Methods for reducing host cell protein content in antibody purification processes and antibody compositions having reduced host cell protein content

Abstract

To provide a method for reducing a host cell protein content in an antibody preparation recombinantly produced in a host cell in the manufacturing process of antibodies intended for administration to a patient, and to provide a therapeutic antibody preparation having reduced host cell protein.SOLUTION: There is provided a method of reducing a host cell protein content in a protein preparation comprising an anti-N3pGlu Aβ antibody recombinantly produced in a mammalian host cell. The method comprises the steps of: a. subjecting the protein preparation to an affinity chromatography column; b. eluting the anti-N3pGlu Aβ antibody from the chromatography column, by using a combination of acids comprising a weak acid and a strong acid to obtain an eluate comprising the anti-N3pGlu Aβ antibody; c. raising pH of the eluate to above about pH5.0; and d. subjecting the eluate to a depth filter and obtaining a filtered protein preparation.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the field of recombinant protein production. More specifically, the present invention provides methods for reducing host cell protein content in protein preparations recombinantly produced in host cells in processes for producing proteins intended for administration to patients, such as therapeutic or diagnostic antibodies or antigen-binding fragments thereof. The disclosed methods can be implemented to produce antibody compositions with reduced host cell protein content. [Background technology]

[0002] Host cell proteins (HCPs) are host cell proteins involved in cellular maintenance and growth, as well as protein synthesis and processing. However, in the field of therapeutic or diagnostic proteins, the presence of HCPs can cause problems such as aggregation, product fragmentation due to catalytic activity, and / or immunogenicity, threatening product quality and patient safety. Therefore, HCPs have been identified as critical quality attributes (CQAs) for protein pharmaceuticals. The formation of undesirable aggregates and product fragmentation necessitates additional purification steps to reduce / remove HCPs, which often reduce the yield of the desired protein and increase overall production costs.

[0003] The challenges of eliminating HCPs from manufacturing processes and attempts to improve methods for reducing HCPs have been disclosed, for example, in Gilgunn et al.; Goey et al., Biotechnology Advances 36 (2018) 1223-1237, and Current Opinion in Chemical Engineering 2018, 22:98-106. However, these methods for removing HCPs have limitations. For example, in some cases, these disclosures demonstrate one or more of the following: incomplete removal of HCPs; inconsistencies in methods for removing HCPs leading to aggregation; co-purification of the desired protein and HCPs; reduced product function; immunogenicity concerns in patients; and / or reduced pharmacokinetic properties such as half-life. Furthermore, methods developed for removing HCPs often require, for example, high labor volume and additional purification steps, increasing production costs and reducing yields. In some cases, the applicability of these methods is limited to certain molecules and / or forms. Therefore, alternative methods for reducing HCPs in therapeutic or diagnostic protein purification processes are needed. Such alternative methods preferably reduce HCP without affecting product stability, yield, or cost, ultimately maintaining product quality, are amenable to large-scale manufacturing, and ensure patient safety. Summary of the Invention

[0004] Thus, the present invention addresses one or more of the above-mentioned problems by providing an alternative method for reducing HCPs in preparations of therapeutic or diagnostic proteins or antigen-binding fragments thereof. The methods of the present invention provide a reproducible method that is highly effective at removing HCPs while maintaining antibody stability, reducing aggregation, maintaining product yield, and potentially reducing the risk of immunogenicity. Such methods can effectively remove HCPs without the need to increase the volume of the antibody preparation. Surprisingly, the methods of the present invention achieved HCP counts well below the industry acceptance standard of <100 ppm. Surprisingly, other embodiments of the present invention achieved HCP counts of <50 ppm while maintaining protein stability, reducing aggregation, and maintaining product yield. Even more surprisingly, other embodiments of the present invention achieved HCP counts of <20 ppm, <10 ppm, <5 ppm, <1 ppm, or about 0 ppm while maintaining protein stability, reducing aggregation, and maintaining product yield. Furthermore, embodiments of the present invention provide a method of HCP removal that is applicable to a wide range of molecules. Other embodiments of the invention allow for the elimination of additional purification steps, resulting in shorter batch processing times and reduced manufacturing costs. The disclosed methods can be practiced to produce antibody compositions with reduced host cell content, where the host cell content of the antibody composition is <100 ppm, <50 ppm, <10 ppm, <5 ppm, <1 ppm, or about 0 ppm. DETAILED DESCRIPTION OF THE INVENTION

[0005] Thus, provided are methods for reducing host cell protein content in anti-N3pGlu Aβ antibody ("anti-N3pG antibody") preparations. In some embodiments, the anti-N3pG antibody is recombinantly produced in a mammalian host cell, such as a Chinese hamster ovary cell host cell.

[0006] Accordingly, certain embodiments provide methods for reducing host cell protein content in a protein preparation comprising an anti-N3pGlu Aβ antibody recombinantly produced in mammalian host cells, the method comprising: subjecting the protein preparation recombinantly produced in host cells to an affinity chromatography column; eluting the anti-N3pGlu Aβ antibody from the chromatography column with a buffer comprising a combination of a weak acid and a strong acid; raising the pH of the eluate to about pH 5.0 or higher (e.g., about pH 6.0 or higher, or about pH 7.0 or higher); subjecting the eluate containing the anti-N3pGlu Aβ antibody to a depth filter; and obtaining a filtered protein preparation containing the anti-N3pGlu Aβ antibody. In some embodiments, the ionic strength of the eluate from the step of raising the pH to about pH 5.0 or higher is about 10 mM to about 45 mM. Preferably, the host cell protein content in the protein preparation comprising the anti-N3pGlu Aβ antibody is reduced. More preferably, the host cell protein content in a protein preparation comprising an anti-N3pGlu Aβ antibody is reduced to less than about 100 ppm, less than about 50 ppm, less than about 20 ppm, less than about 10 ppm, less than about 5 ppm, or less than about 1 ppm.

[0007] Accordingly, certain embodiments provide methods for reducing host cell protein content in a protein preparation comprising an anti-N3pGlu Aβ antibody recombinantly produced in mammalian host cells, the method comprising: subjecting the protein preparation recombinantly produced in host cells to an affinity chromatography column; eluting the anti-N3pGlu Aβ antibody from the chromatography column with a buffer comprising a combination of a weak acid and a strong acid; performing viral inactivation; raising the pH of the eluate to about pH 5.0 or higher (e.g., about pH 6.0 or higher, or about pH 7.0 or higher); subjecting the protein-containing eluate to a depth filter; and obtaining a filtered protein preparation comprising the anti-N3pGlu Aβ antibody. In some embodiments, the ionic strength of the eluate from the step of raising the pH to about pH 5.0 or higher is about 10 mM to about 45 mM. Preferably, the host cell protein content in the protein preparation comprising the anti-N3pGlu Aβ antibody is reduced. More preferably, the host cell protein content in a protein preparation comprising an anti-N3pGlu Aβ antibody is reduced to less than about 100 ppm, less than about 50 ppm, less than about 20 ppm, less than about 10 ppm, less than about 5 ppm, or less than about 1 ppm.

[0008] Thus, in certain embodiments, a method for reducing host cell protein content in a protein preparation comprising an anti-N3pGlu Aβ antibody recombinantly produced in a mammalian host cell is provided, the method comprising: applying the protein preparation comprising an anti-N3pGlu Aβ antibody recombinantly produced in a mammalian host cell to an affinity chromatography column; eluting the anti-N3pGlu Aβ antibody from the chromatography column with a buffer comprising a combination of a weak acid and a strong acid, wherein the weak acid is acetic acid and the strong acid is phosphoric acid or lactic acid; adjusting the pH of the eluate comprising the anti-N3pGlu Aβ antibody from the step of eluting the anti-N3pGlu Aβ antibody from the chromatography column to less than about pH 4.0, wherein the eluate is maintained at less than about pH 4.0 for between about 0 and about 180 minutes; raising the pH of the eluate to about pH 5.0 or greater (e.g., about pH 6.0 or greater, or about pH 7.0 or greater); applying the eluate comprising the anti-N3pGlu Aβ antibody to a depth filter; and obtaining a filtered protein preparation comprising an Aβ antibody. In some embodiments, the ionic strength of the eluate from the step of increasing the pH to about pH 5.0 or greater is about 10 mM to about 45 mM. Preferably, the host cell protein content in the protein preparation comprising the anti-N3pGlu Aβ antibody is reduced. More preferably, the host cell protein content in the protein preparation comprising the anti-N3pGlu Aβ antibody is reduced to less than about 100 ppm, less than about 50 ppm, less than about 20 ppm, less than about 10 ppm, less than about 5 ppm, or less than about 1 ppm.

[0009] In some embodiments of the present invention, the present disclosure provides a method for reducing host cell protein content in a protein preparation comprising an anti-N3pGlu Aβ antibody recombinantly produced in a mammalian host cell, the method comprising: subjecting the protein preparation comprising an anti-N3pGlu Aβ antibody recombinantly produced in a mammalian host cell to an affinity chromatography column; and eluting the anti-N3pGlu Aβ antibody from the chromatography column with a buffer comprising a combination of a weak acid and a strong acid, wherein the weak acid is acetic acid and the strong acid is phosphoric acid, the acetic acid having a concentration of about 20 mM and the phosphoric acid having a concentration of about 5 mM to about 10 mM; and recovering the anti-N3pGlu Aβ antibody from the step of eluting the anti-N3pGlu Aβ antibody from the chromatography column. The method includes adjusting the pH of an eluate containing an Aβ antibody to less than about pH 4.0, where the eluate is maintained at less than about pH 4.0 for about 0 to about 180 minutes; raising the pH of the eluate to about pH 5.0 or higher (e.g., about pH 6.0 or higher, or about pH 7.0 or higher); subjecting the eluate containing an anti-N3pGlu Aβ antibody to a depth filter; and obtaining a filtered protein preparation containing an anti-N3pGlu Aβ antibody. In some embodiments, the ionic strength of the eluate from the step of raising the pH to about pH 5.0 or higher is about 10 mM to about 45 mM. Preferably, the host cell protein content in the protein preparation containing an anti-N3pGlu Aβ antibody is reduced. More preferably, the host cell protein content in a protein preparation comprising an anti-N3pGlu Aβ antibody is reduced to less than about 100 ppm, less than about 50 ppm, less than about 20 ppm, less than about 10 ppm, less than about 5 ppm, or less than about 1 ppm.

[0010] In some embodiments of the present disclosure, the present disclosure provides a method for reducing host cell protein content in a protein preparation comprising an anti-N3pGlu Aβ antibody recombinantly produced in a mammalian host cell, the method comprising: subjecting the protein preparation comprising an anti-N3pGlu Aβ antibody recombinantly produced in a mammalian host cell to an affinity chromatography column; eluting the anti-N3pGlu Aβ antibody from the chromatography column with a buffer comprising a combination of a weak acid and a strong acid, wherein the weak acid is acetic acid and the strong acid is lactic acid, the acetic acid having a concentration of about 20 mM and the lactic acid having a concentration of about 5 mM; adjusting the pH of the eluate comprising the anti-N3pGlu Aβ antibody from the step of eluting the anti-N3pGlu Aβ antibody from the chromatography column to less than about pH 4.0, wherein the eluate is maintained at less than about pH 4.0 for about 0 to about 180 minutes; Provided are methods comprising: subjecting an eluate containing an Aβ antibody to a depth filter; and obtaining a filtered protein preparation containing an anti-N3pGlu Aβ antibody. In some embodiments, the ionic strength of the eluate from the step of increasing the pH to about pH 5.0 or greater is about 10 mM to about 45 mM. Preferably, the host cell protein content in the protein preparation containing the anti-N3pGlu Aβ antibody is reduced. More preferably, the host cell protein content in the protein preparation containing the anti-N3pGlu Aβ antibody is reduced to less than about 100 ppm, less than about 50 ppm, less than about 20 ppm, less than about 10 ppm, less than about 5 ppm, or less than about 1 ppm.

[0011] In some embodiments, the present disclosure provides a method for reducing host cell protein content in a protein preparation comprising an anti-N3pGlu Aβ antibody recombinantly produced in a mammalian host cell, the method comprising: subjecting the protein preparation comprising an anti-N3pGlu Aβ antibody recombinantly produced in a mammalian host cell to an affinity chromatography column; eluting the anti-N3pGlu Aβ antibody from the chromatography column with a buffer comprising a combination of a weak acid and a strong acid, wherein the weak acid is acetic acid and the strong acid is phosphoric acid or lactic acid; and recovering the anti-N3pGlu Aβ antibody from the step of eluting the anti-N3pGlu Aβ antibody from the chromatography column. The method includes adjusting the pH of an eluate containing an Aβ antibody, the step of adjusting the pH of the eluate comprising adding about 20 mM HCl to the eluate, adjusting the pH of the eluate to about pH 3.3 to about pH 3.7, and maintaining the eluate at about pH 3.3 to about pH 3.7 for about 0 to about 180 minutes; raising the pH of the eluate to about pH 5.0 or higher (e.g., about pH 6.0 or higher, or about pH 7.0 or higher); subjecting the eluate containing the anti-N3pGlu Aβ antibody to a depth filter; and obtaining a filtered protein preparation containing the anti-N3pGlu Aβ antibody. In some embodiments, the ionic strength of the eluate from the step of raising the pH to about pH 5.0 or higher is about 10 mM to about 45 mM. Preferably, the host cell protein content in the protein preparation containing the anti-N3pGlu Aβ antibody is reduced. More preferably, the host cell protein content in a protein preparation comprising an anti-N3pGlu Aβ antibody is reduced to less than about 100 ppm, less than about 10 ppm, less than about 5 ppm, or less than about 1 ppm.

[0012] In some embodiments, the present disclosure provides a method for reducing host cell protein content in a protein preparation comprising an anti-N3pGlu Aβ antibody recombinantly produced in a mammalian host cell, the method comprising: subjecting the protein preparation comprising an anti-N3pGlu Aβ antibody recombinantly produced in a mammalian host cell to an affinity chromatography column; eluting the anti-N3pGlu Aβ antibody from the chromatography column with a buffer comprising a combination of a weak acid and a strong acid, wherein the weak acid is acetic acid and the strong acid is phosphoric acid or lactic acid; and recovering the anti-N3pGlu Aβ antibody from the step of eluting the anti-N3pGlu Aβ antibody from the chromatography column. The method includes adjusting the pH of an eluate containing an Aβ antibody, the step of adjusting the pH of the eluate comprising adding about 20 mM HCl to the eluate, adjusting the pH of the eluate to about pH 3.5, and maintaining the eluate at about pH 3.5 for about 0 to about 180 minutes; raising the pH of the eluate to about pH 5.0 or higher (e.g., about pH 6.0 or higher, or about pH 7.0 or higher); subjecting the eluate containing the anti-N3pGlu Aβ antibody to a depth filter; and obtaining a filtered protein preparation containing the anti-N3pGlu Aβ antibody. In some embodiments, the ionic strength of the eluate from the step of raising the pH to about pH 5.0 or higher is about 10 mM to about 45 mM. Preferably, the host cell protein content in the protein preparation containing the anti-N3pGlu Aβ antibody is reduced. More preferably, the host cell protein content in a protein preparation comprising an anti-N3pGlu Aβ antibody is reduced to less than about 100 ppm, less than about 10 ppm, less than about 5 ppm, or less than about 1 ppm.

[0013] In certain embodiments, the present disclosure provides a method for reducing host cell protein content in a protein preparation comprising an anti-N3pGlu Aβ antibody recombinantly produced in a host cell, the method comprising: subjecting the protein preparation comprising an anti-N3pGlu Aβ antibody recombinantly produced in a host cell to an affinity chromatography column; eluting the anti-N3pGlu Aβ antibody from the chromatography column with a buffer comprising a combination of a weak acid and a strong acid, wherein the weak acid is acetic acid and the strong acid is phosphoric acid or lactic acid; adjusting the pH of the eluate comprising the anti-N3pGlu Aβ antibody from the step of eluting the anti-N3pGlu Aβ antibody from the chromatography column to less than about pH 4.0, wherein the eluate is maintained at less than about pH 4.0 for between about 0 and about 180 minutes; and raising the pH of the eluate to between about pH 5.0 and about pH 7.5, wherein the pH comprises adding about 250 mM Tris buffer to the eluate. Provided are methods comprising: subjecting an eluate containing an Aβ antibody to a depth filter; and obtaining a filtered protein preparation containing an anti-N3pGlu Aβ antibody. In some embodiments, increasing the pH of the eluate to about pH 5.0 to about pH 7.5 comprises adding about 100 mM to about 1000 mM Tris buffer to the eluate. In some embodiments, the ionic strength of the eluate from the step of increasing the pH to greater than about pH 5.0 to about pH 7.5 is about 10 mM to about 45 mM. Preferably, the host cell protein content in the protein preparation containing the anti-N3pGlu Aβ antibody is reduced. More preferably, the host cell protein content in the protein preparation containing the anti-N3pGlu Aβ antibody is reduced to less than about 100 ppm, less than about 10 ppm, less than about 5 ppm, or less than about 1 ppm.

[0014] In some embodiments, the disclosure provides a method for reducing host cell protein content in a protein preparation comprising an anti-N3pGlu Aβ antibody recombinantly produced in mammalian host cells, the method comprising: applying the protein preparation comprising an anti-N3pGlu Aβ antibody recombinantly produced in mammalian host cells to an affinity chromatography column; eluting the anti-N3pGlu Aβ antibody from the chromatography column with a buffer comprising a combination of a weak acid and a strong acid, wherein the weak acid is acetic acid and the strong acid is phosphoric acid or lactic acid; adjusting the pH of the eluate comprising the anti-N3pGlu Aβ antibody from the chromatography column to less than about pH 4.0, wherein the eluate is maintained at less than about pH 4.0 for about 0 to about 180 minutes; raising the pH of the eluate to about pH 7.0, comprising adding about 250 mM Tris buffer to the eluate; applying the antibody-containing eluate to a depth filter; and obtaining a filtered antibody preparation. In some embodiments, increasing the pH of the eluate to about pH 6.5 to about pH 7.5 (e.g., about pH 7.0) comprises adding about 100 mM to about 1000 mM Tris buffer to the eluate. In some embodiments, the ionic strength of the eluate from the step of increasing the pH to about pH 6.5 to about pH 7.5 (e.g., about pH 7.0) is about 10 mM to about 45 mM. Preferably, the host cell protein content in the protein preparation comprising the anti-N3pGlu Aβ antibody is reduced. More preferably, the host cell protein content in the protein preparation comprising the anti-N3pGlu Aβ antibody is reduced to less than about 100 ppm, less than about 10 ppm, less than about 5 ppm, or less than about 1 ppm.

[0015] In some embodiments, the disclosure provides a method for reducing host cell protein content in a protein preparation comprising an anti-N3pGlu Aβ antibody recombinantly produced in a mammalian host cell, the method comprising: applying the protein preparation comprising an anti-N3pGlu Aβ antibody recombinantly produced in a mammalian host cell to an affinity chromatography column; eluting the anti-N3pGlu Aβ antibody from the chromatography column with a buffer comprising a combination of a weak acid and a strong acid, wherein the weak acid is acetic acid and the strong acid is phosphoric acid or lactic acid; adjusting the pH of the eluate comprising the anti-N3pGlu Aβ antibody from the step of eluting the anti-N3pGlu Aβ antibody from the chromatography column to less than about pH 4.0, wherein the eluate is maintained at less than about pH 4.0 for between about 0 and about 180 minutes; raising the pH of the eluate to about pH 5.0 or greater (e.g., about pH 6.0 or greater, or about pH 7.0 or greater); applying the eluate comprising the anti-N3pGlu Aβ antibody to a depth filter; and and obtaining a filtered protein preparation comprising an Aβ antibody, wherein the eluate applied to the depth filter has an ionic strength of about 10 mM to about 45 mM. Preferably, the host cell protein content in the protein preparation comprising the anti-N3pGlu Aβ antibody is reduced. More preferably, the host cell protein content in the protein preparation comprising the anti-N3pGlu Aβ antibody is reduced to less than about 100 ppm, less than about 10 ppm, less than about 5 ppm, or less than about 1 ppm.

