication No. WO 2016 / 010927 with the title "CRYSTALLINE ANTIBODY FORMULATIONS" which is incorporated herein by reference in its entirety.

[0083] All samples were washed in a formulation buffer of 27 mM succinic acid, 15% PEG 3350, and 0.1% Tw - 80, pH = 5.5. 1 mL of the crystal or amorphous material was combined with 1 mL of the formulation buffer in an Eppendorf tube and mixed by gentle inversion. Next, the mixture was centrifuged at 2000 RPM = 376 rcf for 5 minutes and the supernatant was removed. A further 1 mL of the formulation buffer was added, the pellet was resuspended, gently mixed, and then centrifuged. This was repeated once.

[0084] Sample filling: All samples were filled into 4.0 mm Bruker rotors. A 1 mL pipette tip was cut to serve as a funnel for the rotor, and the size was adjusted so that the rotor and the funnel fit inside a 2.0 mL Eppendorf tube. The sample was filled by adding 100 μL of the suspension to the funnel and then centrifuged at 10 k rcf for 2 minutes. This was repeated 4 - 8 times until the rotor was sufficiently filled with the solid sample.

[0085] SSNMR: Operated at a 1 500 MHz 1H resonance frequency, a Bruker Ascend Avance III wide - bore spectrometer was used for analysis. Unless otherwise stated, for all experiments, a 4 mm H / F / X MAS probe operating at a spinning frequency of 4 kHz was used. Except when performing temperature experiments, a BCU II - 80 / 60 temperature unit was used to adjust the temperature to 300 K. For a 90 - degree pulse, 1 160 W of H2.5us pi / 2 was used. For a total 10 ms CPMG pulse to suppress signals from solid substances, with 20 5 - μs pulses separated by 500 μs, 1 H the CPMG spectrum was acquired. After measuring the CPMG spectrum, the total solid material in the rotor was calibrated, and to account for differences in filling between the sample and the rotor, the sample was rotated at frequencies below 14 kHz and 13C cross - polarization (CP) was measured. The CP spectrum was acquired in 13C using a 2 ms contact time and a 50 kHz RF spin - lock pulse, 1 and was appropriately matched with the ramp pulse in 1H.

[0086] Observation The CPMG spectrum was used to suppress the proton signals arising from the portion of the sample that was in the solid phase and not in the mobile phase. In the CPMG spectra of the mAb crystals and the amorphous products, there were several signals that appeared in the crystalline material and not in the amorphous material (see Figure 5). These signals could be used to quantify the amount of the crystalline content, as shown by the spectra from samples to which either 5% crystalline or 5% amorphous mAb was added (Figure 6). The signals in the crystalline spectrum also increased with increasing temperature, indicating that these portions of the molecules were more mobile (Figure 7).

[0087] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually incorporated by reference and set forth in its entirety herein.

[0088] The use of the terms “a,” “an,” and “the” and similar references in the description of the present disclosure (especially in the context of the following claims) are to be construed to include both the singular and the plural unless otherwise indicated herein or clearly contradicted by the context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted.

[0089] The recitation of a range of values herein is merely a shorthand method of referring individually to each separate value falling within the range and each endpoint is hereby incorporated into the specification as if it were individually recited herein.

[0090] All of the methods described in this specification may be performed in any suitable order, unless otherwise indicated herein or clearly inconsistent with the content. The use of any examples or representative language provided herein (e.g., "such as") is merely to clarify the disclosure in more detail and does not raise a limitation in the scope of the disclosure, unless otherwise claimed. Nothing in the language of this application should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0091] Preferred embodiments of the disclosure are described herein, including the best mode known to the inventors for practicing the disclosure. Variations of these preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to use such variations as appropriate, and the inventors intend for the disclosure to be used in addition to those specifically described herein. Accordingly, this disclosure includes all alterations and equivalents of the subject matter shown in the claims appended hereto, as permitted by applicable law. Further, unless otherwise indicated herein or clearly inconsistent with the content, any combination of the above elements in all possible variations thereof is encompassed by the disclosure.

Claims

[Claim 1] The invention described in this specification.

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