[0016] In certain embodiments, the present disclosure provides a method for reducing host cell protein content in a protein preparation comprising an anti-N3pGlu Aβ antibody recombinantly produced in a mammalian host cell, the method comprising: applying the protein preparation comprising an anti-N3pGlu Aβ antibody recombinantly produced in a mammalian host cell to an affinity chromatography column; eluting the anti-N3pGlu Aβ antibody from the chromatography column with a buffer comprising a combination of a weak acid and a strong acid, wherein the weak acid is acetic acid and the strong acid is phosphoric acid or lactic acid; and adjusting the pH of the eluate comprising the anti-N3pGlu Aβ antibody from the chromatography column to less than about pH 4.0, wherein the eluate is maintained at less than about pH 4.0 for between about 0 and about 180 minutes to achieve viral inactivation.

[0017] The present disclosure provides a method for reducing host cell protein content in a protein preparation comprising an anti-N3pGlu Aβ antibody recombinantly produced in a mammalian host cell, the method comprising: subjecting the protein preparation comprising an anti-N3pGlu Aβ antibody recombinantly produced in a mammalian host cell to an affinity chromatography column; eluting the anti-N3pGlu Aβ antibody from the chromatography column with a buffer comprising a combination of a weak acid and a strong acid, wherein the weak acid comprises acetic acid at a concentration of about 20 mM and the strong acid comprises one of phosphoric acid, formic acid, or lactic acid, and the strong acid has a concentration of about 5 mM to about 10 mM; and recovering the anti-N3pGlu Aβ antibody from the step of eluting the anti-N3pGlu Aβ antibody from the chromatography column. and (c) adjusting the pH of an eluate containing an Aβ antibody, the step of adjusting the pH of the eluate comprising adding HCl, phosphoric acid, citric acid, acetic acid, or a combination thereof (e.g., a combination of acetic acid and phosphoric acid, or a combination of acetic acid and citric acid) to the eluate to adjust the pH to less than about pH 4.0, and maintaining the eluate at less than about pH 4.0 for about 0 to about 180 minutes. The method also includes: increasing the pH of the eluate to about pH 5.0 to about pH 7.5; subjecting the eluate containing the anti-N3pGlu Aβ antibody to a depth filter; and obtaining a filtered protein preparation containing the anti-N3pGlu Aβ antibody. In some embodiments, the ionic strength of the eluate from the step of increasing the pH to about pH 5.0 to about pH 7.5 is about 10 mM to about 45 mM.

[0018] Preferably, the host cell protein content in a protein preparation comprising an anti-N3pGlu Aβ antibody is reduced, more preferably to less than about 100 ppm, less than about 10 ppm, less than about 5 ppm, or less than about 1 ppm.

[0019] In further embodiments, the elution step comprises an elution buffer comprising any one of a combination of acetic acid and phosphoric acid, acetic acid and lactic acid, or acetic acid and formic acid, and adjusting the pH to less than about pH 4.0 comprises adding any one of HCl, phosphoric acid, citric acid, acetic acid, or a combination thereof (e.g., a combination of acetic acid and phosphoric acid, or a combination of acetic acid and citric acid). In further embodiments, the elution step comprises an elution buffer comprising any one of a combination of about 20 mM acetic acid and about 10 mM phosphoric acid, about 20 mM acetic acid and about 5 mM phosphoric acid, or about 20 mM acetic acid and about 5 mM formic acid, and adjusting the pH to less than about pH 4.0 comprises adding any one of about 20 mM HCl, about 15 mM to about 200 mM phosphoric acid, about 1000 mM citric acid, or a combination of about 20 mM acetic acid and about 10 mM phosphoric acid. In such embodiments, the ionic strength of the eluate from the step of increasing the pH to a pH greater than about 6.0 is from about 10 mM to about 45 mM.

[0020] In one aspect of the invention, the invention provides a method for reducing host cell protein content in a protein preparation comprising an anti-N3pGlu Aβ antibody recombinantly produced in a mammalian host cell, comprising: applying a protein preparation comprising anti-N3pGlu Aβ antibodies recombinantly produced in mammalian host cells to an affinity chromatography column; eluting the anti-N3pGlu Aβ antibody from the chromatography column using a buffer comprising a combination of a weak acid and a strong acid, wherein the weak acid is acetic acid and the strong acid is phosphoric acid or lactic acid; adjusting the pH of the eluate containing the anti-N3pGlu Aβ antibodies from the chromatographic column to less than about pH 4.0, wherein the eluate is maintained at a pH less than about 4.0 for between about 0 and about 180 minutes; increasing the pH of the eluate to about pH 5.0 or higher (e.g., about pH 6.0 or higher, or about pH 7.0 or higher); applying the eluate containing anti-N3pGlu Aβ antibodies to a depth filter; obtaining a filtered protein preparation comprising anti-N3pGlu Aβ antibodies.

[0021] Preferably, the host cell protein content in a protein preparation comprising an anti-N3pGlu Aβ antibody is reduced, more preferably to less than about 100 ppm, less than about 10 ppm, less than about 5 ppm, or less than about 1 ppm.

[0022]

[0023] In a further embodiment of the invention there is provided a method for reducing host cell protein content in a protein preparation comprising an anti-N3pGlu Aβ antibody recombinantly produced in a mammalian host cell, the method comprising: a) subjecting a protein preparation to an affinity chromatography column; b) eluting the anti-N3pGlu Aβ antibodies from the chromatography column to obtain an eluate containing anti-N3pGlu Aβ antibodies; and c) optionally adjusting the pH of the eluate to between pH 5.0 and pH 7.5 and applying the eluate to a depth filter to obtain a filtered protein preparation comprising anti-N3pGlu Aβ antibodies, wherein the depth filter is a fully synthetic depth filter.

[0023] Preferably, the chromatography column comprises a Protein A, Protein G, or Protein L affinity chromatography column. More preferably, the pore size of the depth filter is at least about 9 μm (microns) to about 0.1 μm. Even more preferably, the pore size of the depth filter is at least about 2 μm to about 0.1 μm. Even more preferably, the pore size of the depth filter is about 0.1 μm. Even more preferably, the depth filter is an XOSP filter. In an alternative embodiment of the invention, the pH of the eluate on the depth filter is about 5.0. In a further alternative embodiment of the invention, the pH of the eluate on the depth filter is about 6.0. In a further alternative embodiment of the invention, the pH of the eluate on the depth filter is about 7.0.

[0024] This particular embodiment encompasses a method of eluting anti-N3pG antibodies from an affinity chromatography column using any commonly used weak or strong acid, including, but not limited to, acetic acid, citric acid, phosphoric acid, hydrochloric acid, formic acid, and lactic acid.

[0025] The use of fully synthetic filters with a pH of the solution above the filter between 5.0 and 7.0 has been found to be highly effective in reducing and / or removing HCPs compared to many conventional cellulose / diatomaceous earth-based filters.

[0026] The disclosed methods can be performed to reduce host cell proteins (HCPs) in preparations comprising anti-N3pGlu Aβ antibodies or antigen-binding fragments thereof to obtain antibody compositions with reduced HCP content. In some embodiments, the anti-N3pGlu Aβ antibodies are monoclonal, chimeric, humanized, human, bispecific, or antibody fragments. In some embodiments, the anti-N3pGlu Aβ antibodies are IgG1 antibodies or contain the Fc portion of an IgG1 antibody. Disclosed herein are anti-SARS-COV-2 antibodies.

[0027] In some embodiments of the disclosed methods and compositions produced by the disclosed methods, the anti-N3pG antibody is donanemab. In some embodiments, the anti-N3pG antibody comprises a light chain variable region (LH) comprising an LH complementarity determining region 1 (LCDR1), an LCDR2, and an LCDR3 present in the amino acid sequence of DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSRGKTYLNWLLQKPGQSPQLLIYAVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHYPFTFGQGTKLEIK (SEQ ID NO: 13), and the anti-N3pG antibody comprises a heavy chain variable region (VH) comprising a VH complementarity determining region 1 (HCDR1), an HCDR2, and an HCDR3 present in the amino acid sequence of QVQLVQSGAEVKKPGSSVKVSCKASGYDFTRYYINWVRQAPGQGLEWMGWINPGSGNTKYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCAREGITVYWGQGTTVTVSS (SEQ ID NO: 14).

[0028] In some embodiments, the anti-N3pG antibody comprises an LCDR1 of KSSQSLLYSRGKTYLN (SEQ ID NO: 17), an LCDR2 of AVSKLDS (SEQ ID NO: 18), an LCDR3 of VQGTHYPFT (SEQ ID NO: 19), an HCDR1 of GYDFTRYYIN (SEQ ID NO: 20), an HCDR2 of WINPGSGNTKYNEKFKG (SEQ ID NO: 21), and an HCDR3 of EGITVY (SEQ ID NO: 22).

[0029] In some embodiments, the anti-N3pG antibody comprises a variable light chain (LC) comprising the amino acid sequence of DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSRGKTYLNWLLQKPGQSPQLLIYAVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHYPFTFGQGTKLEIK (SEQ ID NO: 13), and a variable heavy chain (HC) comprising the amino acid sequence of QVQLVQSGAEVKKPGSSVKVSCKASGYDFTRYYINWVRQAPGQGLEWMGWINPGSGNTKYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCAREGITVYWGQGTTVTVSS (SEQ ID NO: 14).

[0030] In some embodiments, the anti-N3pG antibody comprises a light chain (LC) comprising the amino acid sequence of DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSRGKTYLNWLLQKPGQSPQLLIYAVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 15), and a light chain (LC) comprising the amino acid sequence of QVQLVQSGAEVKKPGSSVKVSCKASGYDFTRYYINWVRQAPGQGLEWMGWINPGSGNTKYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCAREGIT VYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 16).

[0031]

[0032] In some embodiments of the disclosed methods and compositions produced by the disclosed methods, the anti-N3pG antibody is the antibody referred to as "antibody 201c" in U.S. Patent No. 10,647,759, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the anti-N3pG antibody comprises a light chain variable region (LH) comprising an LH complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 present in the amino acid sequence of DIQMTQSPSTLSASVGDRVTITCRASQSLGNWLAWYQQKPGKAPKLLIYQASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYKGSFWTFGQGTKVEIK (SEQ ID NO: 23), and the anti-N3pG antibody comprises a heavy chain variable region (VH) comprising a VH complementarity determining region 1 (HCDR1), HCDR2, and HCDR3 present in the amino acid sequence of EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGSGSYYNGFDYWGQGTLVTVSS (SEQ ID NO: 24).

[0033] In some embodiments, the anti-N3pG antibody comprises an LCDR1 of RASQSLGNWLA (SEQ ID NO: 27), an LCDR2 of YQASTLES (SEQ ID NO: 28), an LCDR3 of QHYKGSFWT (SEQ ID NO: 29), an HCDR1 of AASGFTFSSYPMS (SEQ ID NO: 30), an HCDR2 of AISGSGGGSTYYADSVKG (SEQ ID NO: 31), and an HCDR3 of AREGGSGSYYNGFDY (SEQ ID NO: 32).

[0034] In some embodiments, the anti-N3pG antibody comprises a variable light chain (VL) comprising the amino acid sequence of DIQMTQSPSTLSASVGDRVTITCRASQSLGNWLAWYQQKPGKAPKLLIYQASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYKGSFWTFGQGTKVEIK (SEQ ID NO: 23), and a variable heavy chain (VH) comprising the amino acid sequence of EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGSGSYYNGFDYWGQGTLVTVSS (SEQ ID NO: 24).

[0035] In some embodiments, the anti-N3pG antibody comprises a light chain (LC) comprising the amino acid sequence of: DIQMTQSPSTLSASVGDRVTITCRASQSLGNWLAWYQQKPGKAPKLLIYQASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYKGSFWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 25), and The heavy chain (HC) comprises the amino acid sequence of FDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 26).

[0036]

[0037] In another aspect of the invention, the invention provides a method for reducing host cell protein content in an anti-N3pG antibody preparation recombinantly produced in a host cell, comprising: subjecting the anti-N3pG antibody preparation recombinantly produced in the host cells to an affinity chromatography column, e.g., a Protein A affinity chromatography column; eluting the anti-N3pG antibodies with a buffer comprising a combination of acetic acid and phosphoric acid or a combination of acetic acid and lactic acid; adjusting the pH of the eluate containing the anti-N3pG antibody by adding about 20 mM HCl, wherein the pH is adjusted to about pH 3.3 to about pH 3.7, and the eluate is maintained at about pH 3.3 to about pH 3.7 for about 0 to about 180 minutes; increasing the pH of the eluate containing the anti-N3pG antibody by adding about 250 mM Tris buffer, whereby the pH is increased to about pH 5.0 to about pH 7.5; and applying the eluate containing the anti-N3pG antibody to a depth filter to obtain a filtered anti-N3pG antibody preparation. The method provides that the host cell protein content in the anti-N3pG antibody preparation after depth filtration is reduced to less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm, and the anti-N3pG antibody is an IgG1 antibody.

[0038] In some embodiments of the present invention, the disclosure provides a method for reducing host cell protein content in an anti-N3pG antibody preparation recombinantly produced in a host cell, comprising: subjecting the anti-N3pG antibody preparation recombinantly produced in a host cell to a Protein A chromatography column; eluting the anti-N3pG antibody from the chromatography column with a buffer comprising a combination of about 20 mM acetic acid and about 5 mM phosphate, or a buffer comprising a combination of about 20 mM acetic acid and about 10 mM phosphate, or a buffer comprising a combination of about 20 mM acetic acid and about 5 mM lactic acid; adjusting the pH of an eluate containing an anti-N3pG antibody by adding HCl, wherein the pH is lowered to about pH 3.3 to about pH 3.7, and the eluate is maintained at about pH 3.3 to about pH 3.7 for about 0 to about 180 minutes; increasing the pH of the eluate containing an anti-N3pG antibody by adding about 250 mM Tris buffer, wherein the pH is increased to about pH 5.0 to about pH 7.5; subjecting the eluate containing an anti-N3pG antibody to a depth filter; and obtaining a filtered anti-N3pG antibody preparation, wherein the host cell protein content in the filtered anti-N3pG antibody preparation is less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm, and the anti-N3pG antibody is an IgG1 antibody. In some embodiments, increasing the pH of the eluate to about pH 5.0 to about pH 7.5 comprises adding about 100 mM to about 1000 mM Tris buffer to the eluate.

[0039] In some embodiments of the present invention, the disclosure provides a method for reducing host cell protein content in an anti-N3pG antibody preparation recombinantly produced in a host cell, comprising: subjecting the anti-N3pG antibody preparation recombinantly produced in a host cell to a Protein A chromatography column; eluting the anti-N3pG antibody from the chromatography column with a buffer comprising a combination of about 20 mM acetic acid and about 5 mM phosphate, or a buffer comprising a combination of about 20 mM acetic acid and about 10 mM phosphate, or a buffer comprising a combination of about 20 mM acetic acid and about 5 mM lactic acid; adjusting the pH of an eluate containing anti-N3pG antibodies with HCl, wherein the pH is adjusted to about pH 3.5, and the eluate is maintained at about pH 3.5 for about 0 to about 180 minutes; increasing the pH of the eluate containing anti-N3pG antibodies with about 250 mM Tris buffer, wherein the pH is increased to about pH 5.0 to about pH 7.5; subjecting the eluate containing anti-N3pG antibodies to a depth filter; and obtaining a filtered anti-N3pG antibody preparation, wherein the host cell protein content in the filtered anti-N3pG antibody is less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm, and the anti-N3pG antibody is an IgG1 antibody. In some embodiments, increasing the pH of the eluate to about pH 5.0 to about pH 7.5 comprises adding about 100 mM to about 1000 mM Tris buffer to the eluate.

[0040] In some embodiments of the present invention, the disclosure provides methods for reducing host cell protein content in an anti-N3pG antibody preparation recombinantly produced in a host cell, the method comprising: subjecting the anti-N3pG antibody preparation recombinantly produced in a mammalian host cell to a Protein A chromatography column; eluting the anti-N3pG antibody from the chromatography column with a buffer comprising a combination of about 20 mM acetic acid and about 5 mM phosphate, or a buffer comprising a combination of about 20 mM acetic acid and about 10 mM phosphate, or a buffer comprising a combination of about 20 mM acetic acid and about 5 mM lactic acid; and adjusting the pH of the eluate containing the anti-N3pG antibody by adding about 20 mM HCl, wherein the pH is lowered to about pH 3.5 and the eluate is maintained at about pH 3.5 for about 0 to about 180 minutes to achieve viral inactivation.

[0041] In some embodiments of the present invention, the disclosure provides a method for reducing host cell protein content in an anti-N3pG antibody preparation recombinantly produced in a host cell, comprising: subjecting the anti-N3pG antibody preparation recombinantly produced in a host cell to a Protein A chromatography column; eluting the anti-N3pG antibody from the chromatography column with a buffer comprising a combination of about 20 mM acetic acid and about 5 mM phosphate, or a buffer comprising a combination of about 20 mM acetic acid and about 10 mM phosphate, or a buffer comprising a combination of about 20 mM acetic acid and about 5 mM lactic acid; adjusting the pH of an eluate containing an anti-N3pG antibody by adding HCl, wherein the pH is lowered to about pH 3.3 to about pH 3.7, and the eluate is maintained at about pH 3.3 to about pH 3.7 for about 0 to about 180 minutes; increasing the pH of the eluate containing an anti-N3pG antibody with about 250 mM Tris buffer, wherein the pH is increased to about pH 7.25; subjecting the eluate containing an anti-N3pG antibody to a depth filter; and obtaining a filtered anti-N3pG antibody preparation, wherein the host cell protein content in the anti-N3pG antibody preparation is less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm, and the anti-N3pG antibody is an IgG1 antibody. In some embodiments, increasing the pH of the eluate to about pH 7.25 comprises adding about 100 mM to about 1000 mM Tris buffer to the eluate.

[0042] In some embodiments of the present invention, the disclosure provides a method for reducing host cell protein content in an anti-N3pG antibody preparation recombinantly produced in a host cell, comprising: subjecting the anti-N3pG antibody preparation recombinantly produced in a host cell to a Protein A chromatography column; eluting the anti-N3pG antibody from the chromatography column with a buffer comprising a combination of about 20 mM acetic acid and about 5 mM phosphate, or a buffer comprising a combination of about 20 mM acetic acid and about 5 mM phosphate, or a buffer comprising a combination of about 20 mM acetic acid and about 5 mM lactic acid; adjusting the pH of an eluate containing anti-N3pG antibodies by adding HCl, whereby the pH is lowered to about pH 3.5 and the eluate is maintained at about pH 3.5 for about 0 to about 180 minutes; increasing the pH of the eluate containing anti-N3pG antibodies by adding about 250 mM Tris buffer, whereby the pH is increased to about pH 7.25; subjecting the eluate containing anti-N3pG antibodies to a depth filter; and obtaining a filtered anti-N3pG antibody preparation, whereby the host cell protein content in the anti-N3pG antibody preparation is less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm, and the anti-N3pG antibody is an IgG1 antibody. In some embodiments, increasing the pH of the eluate to about pH 7.25 comprises adding about 100 mM to about 1000 mM Tris buffer to the eluate.

[0043] In some embodiments, the present invention provides methods for reducing host cell protein content in an anti-N3pG antibody preparation recombinantly produced in a host cell,

[0044] In some embodiments of the disclosed methods and antibody compositions produced by the disclosed methods, the antibody is an antibody against the spike protein of sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In some embodiments, the anti-SARS-CoV-2 antibody is recombinantly produced in a mammalian host cell, such as a Chinese hamster ovary cell. Suitable anti-SARS-CoV-2 antibodies may include, but are not limited to, bamlanivimab, etesevimab, and bebuterovimab. In some embodiments, the anti-SARS-CoV-2 antibody is bamlanivimab. In some embodiments, the anti-SARS-CoV-2 antibody comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 1 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the anti-SARS-CoV-2 antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 3 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 4. In other embodiments, the anti-SARS-COV-2 antibody is etesevimab. In yet other embodiments, the anti-SARS-COV-2 antibody comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO:5 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO:6. In yet further embodiments, the anti-SARS-COV-2 antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO:7 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, the anti-SARS-COV-2 antibody is bebuterovimab. In yet other embodiments, the anti-SARS-COV-2 antibody comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO:9 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO:10. In yet further embodiments, the anti-SARS-COV-2 antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO:11 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:12.

[0045] In some embodiments, the therapeutic or diagnostic antibody is produced in a mammalian cell, hi some embodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell, or a baby hamster kidney (BHK) cell, a mouse hybridoma cell, or a mouse myeloma cell.

[0046] In some embodiments, the present invention provides methods for reducing host cell protein content in antibody preparations recombinantly produced in host cells after depth filtration, which are further subjected to further purification and / or polishing steps to obtain a drug substance preparation. A drug substance is defined by the FDA as an active ingredient intended to produce pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease, or to affect the structure or any function of the human body, but does not include intermediates used in the synthesis of such ingredients. A drug product is generally, but not necessarily, a finished dosage form suitable for administration to a human patient, e.g., a tablet, capsule, or solution, containing the drug substance in association with one or more other ingredients. In some embodiments, the further purification and / or polishing steps include one or more of the following: performing viral inactivation, performing ion exchange chromatography, performing viral filtration, and / or performing tangential flow filtration.

[0047] In some embodiments, the disclosure provides methods for reducing host cell protein content in a protein preparation comprising an anti-N3pG antibody recombinantly produced in a mammalian host cell, wherein the host cell protein content in the protein preparation comprising the anti-N3pG antibody is reduced to less than about 100 ppm. In other embodiments, the host cell protein content in the protein preparation comprising the anti-N3pG antibody is reduced to less than about 50 ppm. In other embodiments, the host cell protein content in the protein preparation comprising the anti-N3pG antibody is reduced to less than about 20 ppm. In other embodiments, the host cell protein content in the protein preparation comprising the anti-N3pG antibody is reduced to less than about 10 ppm, 5 ppm, or 1 ppm. In other embodiments, the host cell protein content in the protein preparation comprising the anti-N3pG antibody is reduced to about 0 ppm.

[0048] In some embodiments, the disclosure provides methods for reducing host cell protein content in a protein preparation comprising an anti-N3pG antibody recombinantly produced in a mammalian host cell, wherein the host cell protein content in the protein preparation comprises PLBL2, and the PLBL2 content is reduced to less than about 100 ppm. In other embodiments, the PLBL2 content is reduced to less than about 50 ppm. In other embodiments, the PLBL2 content is reduced to less than about 20 ppm. In other embodiments, the PLBL2 content is reduced to less than about 10 ppm, 5 ppm, or 1 ppm. In other embodiments, the PLBL2 content is reduced to about 0 ppm.

[0049] In some embodiments, the disclosure provides methods for reducing host cell protein content in a protein preparation comprising an anti-N3pG antibody recombinantly produced in a host cell, wherein the host cell protein content in the protein preparation comprises a lysosomal-protective protein, and the lysosomal-protective protein content is reduced to less than about 100 ppm. In other embodiments, the lysosomal-protective protein content is reduced to less than about 50 ppm. In other embodiments, the lysosomal-protective protein content is reduced to less than about 20 ppm. In other embodiments, the lysosomal-protective protein content is reduced to less than about 10 ppm, 5 ppm, or 1 ppm. In other embodiments, the lysosomal-protective protein content is reduced to about 0 ppm.

[0050] In some embodiments, the disclosure provides methods for reducing host cell protein content in a protein preparation comprising an anti-N3pG antibody recombinantly produced in a host cell, wherein the host cell protein content in the protein preparation includes protein S100-A6, and the protein S100-A6 content is reduced to less than about 100 ppm. In other embodiments, the protein S100-A6 content is reduced to less than about 50 ppm. In other embodiments, the protein S100-A6 content is reduced to less than about 20 ppm. In other embodiments, the protein S100-A6 content is reduced to less than about 10 ppm, 5 ppm, or 1 ppm. In other embodiments, the protein S100-A6 content is reduced to about 0 ppm.

[0051] In some embodiments, the disclosure provides methods for reducing host cell protein content in a protein preparation comprising an anti-N3pG antibody recombinantly produced in a host cell, wherein the host cell protein content in the protein preparation includes protein S100-A11, and the protein S100-A11 content is reduced to less than about 100 ppm. In other embodiments, the protein S100-A11 content is reduced to less than about 50 ppm. In other embodiments, the protein content of protein S100-A11 is reduced to less than about 20 ppm. In other embodiments, the protein S100-A11 content is reduced to less than about 10 ppm, 5 ppm, or 1 ppm. In other embodiments, the protein S100-A11 content is reduced to about 0 ppm.

[0052] In some embodiments, the disclosure provides methods for reducing host cell protein content in a protein preparation comprising an anti-N3pG antibody recombinantly produced in a host cell, wherein the host cell protein content in the protein preparation includes ubiquitin-40S ribosomal protein S27a, and the ubiquitin-40S ribosomal protein S27a content is reduced to less than about 100 ppm. In other embodiments, the ubiquitin-40S ribosomal protein S27a content is reduced to less than about 50 ppm. In other embodiments, the ubiquitin-40S ribosomal protein S27a content is reduced to less than about 20 ppm. In other embodiments, the ubiquitin-40S ribosomal protein S27a content is reduced to less than about 10 ppm, 5 ppm, or 1 ppm. In other embodiments, the ubiquitin-40S ribosomal protein S27a content is reduced to about 0 ppm.

[0053] In some embodiments, the disclosure provides methods for reducing host cell protein content in a protein preparation comprising an anti-N3pG antibody recombinantly produced in a host cell, wherein the host cell protein content in the protein preparation comprises kallikrein-11, and the kallikrein-11 content is reduced to less than about 100 ppm. In other embodiments, the kallikrein-11 content is reduced to less than about 50 ppm. In other embodiments, the kallikrein-11 content is reduced to less than about 20 ppm. In other embodiments, the kallikrein-11 content is reduced to less than about 10 ppm, 5 ppm, or 1 ppm. In other embodiments, the kallikrein-11 content is reduced to about 0 ppm.

[0054] In some embodiments, the disclosure provides methods for reducing host cell protein content in a protein preparation comprising an anti-N3pG antibody recombinantly produced in a host cell, wherein the host cell protein content in the protein preparation includes serine protease HTRA1 isoform X1, and the serine protease HTRA1 isoform X1 content is reduced to less than about 100 ppm. In other embodiments, the serine protease HTRA1 isoform X1 content is reduced to less than about 50 ppm. In other embodiments, the serine protease HTRA1 isoform X1 content is reduced to less than about 20 ppm. In other embodiments, the serine protease HTRA1 isoform X1 content is reduced to less than about 10 ppm, 5 ppm, or 1 ppm. In other embodiments, the serine protease HTRA1 isoform X1 content is reduced to about 0 ppm.

[0055] In some embodiments, the disclosure provides methods for reducing host cell protein content in a protein preparation comprising an anti-N3pG antibody recombinantly produced in a host cell, wherein the host cell protein content in the protein preparation includes the C1r subcomponent of complement, and the C1r subcomponent content is reduced to less than about 100 ppm. In other embodiments, the C1r subcomponent content is reduced to less than about 50 ppm. In other embodiments, the C1r subcomponent content is reduced to less than about 20 ppm. In other embodiments, the C1r subcomponent content is reduced to less than about 10 ppm, 5 ppm, or 1 ppm. In other embodiments, the C1r subcomponent content is reduced to about 0 ppm.

[0056] In some embodiments, the disclosure provides methods for reducing host cell protein content in a protein preparation comprising an anti-N3pG antibody recombinantly produced in a host cell, wherein the host cell protein content in the protein preparation includes actin, aortic smooth muscle isoform XI, and the actin, aortic smooth muscle isoform XI content is reduced to less than about 100 ppm. In other embodiments, the actin, aortic smooth muscle isoform XI content is reduced to less than about 50 ppm. In other embodiments, the actin, aortic smooth muscle isoform XI content is reduced to less than about 20 ppm. In other embodiments, the actin, aortic smooth muscle isoform XI content is reduced to less than about 10 ppm, 5 ppm, or 1 ppm. In other embodiments, the actin, aortic smooth muscle isoform XI content is reduced to about 0 ppm.

[0057] In some embodiments, the disclosure provides methods for reducing host cell protein content in a protein preparation comprising an anti-N3pG antibody recombinantly produced in a host cell, wherein the host cell protein content in the protein preparation comprises a heat shock cognate 71 kDa protein, and the heat shock cognate 71 kDa protein content is reduced to less than about 100 ppm. In other embodiments, the heat shock cognate 71 kDa protein content is reduced to less than about 50 ppm. In other embodiments, the heat shock cognate 71 kDa protein content is reduced to less than about 20 ppm. In other embodiments, the heat shock cognate 71 kDa protein content is reduced to less than about 10 ppm, 5 ppm, or 1 ppm. In other embodiments, the heat shock cognate 71 kDa protein content is reduced to about 0 ppm.

[0058] In some embodiments, the disclosure provides methods for reducing host cell protein content in a protein preparation comprising an anti-N3pG antibody recombinantly produced in a mammalian host cell, wherein the host cell protein content in the protein preparation comprises polyubiquitin, and the polyubiquitin content is reduced to less than about 100 ppm. In other embodiments, the polyubiquitin content is reduced to less than about 50 ppm. In other embodiments, the polyubiquitin content is reduced to less than about 20 ppm. In other embodiments, the polyubiquitin content is reduced to less than about 10 ppm, 5 ppm, or 1 ppm. In other embodiments, the polyubiquitin content is reduced to about 0 ppm.

[0059] In some embodiments, the disclosure provides methods for reducing host cell protein content in a protein preparation comprising an anti-N3pG antibody recombinantly produced in a host cell, wherein the host cell protein content in the protein preparation comprises peroxiredoxin-1, and the peroxiredoxin-1 content is reduced to less than about 100 ppm. In other embodiments, the peroxiredoxin-1 content is reduced to less than about 50 ppm. In other embodiments, the peroxiredoxin-1 content is reduced to less than about 20 ppm. In other embodiments, the peroxiredoxin-1 content is reduced to less than about 10 ppm, 5 ppm, or 1 ppm. In other embodiments, the peroxiredoxin-1 content is reduced to about 0 ppm.

[0060] In some embodiments, the disclosure provides methods for reducing host cell protein content in a protein preparation comprising an anti-N3pG antibody recombinantly produced in a host cell, wherein the host cell protein content in the protein preparation comprises glutathione S-transferase Y1, and the glutathione S-transferase Y1 content is reduced to less than about 100 ppm. In other embodiments, the glutathione S-transferase Y1 content is reduced to less than about 50 ppm. In other embodiments, the glutathione S-transferase Y1 content is reduced to less than about 20 ppm. In other embodiments, the glutathione S-transferase Y1 content is reduced to less than about 10 ppm, 5 ppm, or 1 ppm. In other embodiments, the glutathione S-transferase Y1 content is reduced to about 0 ppm.

[0061] In some embodiments, the disclosure provides methods for reducing host cell protein content in a protein preparation comprising an anti-N3pG antibody recombinantly produced in a host cell, wherein the host cell protein content in the protein preparation includes 40S ribosomal protein S28, and the 40S ribosomal protein S28 content is reduced to less than about 100 ppm. In other embodiments, the 40S ribosomal protein S28 content is reduced to less than about 50 ppm. In other embodiments, the 40S ribosomal protein S28 content is reduced to less than about 20 ppm. In other embodiments, the 40S ribosomal protein S28 content is reduced to less than about 10 ppm, 5 ppm, or 1 ppm. In other embodiments, the 40S ribosomal protein S28 content is reduced to about 0 ppm.

[0062] In some embodiments, the disclosure provides methods for reducing host cell protein content in a protein preparation comprising an anti-N3pG antibody recombinantly produced in a host cell, wherein the host cell protein content in the protein preparation comprises thioredoxin isoform X1, and the thioredoxin isoform X1 content is reduced to less than about 100 ppm. In other embodiments, the thioredoxin isoform X1 content is reduced to less than about 50 ppm. In other embodiments, the thioredoxin isoform X1 content is reduced to less than about 20 ppm. In other embodiments, the thioredoxin isoform X1 content is reduced to less than about 10 ppm, 5 ppm, or 1 ppm. In other embodiments, the thioredoxin isoform X1 content is reduced to about 0 ppm.

[0063] In some embodiments, the disclosure provides methods for reducing host cell protein content in a protein preparation comprising an anti-N3pG antibody recombinantly produced in a host cell, wherein the host cell protein content in the protein preparation comprises basement membrane-specific heparan sulfate proteoglycan core protein isoform X1, and the basement membrane-specific heparan sulfate proteoglycan core protein isoform X1 content is reduced to less than about 100 ppm. In other embodiments, the basement membrane-specific heparan sulfate proteoglycan core protein isoform X1 content is reduced to less than about 50 ppm. In other embodiments, the basement membrane-specific heparan sulfate proteoglycan core protein isoform X1 content is reduced to less than about 20 ppm. In other embodiments, the basement membrane-specific heparan sulfate proteoglycan core protein isoform X1 content is reduced to less than about 10 ppm, 5 ppm, or 1 ppm. In other embodiments, the basement membrane-specific heparan sulfate proteoglycan core protein isoform X1 content is reduced to about 0 ppm.

[0064] In some embodiments, the disclosure provides methods for reducing host cell protein content in a protein preparation comprising an anti-N3pG antibody recombinantly produced in a host cell, wherein the host cell protein content in the protein preparation comprises a tubulointerstitial nephritis antigen-like protein, and the tubulointerstitial nephritis antigen-like protein content is reduced to less than about 100 ppm. In other embodiments, the tubulointerstitial nephritis antigen-like protein content is reduced to less than about 50 ppm. In other embodiments, the tubulointerstitial nephritis antigen-like protein content is reduced to less than about 20 ppm. In other embodiments, the tubulointerstitial nephritis antigen-like protein content is reduced to less than about 10 ppm, 5 ppm, or 1 ppm. In other embodiments, the tubulointerstitial nephritis antigen-like protein content is reduced to about 0 ppm.

[0065] In some embodiments, the disclosure provides methods for reducing host cell protein content in a protein preparation comprising an anti-N3pG antibody recombinantly produced in a host cell, wherein the host cell protein content in the protein preparation comprises galectin-1, and the galectin-1 content is reduced to less than about 100 ppm. In other embodiments, the galectin-1 content is reduced to less than about 50 ppm. In other embodiments, the galectin-1 content is reduced to less than about 20 ppm. In other embodiments, the galectin-1 content is reduced to less than about 10 ppm, 5 ppm, or 1 ppm. In other embodiments, the galectin-1 content is reduced to about 0 ppm.

[0066] In some embodiments, the disclosure provides methods for reducing host cell protein content in a protein preparation comprising an anti-N3pG antibody recombinantly produced in a host cell, wherein the host cell protein content in the protein preparation comprises cornifin alpha, and the cornifin alpha content is reduced to less than about 100 ppm. In other embodiments, the cornifin alpha content is reduced to less than about 50 ppm. In other embodiments, the cornifin alpha content is reduced to less than about 20 ppm. In other embodiments, the cornifin alpha content is reduced to less than about 10 ppm, 5 ppm, or 1 ppm. In other embodiments, the cornifin alpha content is reduced to about 0 ppm.

[0067] In some embodiments, the invention provides methods for reducing host cell protein content of a protein preparation comprising an anti-N3pG antibody recombinantly produced in a host cell, wherein the protein preparation is subjected to depth filtration. In some embodiments, the protein preparation comprising anti-N3pG antibodies is subjected to a depth filter, which is one or more of a B1HC filter, an X0SP filter, a C0SP filter, an X0HC filter, an Emphaze™ AEX Hybrid Purifier filter, or a Zeta Plus (ZB Media) filter (such as a Zeta Plus (60ZB05A) filter, a Zeta Plus (90ZB05A) filter, or a Zeta Plus (90ZB08A) filter), or a depth filter having the same performance characteristics as any of a B1HC filter, an X0SP filter, a C0SP filter, an X0HC filter, an Emphaze™ AEX Hybrid Purifier filter, or a Zeta Plus (ZB Media) filter (such as a Zeta Plus (60ZB05A) filter, a Zeta Plus (90ZB05A) filter, or a Zeta Plus (90ZB08A) filter).

[0068] In some embodiments, the protein preparation comprising anti-N3pG antibodies is subjected to a depth filter, which is one or more of a B1HC filter, an X0HC filter, or a Zeta Plus (ZB Media) filter (such as a Zeta Plus (60ZB05A) filter, a Zeta Plus (90ZB05A) filter, or a Zeta Plus (90ZB08A) filter), or a depth filter having the same performance characteristics as any of a B1HC filter, an X0HC filter, or a Zeta Plus (ZB Media) filter (such as a Zeta Plus (60ZB05A) filter, a Zeta Plus (90ZB05A) filter, or a Zeta Plus (90ZB08A) filter).

[0069] In some embodiments, the protein preparation comprising anti-N3pG antibodies is subjected to a depth filter, which is one or more of an X0SP filter, a C0SP filter, an X0HC filter, or an Emphaze™ AEX Hybrid Purifier filter, or a depth filter having the same performance characteristics as any of an X0SP filter, a C0SP filter, or an Emphaze™ AEX Hybrid Purifier filter.

[0070] In some embodiments of the disclosed methods, the depth filter utilized in the methods is a fully synthetic depth filter comprising a fully synthetic filter media. In some embodiments, the pore size of the depth filter is from about 9 microns to about 0.1 microns. In some embodiments, the pore size of the depth filter is from about 2 microns to about 0.1 microns. In some embodiments, the pore size of the depth filter is about 0.1 microns.

[0071] In some embodiments of the disclosed methods, the pH of the protein preparation comprising anti-N3pG antibodies that is subjected to depth filtration is about 5.0, and / or the pH of the eluate comprising anti-N3pG antibodies after depth filtration is about 5.0. In other embodiments, the pH of the protein preparation comprising anti-N3pG antibodies that is subjected to depth filtration is about 6.0, and / or the pH of the eluate comprising anti-N3pG antibodies after depth filtration is about 6.0. In other embodiments, the pH of the protein preparation comprising anti-N3pG antibodies that is subjected to depth filtration is about 7.0, and / or the pH of the eluate comprising anti-N3pG antibodies after depth filtration is about 7.0.

[0072] In some embodiments, the disclosure provides methods for reducing host cell protein content in a protein preparation comprising an anti-N3pG antibody recombinantly produced in a mammalian host cell, wherein the ionic strength of the eluate from the step of increasing the pH to about 5.0 or greater (e.g., about 6.0 or about 7.0) is between about 10 mM and about 45 mM. In some embodiments, the ionic strength is less than about 30 mM. In some embodiments, the ionic strength is less than about 20 mM. In other embodiments, the ionic strength is less than about 15 mM.

[0073] In some embodiments, the invention provides methods in which a protein preparation comprising an anti-N3pG antibody recombinantly produced in a mammalian host cell is subjected to a chromatography column. In some embodiments, the chromatography column is one or more of an affinity column, an ion exchange column, a hydrophobic interaction column, a hydroxyapatite column, or a mixed-mode column. In some embodiments, the affinity chromatography column is a Protein A column, a Protein G column, or a Protein L column. In other embodiments, the ion exchange chromatography column is an anion exchange column or a cation exchange column. In some embodiments, the invention provides methods in which HCPs are substantially removed from the final product.

[0074] In some embodiments, the invention provides methods for reducing host cell protein content in a protein preparation comprising an anti-N3pG antibody recombinantly produced in a host cell, wherein the anti-N3pG antibody is a therapeutic or diagnostic antibody. In further embodiments, the therapeutic or diagnostic anti-N3pG antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a bispecific antibody, or an antibody fragment.

[0075] In another aspect, provided herein is a pharmaceutical composition comprising a protein preparation comprising an anti-N3pG antibody. In a further aspect, the present disclosure provides a composition produced by a method described herein. In yet another embodiment, the present disclosure provides a composition produced by a method described herein, wherein the host cell protein content of the composition is less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm.

[0076] The term "host cell protein" (HCP) refers to host cell proteins involved in cellular maintenance and proliferation, as well as protein synthesis and processing. Certain HCPs are associated with immunogenicity concerns in patients, and regulatory agencies desire to reduce HCPs to minimize these concerns. One powerful approach to immunogenicity analysis relies on immunoinformatics tools, which have been shown to provide validated and reliable predictions useful in both biopharmaceutical and vaccine design. Particularly relevant to HCP-driven immunogenicity is the T cell pathway, in which antigen-presenting cells process foreign proteins into constituent peptides, some of which ("epitopes") are recognized by major histocompatibility complex (MHC) class II proteins and presented to the cell surface for examination by T cells. The formation of the ternary MHC:epitope:T cell receptor complex can drive the initial naive response and stimulate subsequent B cell activation and maturation. Therefore, much immunoinformatics research has been directed toward the reliable prediction of putative T cell epitopes (De Groot and Martin, Clin Immunol. 2009 May;131(2):189-201, which is incorporated herein by reference in its entirety), and the EpiMatrix system is one highly validated method based on peptide:MHC binding profiles. EpiMatrix not only identifies individual epitopes within a protein, but can also then assess the overall immunogenic risk of the protein according to its epitope density compared to benchmark proteins (De Groot and Martin, 2009). A general rule of thumb when using the EpiMatrix tool to predict immunogenicity is that scores of +20 or higher indicate a high risk of immunogenicity, and it is therefore desirable to reduce or eliminate such HCPs from the final preparation.

[0077] Such HCPs include, for example, those derived from Chinese hamster ovary (CHO) cells, such as phospholipase B-like 2 protein (PLBL2) (GenBank accession number 354497505), S100-A6 (GenBank accession number 354478978), protein S100-A11 (GenBank accession number 354490016), lysosomal protection protein (GenBank accession number 354476738), ubiquitin-40S ribosome Protein S27a (GenBank accession number 354483686), kallikrein-11 (GenBank accession number 625217455), serine protease HTRA1 isoform X1 (GenBank accession number 625222219), complement C1r small component (GenBank accession number 625183025), actin, aortic smooth muscle isoform X1 (GenBank accession number 625206860), heat shock cognate 71 kDa protein (GenBank accession number 625206860), and α-amyloid β ... ank accession number 350539823), peroxiredoxin-1 (GenBank accession number 350537945), polyubiquitin (GenBank accession number 346986309), glutathione S-transferase Y1 (GenBank accession number 354505868), 40S ribosomal protein S28 (GenBank accession number 625218224), thioredoxin isoform X1 (GenBank accession number 625209431), and basement membrane These include specific heparin sulfate proteoglycan core protein isoform X1 (GenBank accession number 625201352), tubulointerstitial nephritis antigen-like protein (GenBank accession number 625188472), actin, partial cytoplasmic 2 isoform X2 (GenBank accession number 354497282), galectin-1 (GenBank accession number 354496408), and cornifin alpha (GenBank accession number 354504887).In some embodiments of the disclosed methods, the content of an HCP that is reduced in the antibody preparation is the content of an HCP selected from S100-A6, protein S100-A11, phospholipase B-like 2 protein, lysosomal protective protein, ubiquitin-40S ribosomal protein S27a, kallikrein-11, serine protease HTRA1 isoform X1, complement C1r small component, actin, aortic smooth muscle isoform X1, heat shock cognate 71 kDa protein, and peroxiredoxin-1, and combinations thereof. The disclosed methods can be used to prepare antibody compositions having a content of one or more of S100-A6, protein S100-A11, phospholipase B-like 2 protein, lysosomal protective protein, ubiquitin-40S ribosomal protein S27a, kallikrein-11, serine protease HTRA1 isoform X1, complement C1r small component, actin, aortic smooth muscle isoform X1, heat shock cognate 71 kDa protein, and peroxiredoxin-1 that is less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, 2 ppm, and 1 ppm.

[0078] Due to their high risk of immunogenicity, it is particularly desirable to remove those HCPs with an EpiMatrix score of +20, such as phospholipase B-like 2 protein (PLBL2) (GenBank Accession No. 354497505), S100-A6 (GenBank Accession No. 354478978), protein S100-A11 (GenBank Accession No. 354490016), and lysosomal protection protein (GenBank Accession No. 354476738). Other HCPs, such as periredoxin-1, are highly persistent and difficult to remove due to their tendency to co-elute with the protein or antibody of interest.

[0079] The term "weak acid" refers to an acid having a minimum pKa of greater than about 4. Examples of weak acids include, but are not limited to, acetic acid, succinic acid, and 2-(N-morpholino)ethanesulfonic acid.

[0080] The term "strong acid" refers to an acid having a minimum pKa of less than about 4. Examples of strong acids include, but are not limited to, phosphoric acid, lactic acid, formic acid, malic acid, malonic acid, glycolic acid, citric acid, tartaric acid, and hydrochloric acid.

[0081] The term "valence" refers to an atom's ability to bond. The number of bonds an atom can form as part of a compound is expressed by the element's valence. The term "monovalent" refers to an atom, ion, or chemical group that has a valence of one and can therefore form one covalent bond.

[0082] The term "depth filter" refers to a filter element that uses a porous filtration medium to retain particles throughout the medium (within and on the medium), not just on the surface of the medium. Depth filters may also have adsorption capabilities due to the chemical properties of the materials from which they are constructed. Examples of commercially available depth filters include, but are not limited to, B1HC filters, X0SP filters, C0SP filters, X0HC filters, Emphaze™ AEX Hybrid Purifiers, Zeta Plus (60ZB05A) filters, Zeta Plus (90ZB05A) filters, and Zeta Plus (90ZB08A) filters. Depth filters may also be fully synthetic depth filters containing fully synthetic filter media. Depth filters may have pore sizes of about 9 microns to about 0.1 microns, about 2 microns to about 0.1 microns, or about 0.1 microns. The term "depth filtration" refers to the act of passing a liquid material, which may be heterogeneous or homogeneous, through a depth filter.

[0083] The term "ionic strength" when referring to a solution is a measure of the concentration of ions in that solution. Ionic strength (I) is the ratio of the species concentration c i and net charge z i To determine the ionic strength, Equation I is used:

number

[0084] An "antibody preparation" is a material or solution provided for a purification process or method that contains a therapeutic or diagnostic antibody or antigen-binding fragment thereof of interest and may also contain various impurities. Non-limiting examples include, for example, harvested cell culture fluid (HCCF), harvested cell culture material, clarified cell culture fluid, clarified cell culture material, capture pools, recovery pools, and / or collection pools that contain the therapeutic or diagnostic antibody of interest after one or more centrifugation and / or filtration steps, which capture pools, recovery pools, and / or collection pools contain the therapeutic or diagnostic antibody of interest after one or more purification steps.

[0085] The term "impurities" refers to materials that differ from the desired anti-N3pG antibody product. Impurities include, but are not limited to, host cell materials such as host cell proteins and CHOP; leached Protein A; nucleic acids; mutants, size variants, fragments, aggregates, or derivatives of the desired antibody; endotoxins; viral contaminants; cell culture media components; and the like.

[0086] The terms "protein" and "polypeptide" are used interchangeably herein to refer to polymers of amino acids of any length. A polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The term also encompasses amino acid polymers that are modified, either naturally or by intervention, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are proteins containing, for example, one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art. Examples of proteins include, but are not limited to, antibodies, peptides, enzymes, receptors, hormones, regulatory factors, antigens, binding agents, cytokines, Fc fusion proteins, immunoadhesin molecules, and the like.

[0087] As used herein, the term "antibody" refers to an immunoglobulin molecule that binds to an antigen. Antibody embodiments include monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific or multispecific antibodies, or conjugated antibodies. The antibody may be of any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4).

[0088] An exemplary antibody of the present disclosure is an immunoglobulin G (IgG)-type antibody composed of four polypeptide chains: two heavy chains (HC) and two light chains (LC) cross-linked via interchain disulfide bonds. The amino-terminal portion of each of the four polypeptide chains contains a variable region of about 100 to 125 amino acids or more that is primarily responsible for antigen recognition. The carboxy-terminal portion of each of the four polypeptide chains contains a constant region that is primarily responsible for effector function. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain is composed of a light chain variable region (VL) and a light chain constant region. IgG isotypes can be further divided into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4).

[0089] The VH and VL regions can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). The CDRs are exposed on the surface of the protein and are critical regions of the antibody for antigen-binding specificity. Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Herein, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3," and the three CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3." The CDRs contain most of the residues that form specific interactions with the antigen. The assignment of amino acid residues to CDRs has been described by Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins", Journal of Molecular Biology, 196, 901-917 (1987); Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), North (North et al., "A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, Biology, 406, 228-256 (2011)), or IMGT (the international ImMunoGeneTics database available at www.imgt.org; see Lefranc et al., Nucleic Acids Res. 1999;27:209-212).

[0090] As used herein, embodiments of the present disclosure also include antibody fragments or antigen-binding fragments that comprise at least a portion of an antibody that retains the ability to specifically interact with an antigen or an epitope of an antigen, such as Fab, Fab', F(ab')2, Fv fragment, scFv antibody fragment, scFab, disulfide-linked Fv (sdFv), Fd fragment, etc.

[0091] The disclosed methods can be practiced to prepare drug substance preparations.

[0092] The disclosed methods and compositions may utilize or include antibodies against the Np3Glu amyloid beta peptide ("anti-Np3G antibody"). Anti-Np3G antibodies can be used to treat diseases associated with amyloid beta (Aβ) peptide aggregation. Cleavage of the amyloid precursor protein (APP) yields Aβ peptides ranging in size from 38 to 43 amino acids. The conversion of Aβ from a soluble form to an insoluble form with a high β-sheet content, and the deposition of these insoluble forms as neurites and cerebrovascular plaques in the brain, are associated with many diseases, including Alzheimer's disease (AD), Down's syndrome, and cerebral amyloid angiopathy (CAA). The deposits found in plaques are composed of a heterogeneous mixture of Aβ peptides. N3pE, pE3-X, or Aβ p3-XN3pGlu Aβ, also known as N3pGlu Aβ, is an N-terminal truncated form of the Aβ peptide and is primarily found in plaques. N3pGlu Aβ lacks the first two amino acid residues at the N-terminus of human Aβ and has a pyroglutamate-derived glutamate at the third amino acid position. N3pGlu Aβ peptides are a minor component of Aβ deposited in the brain, but studies have shown that they have strong aggregation properties and accumulate early in the deposition cascade. Antibodies against N3pGlu Aβ are known in the art. For example, U.S. Patent No. 8,679,498 discloses a human N3pGlu Aβ antibody (e.g., B12L, also known as LY3002813) and methods for treating diseases such as Alzheimer's disease with the antibody. U.S. Patent No. 10,647,759 discloses N3pG Ab antibodies, including "antibody 201c," and methods for treating diseases such as Alzheimer's disease using the antibody. The anti-Np3Glu antibodies of the disclosed methods and compositions can specifically bind to an epitope present within the Ab that is Pyr-EFRHDSGYEVHHQK (ie, pE3-16).

[0093] The disclosed methods and compositions can utilize or include antibodies against the spike protein of sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As used herein, the term "anti-SARS-CoV2 antibody" refers to an antibody that binds to the spike (S) protein of SARS-CoV-2. The amino acid sequence of the SARS-CoV-2 spike (S) protein is set forth, for example, in GenBank Accession No. YP_009724390.1.

[0094] The terms "ultrafiltration" or "filtration" refer to a form of membrane filtration in which hydrostatic pressure forces a liquid through a semipermeable membrane. Higher molecular weight suspended solids and solutes are retained while water and lower molecular weight solutes pass through the membrane. In some instances, ultrafiltration membranes have pore sizes ranging from 1 μm to 100 μm. The terms "ultrafiltration membrane," "ultrafiltration filter," "filtration membrane," and "filtration filter" may be used interchangeably. Examples of filtration membranes include, but are not limited to, polyvinylidene fluoride (PVDF) membranes, cellulose acetate, cellulose nitrate, polytetrafluoroethylene (PTFE, Teflon), polyvinyl chloride, polyethersulfone, glass fiber, or other filtration materials suitable for use in a cGMP manufacturing environment.

[0095] As used herein, numerical ranges are inclusive of the numbers defining the range.

[0096] The art-recognized term "EU numbering" refers to a system for numbering amino acid residues in immunoglobulin molecules. EU numbering is described, for example, in Kabat et al., "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991); Edelman, GM, et al., "Proc. Natl. Acad. USA," 63, 78-85 (1969); and http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html#refs. The term "Kabat numbering" is recognized in the art as referring to a system for numbering amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues within heavy and light chain variable regions (see, e.g., Kabat, et al., Ann. NY Acad. Sci. 190:382-93 (1971); Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242 (1991)). The term "North numbering" refers to a system for numbering amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues within heavy and light chain variable regions, and is based, at least in part, on affinity-propagated clustering with multiple crystal structures as described in (North et al., A New Clustering of Antibody CDR Loop Conformations, Journal of Molecular Biology, 406:228-256 (2011)).

[0097] As used herein, the term "affinity chromatography" refers to a chromatographic method for separating biochemical mixtures (e.g., proteins and undesired biomolecular species) based on specific, reversible interactions between biomolecules. Exemplary embodiments of affinity chromatography include Protein A affinity, Protein G affinity, Protein L affinity, kappa affinity ligand chromatography (CaptureSelect™, KappaXL™, KappaSelect™, KappaXP™, etc.), or lambda affinity ligand chromatography.

[0098] Proteins of the present disclosure can be prepared by methods well known in the art and can be incorporated into pharmaceutical compositions comprising a protein of the present disclosure and one or more pharmaceutically acceptable carriers and / or diluents (see, e.g., Remington, The Science and Practice of Pharmacy, 22, which provides an overview of formulation techniques generally known to the practicing physician). nd Edition, Loyd V., Ed., Pharmaceutical Press, 2012). Carriers suitable for pharmaceutical compositions include any material that, when combined with the protein, retains the activity of the molecule and is non-reactive with the patient's immune system.

[0099] Expression vectors capable of directing the expression of operably linked genes are well known in the art. Expression vectors can encode a signal peptide that facilitates secretion of a polypeptide from a host cell. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide. Each of the expressed polypeptides can be expressed independently from different promoters to which they are operably linked within a single vector, or alternatively, from different promoters to which they are operably linked within multiple vectors. Expression vectors are typically replicable in the host organism either as episomes or as an integral part of the host chromosomal DNA. Expression vectors generally contain selectable markers, such as tetracycline, neomycin, and dihydrofolate reductase, to enable detection of those cells transformed with the desired DNA sequences.

[0100] Host cells refer to cells stably or transiently transfected, transformed, transduced, or infected with one or more expression vectors that express one or more proteins of the present disclosure. Creation and isolation of host cell lines that produce proteins of the present disclosure can be achieved using standard techniques known in the art. Mammalian cells are preferred host cells for expressing the proteins of the present disclosure. Specific mammalian cells include HEK293, NS0, DG-44, and CHO. Preferably, the protein is secreted into the medium in which the host cells are cultured, from which the protein can be recovered or purified, for example, using conventional techniques. For example, the medium can be applied to and eluted from a protein A affinity chromatography column and / or a kappa or lambda affinity ligand chromatography column. Undesirable biomolecular species, including soluble aggregates and multimers, can be efficiently removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The product may be immediately frozen, refrigerated, or lyophilized, e.g., at −70° C. Various methods of protein purification may be employed, and such methods are known in the art and described, for example, in Deutscher, Methods in Enzymology 182:83-89 (1990), and Scopes, Protein Purification: Principles and Practice, 3rd Edition, Springer, NY (1994).

[0101] Also disclosed herein are pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is prepared by a method comprising purifying the antibody from a mammalian host cell. In the disclosed pharmaceutical compositions comprising an antibody, the total content of host cell proteins (HCPs) in the composition is typically less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm (e.g., as measured by LCMS). In some embodiments of the disclosed pharmaceutical compositions, the antibody of the disclosed pharmaceutical compositions binds to human N3pGlu Aβ (anti-N3pGlu Aβ antibody). In some embodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell.

[0102] The disclosed pharmaceutical compositions typically comprise an antibody or antigen-binding fragment thereof, which may be an anti-N3pGlu Aβ antibody. In some embodiments, the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a bispecific antibody, or an antibody fragment. In some embodiments, the antibody is an IgG1 antibody.

[0103] The disclosed pharmaceutical compositions can comprise an anti-N3pGlu Aβ antibody. In some embodiments, the anti-N3pGlu Aβ antibody comprises a heavy chain (HC) and a light chain (LC), wherein the light chain comprises a light chain variable region (LCVR), the heavy chain comprises a heavy chain variable region (HCVR), the LCVR comprises amino acid sequences LCDR1, LCDR2, and LCDR3, and the HCVR comprises amino acid sequences HCDR1, HCDR2, and HCDR3, wherein LCDR1 is KSSQSLLYSRGKTYLN (SEQ ID NO: 17), LCDR2 is AVSKLDS (SEQ ID NO: 18), LCDR3 is VQGTHYPFT (SEQ ID NO: 19), HCDR1 is GYDFTRYYIN (SEQ ID NO: 20), HCDR2 is WINPGSGNTKYNEKFKG (SEQ ID NO: 21), and HCDR3 is EGITVY (SEQ ID NO: 22).

[0104] In some embodiments of the disclosed pharmaceutical compositions, the composition comprises an anti-N3pGlu Aβ antibody, wherein the antibody comprises a LCVR and an HCVR, wherein the LCVR is DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSRGKTYLNWLLQKPGQSPQLLIYAVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHYPFTFGQGTKLEIK (SEQ ID NO: 13) and the HCVR is QVQLVQSGAEVKKPGSSVKVSCKASGYDFTRYYINWVRQAPGQGLEWMGWINPGSGNTKYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCAREGITVYWGQGTTVTVSS (SEQ ID NO: 14).

[0105] In some embodiments of the disclosed pharmaceutical compositions, the composition comprises an anti-N3pGlu Aβ antibody, wherein the LC of the anti-N3pGlu Aβ antibody is DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSRGKTYLNWLLQKPGQSPQLLIYAVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 15); The HC of Aβ antibody is GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 16).

[0106] In some embodiments of the disclosed compositions, the composition comprises donanemab.

[0107] In some embodiments, the disclosed compositions comprise an anti-N3pGlu Aβ antibody comprising a heavy chain (HC) and a light chain (LC), wherein the light chain comprises a light chain variable region (LCVR), the heavy chain comprises a heavy chain variable region (HCVR), the LCVR comprises amino acid sequences LCDR1, LCDR2, and LCDR3, the HCVR comprises amino acid sequences HCDR1, HCDR2, and HCDR3, wherein LCDR1 is RASQSLGNWLA (SEQ ID NO: 27) and LCDR2 is YQASTLES (SEQ ID NO: 28), LCDR3 is QHYKGSFWT (SEQ ID NO: 29), HCDR1 is AASGFTFSSYPMS (SEQ ID NO: 30), HCDR2 is AISGSGGGSTYYADSVKG (SEQ ID NO: 31), and HCDR3 is AREGGSGSYYNGFDY (SEQ ID NO: 32).

[0108] In some embodiments of the disclosed pharmaceutical compositions, the composition comprises an anti-N3pGlu Aβ antibody, wherein the antibody comprises a LCVR and an HCVR, wherein the LCVR is DIQMTQSPSTLSASVGDRVTITCRASQSLGNWLAWYQQKPGKAPKLLIYQASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYKGSFWTFGQGTKVEIK (SEQ ID NO: 23) and the HCVR is EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGSGSYYNGFDYWGQGTLVTVSS (SEQ ID NO: 24).

[0109] In some embodiments of the disclosed pharmaceutical compositions, the composition comprises an anti-N3pGlu Aβ antibody, wherein the LC of the anti-N3pGlu Aβ antibody is DIQMTQSPSTLSASVGDRVTITCRASQSLGNWLAWYQQKPGKAPKLLIYQASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYKGSFWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 25); HC of Aβ antibody is EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGSGSYYNGF DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 26).

[0110] In some embodiments of the disclosed compositions, the composition comprises antibody 201c, referenced in US Pat. No. 10,647,759.

[0111] In the disclosed pharmaceutical compositions comprising an anti-N3pG antibody, which can include an anti-N3pGlu antibody such as donanemab, the pharmaceutical composition can have a reduced total content of host cell proteins (HCPs). In some embodiments, the composition contains less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm of HCPs (e.g., as measured by LCMS). In some embodiments, the composition comprises less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm of an HCP selected from the following HCPs and combinations thereof: protein S100-A6, protein S100-A11, phospholipase B-like 2 protein, lysosomal protective protein, ubiquitin-40S ribosomal protein S27a, kallikrein-11, serine protease HTRA1 isoform X1, complement C1r small component, actin, aortic smooth muscle isoform X1, heat shock cognate 71 kDa protein, peroxiredoxin-1.

[0112] In the disclosed pharmaceutical compositions comprising an anti-N3pG antibody, the composition may contain less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm of protein S100-A6 (e.g., as measured by LCMS). In the disclosed pharmaceutical compositions comprising an anti-N3pG antibody, the composition may contain less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm of protein S100-A11 (e.g., as measured by LCMS). In the disclosed pharmaceutical compositions comprising an anti-N3pG antibody, the composition may contain less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm of phospholipase B-like 2 protein (e.g., as measured by LCMS). In the disclosed pharmaceutical compositions comprising an anti-N3pG antibody, the composition may comprise less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm of lysosomal protective protein (e.g., as measured by LCMS). In the disclosed pharmaceutical compositions comprising an anti-N3pG antibody, the composition may comprise less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm of ubiquitin-40S ribosomal protein S27a (e.g., as measured by LCMS). In the disclosed pharmaceutical compositions comprising an anti-N3pG antibody, the composition may comprise less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm of kallikrein-11 (e.g., as measured by LCMS). In disclosed pharmaceutical compositions comprising an anti-N3pG antibody, the composition may comprise less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm of serine protease HTRA1 isoform X1 (e.g., as measured by LCMS). In disclosed pharmaceutical compositions comprising an anti-N3pG antibody, the composition may comprise less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm of complement C1r subcomponent (e.g., as measured by LCMS). In disclosed pharmaceutical compositions comprising an anti-N3pG antibody, the composition may comprise less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm of actin, aortic smooth muscle isoform X1 (e.g., as measured by LCMS).In the disclosed pharmaceutical compositions comprising an anti-N3pG antibody, the composition may contain less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm of actin, aortic smooth muscle isoform X1 (e.g., as measured by LCMS). In the disclosed pharmaceutical compositions comprising an anti-N3pG antibody, the composition may contain less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm of heat shock cognate 71 kDa protein (e.g., as measured by LCMS). In the disclosed pharmaceutical compositions comprising an anti-N3pG antibody, the composition may contain less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm of peroxiredoxin-1 (e.g., as measured by LCMS).

[0113] In the disclosed pharmaceutical compositions comprising an antibody, which can include an anti-N3pGlu antibody such as antibody 201c, the pharmaceutical composition can have a reduced total content of host cell proteins (HCPs). In some embodiments, the composition contains less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm of HCPs (as measured, for example, by LCMS). In some embodiments, the composition comprises less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm of an HCP selected from the following HCPs and combinations thereof: polyubiquitin, lysosomal protective protein, glutathione S-transferase Y1, 40S ribosomal protein S28, thioredoxin isoform X1, basement membrane-specific heparan sulfate proteoglycan core protein isoform X1, tubulointerstitial nephritis antigen-like protein, actin-partial cytoplasmic 2 isoform X2, galectin-1, peroxiredoxin-1, and cornifin alpha.

[0114] In the disclosed pharmaceutical compositions comprising an anti-N3pG antibody, the composition may comprise less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm of polyubiquitin (e.g., as measured by LCMS). In the disclosed pharmaceutical compositions comprising an anti-N3pG antibody, the composition may comprise less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm of lysosomal protective protein (e.g., as measured by LCMS). In the disclosed pharmaceutical compositions comprising an anti-N3pG antibody, the composition may comprise less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm of glutathione S-transferase Y1 (e.g., as measured by LCMS). In the disclosed pharmaceutical compositions comprising an anti-N3pG antibody, the composition may comprise less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm of glutathione S-transferase Y1 (e.g., as measured by LCMS). In the disclosed pharmaceutical compositions comprising an anti-N3pG antibody, the composition may comprise less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm of 40S ribosomal protein S28 (e.g., as measured by LCMS). In the disclosed pharmaceutical compositions comprising an anti-N3pG antibody, the composition may comprise less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm of thioredoxin isoform X1 (e.g., as measured by LCMS). In the disclosed pharmaceutical compositions comprising an anti-N3pG antibody, the composition may comprise less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm of basement membrane-specific heparan sulfate proteoglycan core protein isoform X1 (e.g., as measured by LCMS).In the disclosed pharmaceutical compositions comprising an anti-N3pG antibody, the composition may comprise less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm of tubulointerstitial nephritis antigen-like protein (e.g., as measured by LCMS).In the disclosed pharmaceutical compositions comprising an anti-N3pG antibody, the composition may comprise less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm of actin-partial cytoplasmic 2 isoform X2 (e.g., as measured by LCMS). In the disclosed pharmaceutical compositions comprising an anti-N3pG antibody, the composition may comprise less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm of galectin-1 (e.g., as measured by LCMS). In the disclosed pharmaceutical compositions comprising an anti-N3pG antibody, the composition may comprise less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm of peroxiredoxin-1 (e.g., as measured by LCMS). In the disclosed pharmaceutical compositions comprising an anti-N3pG antibody, the composition may contain less than about 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, or 1 ppm of cornifin alpha (as measured, for example, by LCMS). [Example]

[0115] Measurement of host cell proteins (HCPs) by LCMS: To assess the impact of purification on host cell protein (HCP) levels in the following examples, samples are analyzed by peptide mapping / LC-MS / MS HCP profiling, e.g., by ultra-performance liquid chromatography (UPLC) coupled to a Thermo Scientific mass spectrometer. Methods for detecting HCPs have been disclosed in the art. (See, e.g., Huang et al., "A Novel Sample Preparation for Shotgun Proteomics Characterization of HCPs in Antibodies," Anal. Chem. 2017, 89, 5436-5444.) In this analysis, samples are subjected to trypsin digestion, reduction / precipitation with dithiothreitol (DTT), and then the supernatant is transferred and acidified into HPLC vials for LC-MS / MS analysis. LC-MS / MS data are analyzed by Proteome Discoverer against a CHO-K1 protein database supplemented with antibody, spike, and control protein sequences. HCP concentrations are reported as the sum of parts per million (ppm) of HCP per sample relative to the total HCP content (e.g., ng of HCP per mg of product). Additionally, concentrations of specific HCPs (e.g., phospholipase B-like 2 protein (PLBL2) and lysosomal protective protein) are also provided.

[0116] HCP Measurement by ELISA: HCP level concentrations in samples are also assessed in the following examples by ELISA assay using Gyrolab® CHO-HCP Kit 1 (Cygnus Technologies, performed according to the manufacturer's instructions). The resulting concentration of HCP is reported as the sum of parts per million (ppm) of HCP per sample relative to the total HCP content.

[0117] Example 1 - Reduction of HCPs in the mAb1 (etesevimab) purification process Protein Capture Step: Equilibrate a sterilized Protein A column (MabSelect SuRe Protein A media) and load the mAb1 (etesevimab) cell-free bioreactor harvest onto the Protein A column. Wash the Protein A column three times with 20 mM Tris (pH 7.0) as the final wash. Elute mAb1 from the column using 5 column volumes (CV) of 20 mM acetic acid + 5 mM phosphate. Peak cuts based on front and back absorbance were used to collect the major product fraction into a single bulk fraction.

[0118] Low-pH viral inactivation and neutralization step: The pH of the main product fraction containing mAb1 (protein capture eluate bulk fraction) is adjusted to a pH of 3.30-3.60 by adding 20 mM HCl for low-pH viral inactivation. The mixture is incubated at 18 °C-25 °C for approximately 180 min. The mixture is then neutralized to pH 7.0 using 250 mM Tris Base pH-unadjusted buffer.

[0119] Depth filtration step: The depth filter (X0SP, Millipore) is rinsed with water for injection (WFI). The mAb1 mixture obtained from the low pH viral inactivation and neutralization steps is filtered at 1200 g / m 2 (Depth filter membrane area 1m 2 A load of 1000 mAb (grams of mAb per 1000 mAb) is applied to a depth filter. The loaded depth filter is rinsed with WFI. The filtrate from the depth filter, optionally including the post-load WFI wash, is neutralized to pH 8.0 using 250 mM Tris-base pH unadjusted buffer.

[0120] Anion exchange (AEX) chromatography step: A sterilized column (Q Sepharose Fast Flow anion exchange chromatography medium, or QFF) is equilibrated with 2 CV of 20 mM Tris (pH 8.0). The mAb1 solution obtained from the depth filtration step is loaded onto the column at a load of 25–100 g per liter of resin, followed by an additional wash with equilibration buffer. mAb1 is recovered by peak cut based on the absorbance at the front and back of the peak area formed by the unbound fraction and additional wash.

[0121] Results: Using the described purification process, total HCP levels measured by LC-MS are as follows: Protein A elution: 23299 ppm ●After X0SP deep filtration 13ppm, • 2 ppm after AEX chromatography.

[0122] Evaluation of Depth Filter Set 1 for mAb1: mAb1 is processed through Protein A, low pH viral inactivation, neutralization, and depth filtration steps essentially as described above. Four different depth filters: Emphaze™ AEX Hybrid Purifier, Zeta Plus BC25-60ZB05A, Zeta Plus BC25-90ZB05A, and Zeta Plus BC25-90ZB08A (3M) are used at 2000 g / m as shown in Table 1. 2 The results in Table 1 show that the four depth filters tested showed a significant reduction in total HCP content after depth filtration by LCMS and / or ELISA when compared to the total HCP content observed after Protein A elution. [Table 1]

[0123] Example 2 - HCP Reduction in mAb2 (Bamlanivimab) Purification Process Comparison of Protein A Elution Buffers: mAb2 is prepared essentially as described for mAb1 in Example 1, with the following exceptions: 1) after low pH viral inactivation and before depth filtration, the solution is neutralized to a pH of 7.25 instead of 7.0 using 250 mM Tris Base pH-unadjusted buffer, 2) mAb2 is eluted from the Protein A capture column using the buffer combinations listed in Table 2, and 3) AEX chromatography is performed using Poros XQ resin. HCP content (both total HCP and PLBL2 levels) is assessed by LCMS after the purification unit operations listed in Tables 2 and 3. The results in Tables 2 and 3 show that after the depth filtration step, the total HCP and PLBL2 content is reduced for all three buffer combinations tested. Specifically, 20 mM acetate + 5 mM phosphate and 20 mM acetate + 5 mM L-lactate showed a significant reduction in total HCPs and PLBL2 to less than 20 ppm after depth filtration compared to the combination of 20 mM acetate + 5 mM citrate. [Table 2] [Table 3]

[0124] Evaluation of Depth Filter Set 2: mAb2 is prepared essentially as described for mAb1 with the following exceptions: 1) after low pH viral inactivation and prior to depth filtration, the pH of the solution is neutralized to a pH of 7.25 instead of 7.0 using 250 mM Tris Base pH unadjusted buffer, and 2) depth filtration is performed using the depth filters shown in Table 4. Table 4 shows the 1500 g / m 2 Figure 1 shows the total HCP and PLBL2 content after depth filtration using various depth filters at loadings of 10 ppm. All three sets of 2 depth filters tested (X0SP, C0SP, X0HC, (Millipore)) show a significant reduction in total HCP and PLBL2 content after depth filtration of less than 20 ppm. [Table 4]

[0125] Example 3. Reduction of HCPs in the mAb3 (bepterovimab) purification process mAb3 is 900g / m 2 The mAb1 was prepared using protein capture, low pH viral inactivation, neutralization, and depth filtration steps essentially as described for mAb1 in Example 1, except using an X0SP depth filter with a loading of 0.05%. Using the described purification process, the total HCP levels measured by LCMS were as follows: ● Protein A elution: 179964 ppm, ●77ppm after X0SP (Millipore) deep filtration.

[0126] Example 4. Reduction of HCPs in a bispecific antibody (mAb4) purification process Bispecific antibody mAb4 is prepared using a protein capture step essentially as described for mAb1 in Example 1, except that a Protein L affinity capture column (Cytiva) is used and eluted with the buffer system shown in Table 5. Total HCP content is measured by ELISA, yielding a range of about 1300 to about 2500 ppm. Protein capture is followed by low pH viral inactivation essentially as described for mAb1 in Example 1, except that the titrants listed in Table 5 are used, followed by neutralization to pH 7.0 using 500 mM Tris Base pH unadjusted buffer. X0SP depth filters are then filtered at 1200 g / m 2 and perform the depth filtration step as described for mAb1 in Example 1. 。 HCP content is measured after depth filtration by ELISA.

[0127] The results in Table 5 show that the total HCP content of entries 1-7 was significantly reduced to less than 50 ppm after depth filtration, even when the ionic strength of the mixture applied to the depth filter was less than 45 mM. Additionally, there is a correlation between the ionic strength of the mixture applied to the depth filter and the total HCP content after depth filtration. Entry 2 further demonstrates that diluting the buffer can reduce the ionic strength, resulting in a lower HCP content after depth filtration, but the increased volume due to dilution can be detrimental to the manufacturing process. [Table 5]

[0128] Example 5. Reduction of HCPs in the mAb5 (donanemab) purification process The mAb5 preparation is prepared using essentially the steps described below: protein capture, low pH viral inactivation and neutralization, depth filtration, anion exchange (AEX) chromatography, cation exchange (CEX) chromatography, viral filtration, and tangential flow filtration (TFF).

[0129] Protein capture step: The antibody is captured and purified by reducing process-related impurities such as residual HCP and residual DNA. A sterilized Protein A column (MabSelect Protein A media) is equilibrated, and the cell-free bioreactor harvest of a monoclonal antibody (mAb5 (donanemab) expressed from CHO cells) is loaded onto the Protein A column. The Protein A column is washed three times, with the final wash being 20 mM Tris, pH 7.0. The antibody is eluted from the column using 5 column volumes (CV) of 20 mM acetic acid + 5 mM citric acid. Peak cuts based on front and back absorbance are used to collect the major product fraction into a single bulk fraction.

[0130] Low pH viral inactivation and neutralization steps: This method inactivates low-pH-sensitive viruses and reduces residual HCP, protein A, DNA, and total aggregates. Viral inactivation is achieved by adjusting the pH of the collected primary product fraction (protein capture eluate bulk fraction) containing the mAb to 3.30-3.60 by adding 20 mM acetic acid and 5 mM citric acid. The mixture is incubated at 18°C-25°C for approximately 180 minutes. The mixture is then neutralized to a pH of 5-7.0, preferably 5.0, using 250 mM Tris Base pH-unadjusted buffer.

[0131] Depth filtration step: For each test condition (pH 5 with B1HC), a separate depth filter (B1HC, Millipore) is flushed with water for injection (WFI). The resulting mAb mixture from the low pH viral inactivation and neutralization steps is filtered at approximately 500-1500 g / m 2 (Depth filter membrane area 1m 2 A target load of mAb (grams of mAb per 1000 ml) is applied to a depth filter. The loaded depth filter is rinsed with WFI. The filtrate from the depth filter, optionally including a post-load WFI wash, is neutralized to pH 7.25 using 250 mM Tris base pH unadjusted buffer. A calculated volume of 20 mM Tris, 1 M NaCl, pH 7.0 buffer is added to achieve a final NaCl concentration of 50 mM.

[0132] Anion Exchange (AEX) Chromatography Step: To reduce potential viral contaminants, a sterilized Poros XQ (or Sartobind Q or Poros HQ) anion exchange (AEX) column is pre-equilibrated with 2 CV of 20 mM Tris, 1 M NaCl, pH 7.0 buffer, followed by 3 CV of equilibration buffer, 20 mM Tris, 50 mM NaCl, pH 7.25. The mAb solution from each depth filter condition is run through the AEX column in separate runs based on the depth filter condition. The mAb solution obtained from the depth filtration step is loaded onto the column at a load of approximately 100 g to 200 g per liter of resin (e.g., approximately 150 g per liter of resin), followed by an additional wash with equilibration buffer. The mAb is collected from the start of the load until the end of the wash.

[0133] Cation Exchange (CEX) Chromatography Steps: The pH of the different AEX intermediates was adjusted to approximately 7.25-5.0 by adding 0.1 N acetic acid, followed by loading onto equilibrated (20% mobile phase B or 20 mM sodium acetate, 200 mM sodium chloride, pH 5.0 equivalent) CEX chromatography resin (POROS™ HS or UNOsphere S). The pH 5.0 AEX process intermediate was mixed with 15% mobile phase B (193 mM sodium chloride equivalent) prior to loading onto the CEX column. The column load was approximately 25 grams of mAb per liter of resin. After loading, the column was washed with 20% mobile phase B (20 mM sodium acetate, 200 mM sodium chloride, pH 5.0 equivalent) to facilitate removal of unbound impurities. The mAb is then eluted from the column using a linear gradient of 20% to 55% mobile phase B over 10 column volumes (200 to 550 mM sodium chloride gradient in 20 mM sodium acetate, pH 5.0 buffer). To ensure complete elution of the product, an isocratic hold at 55% mobile phase B (equivalent to 20 mM sodium acetate, 550 mM sodium chloride, pH 5.0) can be performed after the linear gradient. During elution, a frontal UV-based cut at NLT 4.8 AU / cm initiates collection of the CEX eluate, followed by a peak apex until a backside cut is performed at NLT 2.4 AU / cm. The column is regenerated and sterilized with 1 N sodium hydroxide solution. The column can be stored in 0.01 N sodium hydroxide. The preparation is then analyzed for HCP content using LCMS.

[0134] Viral Filtration: Remove potential viral contaminants. Viral filtration is performed through Viresolve Pro, Planova 20N, or Planova BioEX membranes.

[0135] Tangential Flow Filtration (TFF): The viral filtrate process intermediate is exchanged into an appropriate matrix for preparation of the final drug substance (DS), and the antibody is concentrated to the appropriate range for preparation of the final DS. TFF is performed on a 30 kDa PES or 30 kDa regenerated cellulose membrane.

[0136] Drug Substance Preparation: After TFF, surfactants are added to complete the drug substance formulation, which is then dispensed into approved container closure systems for storage and transportation at the appropriate temperatures prior to drug manufacturing.

[0137] Measurement of HCP content by LC-MS HCP content was measured by LC-MS as described below. For mAb5 batch 1 and mAb5 batch 2, HCP content was measured after the protein capture step, after low pH viral inactivation, after AEX, and after CEX. For mAb5 batches 3-5, HCP content was measured before drug substance formulation. The results are shown in Tables 6a and 6b and Table 7 below.

[0138] Sample preparation Aliquots containing approximately 1 mg of protein from each sample or control were added to 193 mL with purified water. The solutions were mixed with 5.0 mL of 1 M Tris-HCl buffer, pH 8, and a 1.0 mL aliquot of each of the four protein mixtures, then treated with 1 mL of 2.5 mg / mL r-trypsin at 37°C overnight. Each digest was mixed with 2.0 mL of 50 mg / mL DTT solution and heated at 90°C for 15 minutes. A precipitate was observed. The samples were vortexed vigorously for 2 × 30 seconds. Each sample was centrifuged at 13,200 rpm for 3 minutes, and 120 mL of the supernatant was transferred to an HPLC vial. The samples in the HPLC vials were then mixed with 5.0 μL of 20% TFA in HO for LC / MS analysis.

[0139] LC / MS / MS method The prepared tryptic peptides were analyzed using UPLC-MS / MS. Samples were injected in a volume of 50 μL directly onto a Waters Acquity UPLC CSH C18 (Milford, MA, USA) (2.1 × 50 mm, 1.7 μm particle size). The column was heated to 60 °C during analysis. Separation was performed on a Waters Acquity UPLC system using mobile phase A consisting of 0.1% formic acid in water and mobile phase B consisting of 0.1% formic acid in acetonitrile equilibrated with 0% mobile phase B at a flow rate of 200 μL / min for 2 min, followed by a linear increase from 0% to 10% B over 23 min, to 20% B over 57 min, and to 30% B over 30 min at a flow rate of 50 μL / min, followed by multiple zigzag wash cycles at a flow rate of 400 μL / min. Mass spectrometry was performed on a Thermo Scientific Q Exactive Plus mass spectrometer (Bremen, Germany). Data-dependent MS / MS was performed as follows: The first event was a survey positive mass scan (m / z range 230–1500), followed by 10 HCD events (28% NCE) for the 10 most abundant ions from the first event. Ions were generated using a sheath gas flow rate of 15, an auxiliary gas flow rate of 5, a spray voltage of 4 kV, a capillary temperature of 320 °C, and an S-lens RF level of 50. Resolution was set to 35,000 (AGC target of 5E6) and 17,500 (AGC target of 5E4) for the survey scan and MS / MS event, respectively. The maximum ion injection time was 250 ms for the survey scan and 300 ms for other scans. A dynamic exclusion time of 60 s was used with a single repetition rate.

[0140] HCP identification and quantification A customized protein database, consisting of sequences obtained from the CHO-K1_refseq_2014 Protein.fasta database (downloaded from http: / / www.chogenome.org on August 23, 2014), was developed to predict HCP identities from MS / MS data. MS / MS data were searched with a mass tolerance of 10 ppm and 0.02 Da, and a stringent false discovery rate (FDR) of 1% or less was achieved against this database using the Proteome Discoverer software package, version 1.4 or 2.3 (Thermo Scientific, Bremen, Germany) with Sequest HT search. Further peptide / protein filtering was performed by excluding proteins with a score of 0 and a single spectral hit, or proteins with a single spectral hit and a mass of 10 ppm or more, and contaminating human proteins. Protein areas from the top three peptides (if available) for each HCP and areas for the three spiked proteins, r-trypsin, PCSK9, and ADH1, were used to calculate individual HCP concentrations (ppm or ng HCP / mg mAb). [Table 6-1] [Table 6-2] [Table 7] [Table 8]

[0141] Example 6. Reduction of HCPs in mAb7 (U.S. Patent No. 10,647,759 Antibody 201c") Purification Process A mAb7 (antibody 201c" of U.S. Pat. No. 10,647,759) (LC is SEQ ID NO: 25, HC is SEQ ID NO: 26) preparation is prepared using essentially the steps described above for mAb5, with the following minor differences. Protein Capture: Protein A column: MabSelect SuRe Load: 20~40g / L Elution: 20mM acetic acid / 5mM citric acid Low pH viral inactivation and neutralization: Titrant: 20 mM acetic acid / 5 mM citric acid, pH 3.45 Duration: 180 minutes Neutralization: pH 5.0, 500 mM Tris base AEX chromatography: Resin: POROS 50 XQ, Load: 100~200g / L load pH: 7.0 CEX chromatography: Resin: POROS 50 HS Load: 20~40g / L

[0142] HCP content was measured by LC-MS as described in Example 5. For mAb7 Batch 1 and mAb7 Batch 2, HCP content was measured after the protein capture step, after low pH viral inactivation, after AEX, after CEX, and after TFF. The results are shown in Tables 8a and 8b. [Table 9] [Table 10]

[0143] Example 7. Effect of Depth Filter Type and pH on HCP Reduction During Depth Filtration—mAb5 (Donanemab) and mAb6 Part A - Effect of pH on HCP reduction Two antibodies (mAb5 and mAb6) are prepared using a protein capture step essentially as described for mAb1 in Example 1, except that the elution step is performed in the buffer system shown in Table 9. Total HCP content is measured by ELISA, yielding a range of about 2800 to about 3200 ppm. Protein capture is followed by a low pH viral inactivation step essentially as described for mAb1 in Example 1, followed by a neutralization step at either pH 5.0 or pH 7.0 using 500 mM Tris-base pH unadjusted buffer. X0SP depth filters are loaded at 1000 g / m 2 and perform the depth filtration step as described for mAb1 in Example 1. 。 The HCP content after the depth filtration step is measured by ELISA.

[0144] The results in Table 9 show that the total HCP content for both antibodies was significantly reduced to less than 50 ppm after depth filtration when the pH of the mixture applied to the depth filter was pH 7.0. The total HCP content was not significantly reduced when the pH of the mixture applied to the depth filter was pH 5.0. [Table 11]

[0145] Part B: Effect of depth filter and pH on HCP reduction for mAb5 mAb5 is prepared using a protein capture step essentially as described in Example 5. The eluate is subjected to low pH viral inactivation and neutralization essentially as described in Example 5. For the depth filtration step, four different pH and depth filter settings were evaluated. (i) B1HC filter + pH 5.1 (ii) X0SP filter + pH 5.1 (iii) X0SP filter + pH 6.2 (iv) X0SP filter + pH 7.3

[0146] (i) B1HC filter + pH 5.1 mAb5 is prepared using a protein capture step essentially as described in Example 5. 500 ml is placed in a glass beaker and mixed with a Teflon stir bar. The protein concentration of the Protein A eluate is 12.5 mg / ml. Once the beaker is filled with 500 ml, the total protein content is 6250 mg (12.5 mg / ml x 500 ml = 6250 mg).

[0147] The starting pH of the solution in the beaker is 3.98 (temperature = 18.1 C). The pH is adjusted to 3.45 with 20 mM acetic acid / 5 mM citric acid and a low pH viral inactivation step is performed essentially as described in Example 5.

[0148] While the low pH viral inactivation step is in progress, B1HC filters (Micropod or 23 sq cm, lot CP7NA77798, part MB1HC23CL3) are set up using size 14 platinum-cured silicone tubing with a PendoTech Filter Screening Peristaltic Pump System (K434694) along with an OHAUS Scout scale (K434696 to K434699). All filters are flushed at 230 ml per filter, or 100 L / sqm, with a PWTR of 23 ml / min (approximately 600 LMH).

[0149] Neutralization to pH 5.0 is achieved with 0.25M Tris base (EL19562-368, LB213, EXP4 / 15 / 2020). The solution becomes cloudy as the pH reaches 5, and the final pH is measured as 5.09 (5.1). The concentration is calculated to be 7.27 mg / ml (6250 mg / 860 ml at pH 5). While stirring the pH 5 solution, filtration through the B1HC filter begins at a loading of 997 g / sqm (309 ml x 7.27 mg / ml = 2.246 g / 0.0023 sqm = 997 g / sqm). The B1HC filter is subjected to a recovery flush with a 45 ml PWTR. The filter is essentially pumped dry after the recovery flush. The final volume of B1HC is 375.5 ml at 5.13 mg / ml, resulting in an 85.8% yield of 1.926 g.

[0150] (ii) X0SP filter + pH 5.1, pH 6.3, or pH 7.2 mAb5 is prepared using a protein capture step essentially as described in Example 5. 500 ml is placed in a glass beaker and mixed with a Teflon stir bar. The protein concentration of the Protein A eluate is 15.75 mg / ml. Once the beaker is filled with 500 ml, the total protein content is 7875 mg (15.75 mg / ml x 500 ml = 7875 mg).

[0151] The starting pH of the solution in the beaker is 4.05 (temperature = 18.1 C). The pH is adjusted to 3.45 with 20 mM acetic acid / 5 mM citric acid and a low pH viral inactivation step is performed essentially as described in Example 5.

[0152] While the low pH viral inactivation step is in progress, three X0SP filters (micropod or 23 sq cm, lot CP9AA93251, cat MX0SP23CL3) are set up and rinsed separately as above.

[0153] Neutralization achieved using 0.25M Tris base I (EL19562-368, LB213, EXP 4 / 15 / 2020):

[0154] The first beaker was adjusted to pH 5.1 with 20 ml of 250 mM Tris base. The calculated concentration is 9.04 mg / ml.

[0155] The second beaker was adjusted to pH 6.3 with 27 ml of 250 mM Tris base. The calculated concentration is 8.82 mg / ml.

[0156] A third beaker was adjusted to pH 7.2 with 32 ml of 250 mM Tris base. The calculated concentration is 8.67 mg / ml.

[0157] The precipitate at pH 6.3 and 7.2 appears slimy (as it adheres to the bottom of the glass towards the end of the filtration) and may be larger in size than at pH 5.1.

[0158] While stirring the three solutions, begin filtering through the X0SP filter.

[0159] The pH 5.0 X0SP reached 25 psi with a load of 203 ml and then switched to water recovery flush. The load is calculated as 798 g / sq m (9.04 mg / ml x 203 ml = 1.835 g / 0.0023 sq m = 798 g / sq m).

[0160] The filter is reclaim flushed with approximately 45 ml of PWTR. The filter is essentially pumped dry after the reclaim flush.

[0161] Final volume of X0SP pH 5.1 = 278 ml at 5.89 mg / ml = 1.637 g Yield = 1.637 g / 1.835 = 89.2%

[0162] Final volume of X0SP pH 6.3 = 365 ml at 5.76 mg / ml = 1 g yield = 2.102 g / 2.58 = 81.5%

[0163] Final volume of X0SP pH 7.2 = 365 ml at 5.52 mg / ml = 2.015 g / 2.58 = 78.1%

[0164] (iii) AEX chromatography Each depth filtration preparation was subjected to AEX essentially as described in Example 5. For all AEX load preparations, the pH 5 filtrate and the pH 6 filtrate (but not the 7.2 filtrate) were adjusted to pH 7.25 with 250 mM Tris base (Lot EL19562-368, LB213, exp 4-15-20, for development use), and then NaCl was added to a final concentration of 50 mM using 20 mM Tris, 1 M NaCl, pH 7.0 (EL19562-862 LB198, exp 9-30-2020) at 0.0526 x volume at pH 7.25. All load preparations were performed in glass beakers with stir bars. 600 mg of each filtrate was used to load the same amount of AEX. The pH of all AEX loads was 7.1-7.3, and all conductivities were 6.5 + / - 0.2 mS.

[0165] The final AEX MS (at pH 5) volume, mAb5 concentration, total mg, and yield were as follows: 1. B1HC material - 3.91 mg / ml in 155 ml = 606.1 mg or 101% 2. pH 5.1 X0SP - 5.00mg / ml, 120ml = 600mg or 100% 3. X0SP at pH 6.3 - 4.96 mg / ml, 121 ml = 600.2 mg or 100% 4. X0SP at pH 7.2 - 4.79 mg / ml, 126 ml = 603.5 mg or 100.6%

[0166] (iii) CEX chromatography Each of the AEX preparations is subjected to CEX chromatography essentially as described in Example 5. The actual loading on the CEX resin is as follows: (i) 3.91 mg / ml x 130 ml loading volume x 0.85 = 110.5 ml = 432.1 / 17.28 = 25.0 mg / ml of B1HC preparation (ii) 5.00 mg / ml Loading Volume 101.7 ml × 0.85% = 86.4 ml = 432 / 17.28 = 25.0 mg / ml X0SP at pH 5 (iii) 4.96 mg / ml Loading volume = 102.5 × 0.85 = 87.1 ml = 432.0 / 17.28 ml = 25.0 mg / ml X0SP at pH 6.3 (iv) 4.79 mg / ml Loading volume = 106.1 × 0.85 = 90.2 ml = 432.1 / 17.28 = 25.0 mg / ml X0SP at pH 7.2

[0167] The CEX mainstream volume, concentration, and yield for each condition are as follows: (i) 5.83 mg / ml x MS volume = 64.1 ml MS volume = 373 mg / 432.1 mg = 86.3% B1HC at pH 5.0 (ii) 5.83 mg / ml x 64.8 ml MS volume = 377.8 mg / 432 mg = 87.5% X0SP at pH 5 (iii) 5.80 mg / ml = 64.8 ml MS volume = 375.8 mg / 432.0 mg = 87.0% X0SP at pH 6.3 (iv) 5.80 mg / ml = 64.7 ml MS volume = 375.3 mg / 432.1 mg = 86.9% X0SP at pH 7.2

[0168] (v) Analysis of HCP content by LC-MS The CEX preparations are analyzed for HCP content using LC-MS essentially as described in Example 5. The LC-MS data are shown in Table 10. [Table 12]

[0169] The data in Table 10 show that total HCP content was significantly reduced to less than 50 ppm after depth filtration using the X0SP filter at all pH levels tested. This compares favorably with the reduction in HCP content after depth filtration using the B1HC filter. It is also noteworthy that the yield after the depth filtration step was lower at pH 6.3 and 7.2 compared to the lower pH of 5.1. Thus, the reduction in HCP content at higher pH levels may be offset by a loss in yield. Optimal performance was observed with the X0SP filter at pH 5.0.

[0170] Example 8. Method for determining ionic strength during a biomolecule purification process Here we describe a method to estimate ionic strength based on known buffer composition during a biomolecule purification unit process. The ionic strength (I) of a solution is a measure of the concentration of ions in that solution, and for all species, the species concentration c i and net charge z i To determine the ionic strength, Equation I is used:

number

[0171] Strong electrolytes: For strong electrolytes at low concentrations (e.g., less than 50 mM), complete dissociation is assumed. Complete dissociation allows for easy calculation of the composition and ionic strength calculations. For example, if a solution of 50 mM NaCl dissociates, the ionic strength will be 0.5 × [50 mM × 1 2 +50mM×(-1) 2 ] = 50 mM Na + and Cl - As another example, the dissociation of 50 mM Na2SO4 results in 0.5 × [100 mM × 1 2 +50mM×(-2) 2 ] = 150 mM, taking into account the ionic strength of 100 mM Na + and 50 mM SO4 2-In the absence of buffer species, these calculations predict a near-neutral pH, so the ion concentration due to water dissociation does not contribute significantly to the ionic strength. The dissociation constant of water is [H + ]=10 -pH DeK w =[H + ][OH - ]=10 -14 (The brackets indicate the concentration.) For the purposes of this calculation, H (as opposed to, for example, the hydronium ion) + The physical interpretation of the ions is not necessary; similarly, H + There is no need to distinguish between concentration and activity.

[0172] Buffer Systems: In buffer systems, complete dissociation cannot be assumed. The acid dissociation constant of the buffer must be used to determine the proportion of the acid and base forms of the buffer. + and A - For HA, a common acid that dissociates into a and related to species concentration.

number

[0173] Acid dissociation constants are often expressed as pK a =-log 10 (K a ) is used in the logarithmic form of pK a,0 Thermodynamic pK, denoted as a is available in the literature for many buffers of interest. However, the effective pK of a buffer is often inaccurate, except for very dilute solutions, as activity coefficients deviate from 1. a deviates from the thermodynamic value. For the moderately dilute solutions considered in this disclosure, the extended Debye-Huckel equation or Davis equation was used to account for activity coefficients that are not unity. Although values ​​of some of the constants found in the literature may vary slightly, similar results are obtained over the range of ionic strength values ​​of interest in this disclosure. The extended Debye-Huckel equation is provided as Equation 3:

number

[0174] The Davis equation is provided as Equation 4:

number

[0175] pK a Since is a function of ionic strength, the composition and ionic strength cannot be determined independently, but are part of a system of equations. The system of equations includes the aforementioned ionic strength, the acid dissociation constant of each buffer, and the pK of each buffer. a The equations include the electroneutrality condition and total species balance for each buffer solution. This system of equations can be used to estimate several values. For example, the pH of a known solution can be used to estimate the acid / base ratio of a buffer formulation, and conversely, the acid / base ratio can be used to estimate the pH of a solution and the corresponding titer. In any of these applications, ionic strength can be estimated to help rationally select eluent and titrant options.

[0176] To calculate the ionic strength associated with the buffer system of the present disclosure, for example, the ionic strength of a feed material for depth filtration, the buffer composition of the solution is required. This composition can be reasonably estimated based on the amount and composition of the buffer and titrant used in the process. Ion measurement techniques known in the art can also be used to estimate the composition.

[0177] As a starting point for estimating the solution composition, one possible approach is to assume that the affinity column eluate pool has the same buffer composition as the eluent, except that it is buffered at the measured pH of the eluate pool. For example, if a protein of interest is eluted from a Protein A column with 20 mM acetate, 5 mM lactate, and the eluate pool has a measured pH of 4.2, the buffer composition of the eluate pool is assumed to be 20 mM acetate, 5 mM lactate, and enough NaOH to bring the pH to 4.2 (this corresponds to approximately 8.2 mM NaOH). Sodium cations, Na + Since only the total content is important for the calculation, it does not matter whether the sodium content of the eluate is assumed to come from sodium acetate, sodium phosphate, sodium hydroxide, or a combination thereof, and for convenience the convention of attributing sodium to NaOH is used.

[0178] The eluent composition and eluate pH are used to estimate the eluate buffer composition, and then titration of the solution is considered. For example, for an estimated eluate composition of 20 mM acetate, 5 mM lactate, and approximately 8.2 mM NaOH at pH 4.2, if the volume of 20 mM HCl required to lower the pH to a target value of 3.45 for viral inactivation is equal to 0.305 times the starting volume, then the composition of that process intermediate at pH 3.45 will be known from dilution. Acetate, lactate, and NaOH will be present in the titrant at 1 / 1.305 times their respective initial values ​​(i.e., approximately 15.3 mM acetate, approximately 3.8 mM lactate, and approximately 6.2 mM NaOH), and HCl will be present at 0.305 / 1.305 of its value (approximately 4.7 mM HCl). Similarly, for neutralization with 250 mM Tris base, if the ratio to raise the pH to the target pH of 7.0 is 0.0743 times the volume of the pH 3.45 solution, apply the ratios 1 / 1.0743 and 0.0743 / 1.0743 to determine the final concentrations of the neutralization solution (approximately 14.3 mM acetate, approximately 3.6 mM lactate, approximately 5.8 mM NaOH, approximately 4.4 mM HCl, approximately 17.3 mM Tris). All known values ​​are substituted into the system of equations (Equations 5-15) to calculate the ionic strength.

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[0179] As described herein, the buffering capacity of protein products is not directly modeled. Therefore, when using strong acids or bases in titrations, some deviations may occur between calculated and empirical titration results. For example, when titrating Protein A eluate to a low pH for viral inactivation, buffer calculations typically underestimate the empirical amount of 20 mM HCl required. The empirical amount required may be as much as 50% higher than the calculated estimate. One way to account for this difference is to model the affinity column eluate at a higher pH and then empirically adjust the value until the estimated titer matches the experimental value. For example, in the example above, if the amount of 20 mM HCl was 50% higher than the initially estimated ratio of 0.305, the Protein A eluate would be modeled as approximately pH 4.45 rather than pH 4.2. Making this empirical change to the modeling reduces the estimated ionic strength in this example in one direction, but only slightly: from the initial estimate of 22.1 mM to 21.9 mM. Therefore, it is concluded that either approach is sufficient to estimate ionic strength for guiding preferred embodiments of the present disclosure.

[0180] Alternative: Ion content measurement methods can be used to determine the buffer composition of depth filtration feed materials and calculate ionic strength. This requires verifying that measurements yield consistent results with any known quantities, such as the amount of titrant added. The buffer composition of the affinity column eluate is assumed to be equivalent to that of the eluent, but at different pHs, so the actual compositional differences can be determined by ion content measurements. For example, either quantities based on the eluent composition or measurements can be used to calculate the ionic strength of the buffer components in the eluent.

[0181] Incorporation by Reference All patents and publications referenced herein are incorporated by reference in their entirety. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[0182] array The following nucleic acid and / or amino acid sequences are referred to in this disclosure and are provided below for reference. SEQ ID NO: 1 - Bamlanivimab variable heavy chain (VH) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGYYYEARHYYYYYAMDVWGQGTAVTVSS SEQ ID NO:2 - Bamlanivimab variable light chain (VL) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLSWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTITSLQPEDFATYYCQQSYSTPRTFGQGTKVEIK SEQ ID NO:3 - Bamlanivimab heavy chain (HC) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGYYEARHYYYYYAMD VWGQGTAVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 4 - Bamlanivimab light chain (LC) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLSWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTITSLQPEDFATYYCQQSYSTPRTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 5 - Etesevimab variable heavy chain (VH) EVQLVESGGGLVQPGGSLRLSCAASGFTVSSNYMSWVRQAPGKGLEWVSVIYSGGSTFYADSVKGRFTISRDNSMNTLFLQMNSLRAEDTAVYYCARVLPMYGDYLDYWGQGTLVTVSS SEQ ID NO: 6 - Etesevimab variable light chain (VL) DIVMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPEYTFGQGTKLEIKRTV SEQ ID NO:7 - Etesevimab heavy chain (HC) EVQLVESGGGLVQPGGSLRLSCAASGFTVSSNYMSWVRQAPGKGLEWVSVIYSGGSTFYADSVKGRFTISRDNSMNTLFLQMNSLRAEDTAVYYCARVLPMYGDYLDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:8 - Etesevimab light chain (LC) DIVMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPEYTFGQGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 9 - Bebterovimab variable heavy chain (VH) QITLKESGPTLVKPTQTLTLTCTFSGFSLSISGVGVGWLRQPPGKALEWLALIYWDDDKRYSPSLKSRLTISKDTSKNQVVLKMTNIDPVDTATYYCAHHSISTIFDHWGQGTLVTVSS SEQ ID NO: 10 - Bebterovimab variable light chain (VL) QSALTQPASVSGSPGQSITISCTATSSDVGDYNYVSWYQQHPGKAPKLMIFEVSDRPSGISNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTTSSAVFGGGTKLTVL SEQ ID NO: 11 - Bebterovimab heavy chain (HC) QITLKESGPTLVKPTQTLTLTCTFSGFSLSISGVGVGWLRQPPGKALEWLALIYWDDDKRYSPSLKSRLTISKDTSKNQVVLKMTNIDPVDTATYYCAHHSISTIFDHWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 12 - Bebterovimab light chain (LC) QSALTQPASVSGSPGQSITISCTATSSDVGDYNYVSWYQQHPGKAPKLMIFEVSDRPSGISNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTTSSAVFGGGTKLT VLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO: 13 - LCVR of donanemab DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSRGKTYLNWLLQKPGQSPQLLIYAVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHYPFTFGQGTKLEIK SEQ ID NO: 14 - HCVR of donanemab QVQLVQSGAEVKKPGSSVKVSCKASGYDFTRYYINWVRQAPGQGLEWMGWINPGSGNTKYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCAREGITVYWGQGTTVTVSS SEQ ID NO: 15 - LC of donanemab DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSRGKTYLNWLLQKPGQSPQLLIYAVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHYPFTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 16 - HC of donanemab QVQLVQSGAEVKKPGSSVKVSCKASGYDFTRYYINWVRQAPGQGLEWMGWINPGSGNTKYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCAREGITVYWGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 17 - LCDR1 of donanemab KSSQSLLYSRGKTYLN SEQ ID NO: 18 - LCDR2 of donanemab AVSKLDS SEQ ID NO: 19 - LCDR3 of donanemab VQGTHYPFT SEQ ID NO: 20 - HCDR1 of donanemab GYDFTRYYIN SEQ ID NO: 21 - HCDR2 of donanemab WINPGSGNTKYNEKFKG SEQ ID NO: 22 - HCDR3 of donanemab EGITVY SEQ ID NO: 23 - LCVR of antibody 201c (mAb7) DIQMTQSPSTLSASVGDRVTITCRASQSLGNWLAWYQQKPGKAPKLLIYQASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYKGSFWTFGQGTKVEIK SEQ ID NO: 24 - HCVR of antibody 201c (mAb7) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGSGSYYNGFDYWGQGTLVTVSS SEQ ID NO: 25 - LC of antibody 201c (mAb7) DIQMTQSPSTLSASVGDRVTITCRASQSLGNWLAWYQQKPGKAPKLLIYQASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYKGSFWTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 26 - HC of antibody 201c (mAb7) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGSGSYYNGFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 27 - LCDR1 of antibody 201c (mAb7) RASQSLGNWLA SEQ ID NO: 28 - LCDR2 of antibody 201c (mAb7) YQASTLES SEQ ID NO: 29 - LCDR3 of antibody 201c (mAb7) QHYKGSFWT SEQ ID NO: 30 - HCDR1 of antibody 201c (mAb7) AASGFTFSSYPMS SEQ ID NO: 31 - HCDR2 of antibody 201c (mAb7) AISGSGGSTYYADSVKG SEQ ID NO: 32 - HCDR3 of antibody 201c (mAb7) AREGGSGSYYNGFDY SEQ ID NO: 33 - Donanemab LC DNA sequence gatattgtgatgactcagactccactctccctgtccgtcacccctggacagccggcctccatctcctgcaagtcaagtcagagcctcttatatagtcgcggaaaaacctatttgaattggctcctgcagaagccaggccaatctccacagctcctaatttatgc ggtgtctaaactggactctggggtcccagacagattcagcggcagtgggtcaggcacagatttcacactgaaaatcagcagggtggaggccgaagatgttggggtttattactgcgtgcaaggtacacattacccattcacgtttggccaagggaccaagctgg agatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcc caggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgc SEQ ID NO: 34 - Donanemab HC DNA sequence LC DNA sequence of Array No. 35 - Antibody 201c gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgccgggccagtcagagtcttggtaactggttggcctggtatcagcagaaaccagggaaagcccctaaactcctgatctatcaggcgtctactttagaatctggggtcccatcaagattcagcggcagtggatctgggacagagttcactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaacattataaaggttctttttggacgttcggccaagggaccaaggtggaaatcaaacggaccgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgc HC DNA sequence of Array No. 36 - Antibody 201c

Claims

1. 1. A method for reducing host cell protein content in a protein preparation comprising an anti-N3pGlu Aβ antibody recombinantly produced in a mammalian host cell, the method comprising: a. subjecting the protein preparation to an affinity chromatography column; b. eluting the anti-N3pGlu Aβ antibody from the chromatography column using a combination of acids, including a weak acid and a strong acid, to obtain an eluate containing the anti-N3pGlu Aβ antibody; c. increasing the pH of the eluate to above about pH 5.0; and d. subjecting the eluate to a depth filter to obtain a filtered protein preparation.

2. 2. The method of claim 1, wherein the chromatography column comprises a Protein A, Protein G, or Protein L affinity chromatography column.

3. 10. The method of claim 1, wherein the weak acid and the strong acid are monobasic acids up to about pH 7.

3.

4. 2. The method of claim 1, wherein the weak acid is acetic acid and the strong acid is phosphoric acid or lactic acid.

5. 5. The method of claim 4, wherein the concentration of the acetic acid is about 20 mM, the strong acid is phosphoric acid, and the concentration of the phosphoric acid is about 5 mM to about 10 mM.

6. 5. The method of claim 4, wherein the concentration of the acetic acid is about 20 mM, the strong acid is lactic acid, and the concentration of the lactic acid is about 5 mM.

7. The method of claim 1 further comprising the step of performing viral inactivation.

8. 10. The method of claim 1, further comprising a step of performing viral inactivation comprising adjusting the pH of the eluate from the step of eluting protein from the chromatography column to less than about pH 4.0, wherein the eluate is maintained at less than about pH 4.0 for from about 0 minutes to about 180 minutes.

9. 9. The method of claim 8, wherein the step of adjusting the pH of the eluate comprises adjusting the pH of the eluate to about pH 3.3 to about pH 3.

7.

10. 10. The method of claim 9, wherein the pH of the eluate is adjusted to about pH 3.

5.

11. 11. The method of any one of claims 8 to 10, wherein adjusting the pH of the eluate comprises adding any one of HCl, phosphoric acid, or a combination of acetic acid and phosphoric acid.

12. 10. The method of claim 1, wherein the step of increasing the pH of the eluate comprises increasing the pH to about pH 6.5 to about pH 7.

5.

13. 13. The method of claim 12, wherein the pH of the eluate is increased to about pH 7.

0.

14. 14. The method of claim 12 or 13, wherein the step of increasing the pH of the eluate comprises adding Tris.

15. 15. The method of any one of claims 1 to 14, wherein the eluate in the step of increasing the pH to above about 5.0 has an ionic strength of about 10 mM to about 45 mM.

16. 16. The method of any one of claims 1 to 15, further comprising subjecting the depth-filtered protein preparation to one or more of the following purification and / or polishing steps: viral inactivation, ion exchange chromatography, viral filtration, tangential flow filtration to obtain a bulk drug preparation comprising an anti-N3pGlu Aβ antibody.

17. 17. The method of any one of claims 1 to 16, wherein the depth filter is a cellulose / diatomaceous earth based filter.

18. 18. The method of claim 17, wherein the depth filter is a B1HC filter, an X0HC filter, or a Zeta Plus (ZB Media) filter.

19. The method of any one of claims 1 to 16, wherein the depth filter is a synthesis filter.

20. 20. The method of claim 19, wherein the depth filter is a C0SP filter, an X0SP filter, or an Emphaze AEX Hybrid Purifier filter.

21. 21. The method of claim 20, wherein the depth filter is an XOSP filter.

22. 22. The method of any one of claims 17 to 21, wherein the depth filter has a pore size of at least about 9μ to about 0.1μ.

23. 23. The method of claim 22, wherein the depth filter has a pore size of at least about 2 microns to about 0.1 microns.

24. 24. The method of claim 23, wherein the depth filter has a pore size of about 0.1 μm.

25. 25. The method of any one of claims 1 to 24, wherein the pH of the eluate on the depth filter is about 5.

0.

26. 25. The method of any one of claims 1 to 24, wherein the pH of the eluate on the depth filter is about 6.

0.

27. 25. The method of any one of claims 1 to 24, wherein the pH of the eluate on the depth filter is about 7.

0.

28. The method of any one of claims 1 to 27, wherein the mammalian cells are CHO cells.

29. 29. The method of any one of claims 1 to 28, wherein the protein preparation comprises a harvested cell culture medium, a capture pool, or a recovered protein pool.

30. The method of any one of claims 1 to 29, wherein the anti-N3pGlu Aβ antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a bispecific antibody, or an antibody fragment.

31. The method of claim 30, wherein the anti-N3pGlu Aβ antibody is an IgG1 antibody.

32. The anti-N3pGlu Aβ antibody comprises a heavy chain (HC) and a light chain (LC), the light chain comprises a light chain variable region (LCVR), the heavy chain comprises a heavy chain variable region (HCVR), the LCVR comprises amino acid sequences LCDR1, LCDR2, and LCDR3, the HCVR comprises amino acid sequences HCDR1, HCDR2, and HCDR3, and LCDR1 comprises the amino acid sequence KSSQSLLYSRGKTYLN (SEQ ID NO: 1). 7), LCDR2 is AVSKLDS (SEQ ID NO: 18), LCDR3 is VQGTHYPFT (SEQ ID NO: 19), HCDR1 is GYDFTRYYIN (SEQ ID NO: 20), HCDR2 is WINPGSGNTKYNEKFKG (SEQ ID NO: 21), and HCDR3 is EGITVY (SEQ ID NO: 22).

33. the LC of the anti-N3pGlu Aβ antibody comprises an LCVR, the HC of the anti-N3pGlu Aβ antibody comprises an HCVR, and the LCVR is DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSRGKTYLNWLLQKPGQSPQLLIYAVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHYPFTFGQGTKLEIK (SEQ ID NO: 13); 33. The method of claim 32, wherein the HCVR is QVQLVQSGAEVKKPGSSVKVSCKASGYDFTRYYINWVRQAPGQGLEWMGWINPGSGNTKYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCAREGITVYWGQGTTVTVSS (SEQ ID NO: 14).

34. the LC of the anti-N3pGlu Aβ antibody is DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSRGKTYLNWLLQKPGQSPQLLIYAVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 15); The HC of the Aβ antibody is 34. The method of claim 32 or 33, wherein the amino acid sequence of ...

35. 35. The method of claim 34, wherein the anti-N3pGlu Aβ antibody is donanemab.

36. 36. The method of any one of claims 32 to 35, wherein the host cell protein content in the filtered protein preparation is less than 100 ppm (as measured by LCMS).

37. 37. The method of any one of claims 32-36, wherein the filtered protein preparation comprises one, a combination, or all of the following host cell proteins: protein S100-A6, protein S100-A11, phospholipase B-like 2 protein, lysosomal protective protein, ubiquitin-40S ribosomal protein S27a, kallikrein-11, serine protease HTRA1 isoform XI, complement C1r small component, actin, aortic smooth muscle isoform XI, heat shock cognate 71 kDa protein, and peroxiredoxin-1.

38. 38. The method of claim 37, wherein the filtered protein preparation contains less than about 5 ppm of protein S100-A6 (as measured by LCMS).

39. 39. The method of claim 37 or 38, wherein the filtered protein preparation contains less than about 5 ppm of protein S100-A11 (as measured by LCMS).

40. 40. The method of any one of claims 37-39, wherein the filtered protein preparation contains less than about 10 ppm phospholipase B-like 2 protein (as measured by LCMS).

41. 41. The method of any one of claims 37-40, wherein the filtered protein preparation comprises less than about 5 ppm of lysosomal protective protein (as measured by LCMS).

42. 42. The method of any one of claims 37-41, wherein the filtered protein preparation contains less than about 5 ppm ubiquitin-40S ribosomal protein S27a (as measured by LCMS).

43. 43. The method of any one of claims 37-42, wherein the filtered protein preparation comprises less than about 5 ppm kallikrein-11 (as measured by LCMS).

44. 44. The method of any one of claims 37-43, wherein the filtered protein preparation comprises less than about 5 ppm of serine protease HTRA1 isoform X1 (as measured by LCMS).

45. 45. The method of any one of claims 37 to 44, wherein the filtered protein preparation contains less than about 5 ppm of the complement C1r minor component (as measured by LCMS).

46. 46. ​​The method of any one of claims 37-45, wherein the filtered protein preparation contains less than about 5 ppm actin, aortic smooth muscle isoform X1 (as measured by LCMS).

47. 47. The method of any one of claims 37-46, wherein the filtered protein preparation contains less than about 5 ppm actin, aortic smooth muscle isoform X1 (as measured by LCMS).

48. 48. The method of any one of claims 37-47, wherein the filtered protein preparation comprises less than about 5 ppm heat shock cognate 71 kDa protein (as measured by LCMS).

49. 49. The method of any one of claims 37-48, wherein the filtered protein preparation contains less than about 5 ppm peroxiredoxin-1 (as measured by LCMS).

50. 36. The method of any one of claims 32 to 35, wherein the host cell protein content in the drug substance preparation is less than 100 ppm (as measured by LCMS).

51. 51. The method of any one of claims 32-35 and 50, wherein the drug substance preparation comprises one, a combination, or all of the following host cell proteins: protein S100-A6, protein S100-A11, phospholipase B-like 2 protein, lysosomal protective protein, ubiquitin-40S ribosomal protein S27a, kallikrein-11, serine protease HTRA1 isoform XI, complement C1r small component, actin, aortic smooth muscle isoform XI, heat shock cognate 71 kDa protein, and peroxiredoxin-1.

52. 52. The method of claim 51, wherein the drug substance preparation contains less than about 5 ppm protein S100-A6 (as measured by LCMS).

53. 53. The method of claim 51 or 52, wherein the drug substance preparation comprises less than about 5 ppm of protein S100-A11 (as measured by LCMS).

54. 54. The method of any one of claims 51-53, wherein the drug substance preparation comprises less than about 10 ppm phospholipase B-like 2 protein (as measured by LCMS).

55. 55. The method of any one of claims 51-54, wherein the drug substance preparation comprises less than about 5 ppm of lysosomal protective protein (as measured by LCMS).

56. 56. The method of any one of claims 51-55, wherein the drug substance preparation comprises less than about 5 ppm ubiquitin-40S ribosomal protein S27a (as measured by LCMS).

57. 57. The method of any one of claims 51-56, wherein the drug substance preparation comprises less than about 5 ppm kallikrein-11 (as measured by LCMS).

58. 58. The method of any one of claims 51-57, wherein the drug substance preparation comprises less than about 5 ppm serine protease HTRA1 isoform X1 (as measured by LCMS).

59. 59. The method of any one of claims 51-58, wherein the drug substance preparation comprises less than about 5 ppm of complement C1r minor component (as measured by LCMS).

60. 60. The method of any one of claims 51-59, wherein the drug substance preparation comprises less than about 5 ppm actin, aortic smooth muscle isoform X1 (as measured by LCMS).

61. 61. The method of any one of claims 51-60, wherein the drug substance preparation comprises less than about 5 ppm actin, aortic smooth muscle isoform X1 (as measured by LCMS).

62. 62. The method of any one of claims 51-61, wherein the drug substance preparation comprises less than about 5 ppm heat shock cognate 71 kDa protein (as measured by LCMS).

63. 63. The method of any one of claims 51-62, wherein the drug substance preparation comprises less than about 5 ppm peroxiredoxin-1 (as measured by LCMS).

64. The anti-N3pGlu Aβ antibody comprises a heavy chain (HC) and a light chain (LC), the light chain comprises a light chain variable region (LCVR), the heavy chain comprises a heavy chain variable region (HCVR), the LCVR comprises amino acid sequences LCDR1, LCDR2, and LCDR3, the HCVR comprises amino acid sequences HCDR1, HCDR2, and HCDR3, LCDR1 is RASQSLGNWLA (SEQ ID NO: 27), and LCDR2 is YQASTLES (SEQ ID NO: 28).

32. The method of any one of claims 1 to 31, wherein LCDR3 is QHYKGSFWT (SEQ ID NO: 29), HCDR1 is AASGFTFSSYPMS (SEQ ID NO: 30), HCDR2 is AISGSGGSTYYADSVKG (SEQ ID NO: 31), and HCDR3 is AREGGSGSYYNGFDY (SEQ ID NO: 32).

65. The LC of the anti-N3pGlu Aβ antibody comprises an LCVR, and the HC of the anti-N3pGlu Aβ antibody comprises an HCVR, wherein the LCVR is DIQMTQSPSTLSASVGDRVTITCRASQSLGNWLAWYQQKPGKAPKLLIYQASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYKGSFWTFGQGTKVEIK (SEQ ID NO: 23), and the HC 65. The method of claim 64, wherein VR is EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGSGSYYNGFDYWGQGTLVTVSS (SEQ ID NO: 24).

66. the LC of the anti-N3pGlu Aβ antibody is DIQMTQSPSTLSASVGDRVTITCRASQSLGNWLAWYQQKPGKAPKLLIYQASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYKGSFWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 25); The HC of the Aβ antibody is selected from the group consisting of EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGSGSYYNGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC 66. The method of claim 64 or 65, wherein the amino acid sequence is PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 26).

67. 67. The method of any one of claims 64 to 66, wherein the host cell protein content in the filtered protein preparation is less than 10 ppm (as measured by LCMS).

68. 68. The method of any one of claims 64-67, wherein the filtered protein preparation comprises one, a combination, or all of the following host cell proteins: polyubiquitin, lysosomal protection protein, glutathione S-transferase Y1, 40S ribosomal protein S28, thioredoxin isoform X1, basement membrane-specific heparan sulfate proteoglycan core protein isoform X1, tubulointerstitial nephritis antigen-like protein, actin-partial cytoplasmic 2 isoform X2, galectin-1, peroxiredoxin-1, and cornifin alpha.

69. 69. The method of claim 68, wherein the filtered protein preparation contains less than about 1 ppm polyubiquitin (as measured by LCMS).

70. 70. The method of claim 68 or 69, wherein the filtered protein preparation contains less than about 1 ppm of lysosomal protective protein (as measured by LCMS).

71. 71. The method of any one of claims 68-70, wherein the filtered protein preparation comprises less than about 1 ppm glutathione S-transferase Y1 (as measured by LCMS).

72. 72. The method of any one of claims 68-71, wherein the filtered proteins comprise less than about 1 ppm glutathione S-transferase Y1 (as measured by LCMS).

73. 73. The method of any one of claims 68-72, wherein the filtered protein preparation comprises less than about 1 ppm 40S ribosomal protein S28 (as measured by LCMS).

74. 74. The method of any one of claims 68-73, wherein the filtered protein preparation comprises less than about 1 ppm thioredoxin isoform X1 (as measured by LCMS).

75. 75. The method of any one of claims 68-74, wherein the filtered protein preparation comprises less than about 1 ppm basement membrane-specific heparan sulfate proteoglycan core protein isoform X1 (as measured by LCMS).

76. 76. The method of any one of claims 68-75, wherein the filtered protein preparation comprises less than about 1 ppm tubulointerstitial nephritis antigen-like protein (as measured by LCMS).

77. 77. The method of any one of claims 68-76, wherein the filtered protein preparation comprises less than about 1 ppm actin-partial cytoplasmic 2 isoform X2 (as measured by LCMS).

78. 78. The method of any one of claims 68-77, wherein the filtered protein preparation comprises less than about 1 ppm galectin-1 (as measured by LCMS).

79. 79. The method of any one of claims 68-78, wherein the filtered protein preparation comprises less than about 1 ppm peroxiredoxin-1 (as measured by LCMS).

80. 80. The method of any one of claims 68-79, wherein the filtered protein preparation comprises less than about 1 ppm cornifin alpha (as measured by LCMS).

81. 67. The method of any one of claims 64 to 66, wherein the host cell protein content in the drug substance preparation is less than 10 ppm (as measured by LCMS).

82. 82. The method of any one of claims 64-66 and 81, wherein the drug substance preparation comprises one, a combination, or all of the following host cell proteins: polyubiquitin, lysosomal protection protein, glutathione S-transferase Y1, 40S ribosomal protein S28, thioredoxin isoform X1, basement membrane-specific heparan sulfate proteoglycan core protein isoform X1, tubulointerstitial nephritis antigen-like protein, actin-partial cytoplasmic 2 isoform X2, galectin-1, peroxiredoxin-1, and cornifin alpha.

83. 83. The method of claim 82, wherein the drug substance preparation contains less than about 1 ppm polyubiquitin (as measured by LCMS).

84. 84. The method of claim 82 or 83, wherein the drug substance preparation comprises less than about 1 ppm of lysosomal protective protein (as measured by LCMS).

85. 85. The method of any one of claims 82-84, wherein the drug substance preparation comprises less than about 1 ppm glutathione S-transferase Y1 (as measured by LCMS).

86. 86. The method of any one of claims 82-85, wherein the drug substance comprises less than about 1 ppm glutathione S-transferase Y1 (as measured by LCMS).

87. 87. The method of any one of claims 82-86, wherein the drug substance preparation comprises less than about 1 ppm 40S ribosomal protein S28 (as measured by LCMS).

88. 88. The method of any one of claims 82-87, wherein the drug substance preparation comprises less than about 1 ppm thioredoxin isoform X1 (as measured by LCMS).

89. 89. The method of any one of claims 82-88, wherein the drug substance preparation comprises less than about 1 ppm basement membrane-specific heparan sulfate proteoglycan core protein isoform X1 (as measured by LCMS).

90. 90. The method of any one of claims 82-89, wherein the drug substance preparation comprises less than about 1 ppm tubulointerstitial nephritis antigen-like protein (as measured by LCMS).

91. 91. The method of any one of claims 82-90, wherein the drug substance preparation comprises less than about 1 ppm actin-partial cytoplasmic 2 isoform X2 (as measured by LCMS).

92. 92. The method of any one of claims 82-91, wherein the drug substance preparation comprises less than about 1 ppm Galectin-1 (as measured by LCMS).

93. 93. The method of any one of claims 82-92, wherein the drug substance preparation comprises less than about 1 ppm peroxiredoxin-1 (as measured by LCMS).

94. 94. The method of any one of claims 82-93, wherein the drug substance preparation comprises less than about 1 ppm cornifin alpha (as measured by LCMS).

95. 95. A composition produced by the method of any one of claims 1 to 94.

96. 1. A method for reducing host cell protein content in a protein preparation comprising an anti-N3pGlu Aβ antibody recombinantly produced in a mammalian host cell, the method comprising: a) subjecting the protein preparation to an affinity chromatography column; b) eluting the anti-N3pGlu Aβ antibody from the chromatography column to obtain an eluate containing the anti-N3pGlu Aβ antibody; c) optionally adjusting the pH of the eluate to between pH 5.0 and pH 7.5 and subjecting the eluate to a depth filter to obtain a filtered protein preparation, wherein the depth filter is a totally synthetic depth filter.

97. 97. The method of claim 96, wherein the chromatography column comprises a Protein A, Protein G, or Protein L affinity chromatography column.

98. 98. The method of claim 96 or 97, wherein the depth filter has a pore size of at least about 9μ to about 0.1μ.

99. 99. The method of claim 98, wherein the depth filter has a pore size of at least about 2μ to about 0.1μ.

100. 100. The method of claim 99, wherein the depth filter has a pore size of about 0.1 μ.

101. 101. The method of any one of claims 96 to 100, wherein the depth filter is an X0SP filter.

102. 102. The method of any one of claims 96-101, wherein the pH of the eluate on the depth filter is about 5.

0.

103. 102. The method of any one of claims 96-101, wherein the pH of the eluate on the depth filter is about 6.

0.

104. 102. The method of any one of claims 96-101, wherein the pH of the eluate on the depth filter is about 7.

0.

105. 105. The method of any one of claims 96 to 104, wherein the mammalian cells are CHO cells.

106. 106. The method of any one of claims 96 to 105, wherein the protein preparation comprises a harvested cell culture medium, a capture pool, or a recovered protein pool.

107. The method of any one of claims 96 to 106, wherein the anti-N3pGlu Aβ antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a bispecific antibody, or an antibody fragment.

108. The method of claim 107, wherein the anti-N3pGlu Aβ antibody is an IgG1 antibody.

109. The anti-N3pGlu Aβ antibody comprises a heavy chain (HC) and a light chain (LC), the light chain comprises a light chain variable region (LCVR), the heavy chain comprises a heavy chain variable region (HCVR), the LCVR comprises amino acid sequences LCDR1, LCDR2, and LCDR3, the HCVR comprises amino acid sequences HCDR1, HCDR2, and HCDR3, and LCDR1 comprises KSSQSLLYSRGKTYLN (SEQ ID NO: 17). ), LCDR2 is AVSKLDS (SEQ ID NO: 18), LCDR3 is VQGTHYPFT (SEQ ID NO: 19), HCDR1 is GYDFTRYYIN (SEQ ID NO: 20), HCDR2 is WINPGSGNTKYNEKFKG (SEQ ID NO: 21), and HCDR3 is EGITVY (SEQ ID NO: 22).

110. the LC of the anti-N3pGlu Aβ antibody comprises an LCVR, the HC of the anti-N3pGlu Aβ antibody comprises an HCVR, and the LCVR is DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSRGKTYLNWLLQKPGQSPQLLIYAVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHYPFTFGQGTKLEIK (SEQ ID NO: 13); 110. The method of claim 109, wherein the HCVR is QVQLVQSGAEVKKPGSSVKVSCKASGYDFTRYYINWVRQAPGQGLEWMGWINPGSGNTKYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCAREGITVYWGQGTTVTVSS (SEQ ID NO: 14).

111. the LC of the anti-N3pGlu Aβ antibody is DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSRGKTYLNWLLQKPGQSPQLLIYAVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 15); The HC of the Aβ antibody is 111. The method of claim 109 or 110, wherein the amino acid sequence is LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 16).

112. The method of claim 111, wherein the anti-N3pGlu Aβ antibody is donanemab.

113. 113. The method of any one of claims 109 to 112, wherein the host cell protein content in the filtered protein preparation is less than 100 ppm (as measured by LCMS).

114. 114. The method of any one of claims 109-113, wherein the filtered protein preparation comprises one, a combination, or all of the following host cell proteins: protein S100-A6, protein S100-A11, phospholipase B-like 2 protein, lysosomal protective protein, ubiquitin-40S ribosomal protein S27a, kallikrein-11, serine protease HTRA1 isoform XI, complement C1r small component, actin, aortic smooth muscle isoform XI, heat shock cognate 71 kDa protein, and peroxiredoxin-1.

115. 115. The method of claim 114, wherein the filtered protein preparation contains less than about 5 ppm of protein S100-A6 (as measured by LCMS).

116. 116. The method of claim 114 or 115, wherein the filtered protein preparation contains less than about 5 ppm of protein S100-A11 (as measured by LCMS).

117. 117. The method of any one of claims 114-116, wherein the filtered protein preparation comprises less than about 10 ppm phospholipase B-like 2 protein (as measured by LCMS).

118. 118. The method of any one of claims 114-117, wherein the filtered protein preparation comprises less than about 5 ppm of lysosomal protective protein (as measured by LCMS).

119. 119. The method of any one of claims 114-118, wherein the filtered protein preparation contains less than about 5 ppm ubiquitin-40S ribosomal protein S27a (as measured by LCMS).

120. 120. The method of any one of claims 114-119, wherein the filtered protein preparation comprises less than about 5 ppm kallikrein-11 (as measured by LCMS).

121. 121. The method of any one of claims 114-120, wherein the filtered protein preparation comprises less than about 5 ppm of serine protease HTRA1 isoform X1 (as measured by LCMS).

122. 122. The method of any one of claims 114-121, wherein the filtered protein preparation contains less than about 5 ppm of the complement C1r minor component (as measured by LCMS).

123. 123. The method of any one of claims 114-122, wherein the filtered protein preparation contains less than about 5 ppm actin, aortic smooth muscle isoform X1 (as measured by LCMS).

124. 124. The method of any one of claims 114-123, wherein the filtered protein preparation contains less than about 5 ppm actin, aortic smooth muscle isoform X1 (as measured by LCMS).

125. 125. The method of any one of claims 114-124, wherein the filtered protein preparation comprises less than about 5 ppm heat shock cognate 71 kDa protein (as measured by LCMS).

126. 126. The method of any one of claims 114-125, wherein the filtered protein preparation comprises less than about 5 ppm peroxiredoxin-1 (as measured by LCMS).

127. 113. The method of any one of claims 109 to 112, wherein the host cell protein content in the drug substance preparation is less than 100 ppm (as measured by LCMS).

128. 128. The method of any one of claims 109-112 and 127, wherein the drug substance preparation comprises one, a combination, or all of the following host cell proteins: protein S100-A6, protein S100-A11, phospholipase B-like 2 protein, lysosomal protective protein, ubiquitin-40S ribosomal protein S27a, kallikrein-11, serine protease HTRA1 isoform XI, complement C1r small component, actin, aortic smooth muscle isoform XI, heat shock cognate 71 kDa protein, peroxiredoxin-1.

129. 129. The method of claim 128, wherein the drug substance preparation comprises less than about 5 ppm of protein S100-A6 (as measured by LCMS).

130. 130. The method of claim 128 or 129, wherein the drug substance preparation comprises less than about 5 ppm of protein S100-A11 (as measured by LCMS).

131. 131. The method of any one of claims 128-130, wherein the drug substance preparation comprises less than about 10 ppm phospholipase B-like 2 protein (as measured by LCMS).

132. 132. The method of any one of claims 128-131, wherein the drug substance preparation comprises less than about 5 ppm of lysosomal protective protein (as measured by LCMS).

133. 133. The method of any one of claims 128-132, wherein the drug substance preparation comprises less than about 5 ppm ubiquitin-40S ribosomal protein S27a (as measured by LCMS).

134. 134. The method of any one of claims 128-133, wherein the drug substance preparation comprises less than about 5 ppm kallikrein-11 (as measured by LCMS).

135. 135. The method of any one of claims 128-134, wherein the drug substance preparation comprises less than about 5 ppm serine protease HTRA1 isoform X1 (as measured by LCMS).

136. 136. The method of any one of claims 128-135, wherein the drug substance preparation comprises less than about 5 ppm of complement C1r minor component (as measured by LCMS).

137. 137. The method of any one of claims 128-136, wherein the drug substance preparation comprises less than about 5 ppm actin, aortic smooth muscle isoform X1 (as measured by LCMS).

138. 138. The method of any one of claims 128-137, wherein the drug substance preparation comprises less than about 5 ppm actin, aortic smooth muscle isoform X1 (as measured by LCMS).

139. 139. The method of any one of claims 128-138, wherein the drug substance preparation comprises less than about 5 ppm heat shock cognate 71 kDa protein (as measured by LCMS).

140. 140. The method of any one of claims 128-139, wherein the drug substance preparation comprises less than about 5 ppm peroxiredoxin-1 (as measured by LCMS).

141. The anti-N3pGlu Aβ antibody comprises a heavy chain (HC) and a light chain (LC), the light chain comprises a light chain variable region (LCVR), the heavy chain comprises a heavy chain variable region (HCVR), the LCVR comprises amino acid sequences LCDR1, LCDR2, and LCDR3, the HCVR comprises amino acid sequences HCDR1, HCDR2, and HCDR3, LCDR1 is RASQSLGNWLA (SEQ ID NO: 27), and LCDR2 is YQASTLES (SEQ ID NO: 28). The method of any one of claims 96 to 108, wherein LCDR3 is QHYKGSFWT (SEQ ID NO: 29), HCDR1 is AASGFTFSSYPMS (SEQ ID NO: 30), HCDR2 is AISGSGGSTYYADSVKG (SEQ ID NO: 31), and HCDR3 is AREGGSGSYYNGFDY (SEQ ID NO: 32).

142. The LC of the anti-N3pGlu Aβ antibody comprises an LCVR, and the HC of the anti-N3pGlu Aβ antibody comprises an HCVR, wherein the LCVR is DIQMTQSPSTLSASVGDRVTITCRASQSLGNWLAWYQQKPGKAPKLLIYQASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYKGSFWTFGQGTKVEIK (SEQ ID NO: 23), and the HC 142. The method of claim 141, wherein VR is EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGSGSYYNGFDYWGQGTLVTVSS (sequence number 24).

143. the LC of the anti-N3pGlu Aβ antibody is DIQMTQSPSTLSASVGDRVTITCRASQSLGNWLAWYQQKPGKAPKLLIYQASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYKGSFWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 25); The HC of the Aβ antibody is EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSA ISGSGGSTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGSGSYYNGFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP 143. The method of claim 141 or 142, wherein the amino acid sequence is APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 26).

144. 144. The method of any one of claims 141 to 143, wherein the host cell protein content in the filtered protein preparation is less than 10 ppm (as measured by LCMS).

145. 145. The method of any one of claims 141-144, wherein the filtered protein preparation comprises one, a combination, or all of the following host cell proteins: polyubiquitin, lysosomal protection protein, glutathione S-transferase Y1, 40S ribosomal protein S28, thioredoxin isoform X1, basement membrane-specific heparan sulfate proteoglycan core protein isoform X1, tubulointerstitial nephritis antigen-like protein, actin-partial cytoplasmic 2 isoform X2, galectin-1, peroxiredoxin-1, and cornifin alpha.

146. 146. The method of claim 145, wherein the filtered protein preparation contains less than about 1 ppm polyubiquitin (as measured by LCMS).

147. 147. The method of claim 145 or 146, wherein the filtered protein preparation contains less than about 1 ppm of lysosomal protective protein (as measured by LCMS).

148. 148. The method of any one of claims 145-147, wherein the filtered protein preparation contains less than about 1 ppm glutathione S-transferase Y1 (as measured by LCMS).

149. 149. The method of any one of claims 145-148, wherein the composition comprises less than about 1 ppm glutathione S-transferase Y1 (as measured by LCMS).

150. 150. The method of any one of claims 145 to 149, wherein the filtered protein preparation comprises less than about 1 ppm 40S ribosomal protein S28 (as measured by LCMS).

151. 151. The method of any one of claims 145-150, wherein the filtered protein preparation comprises less than about 1 ppm thioredoxin isoform X1 (as measured by LCMS).

152. 152. The method of any one of claims 145-151, wherein the filtered protein preparation comprises less than about 1 ppm basement membrane-specific heparan sulfate proteoglycan core protein isoform X1 (as measured by LCMS).

153. 153. The method of any one of claims 145-152, wherein the filtered protein preparation comprises less than about 1 ppm tubulointerstitial nephritis antigen-like protein (as measured by LCMS).

154. 154. The method of any one of claims 145-153, wherein the filtered protein preparation comprises less than about 1 ppm actin-partial cytoplasmic 2 isoform X2 (as measured by LCMS).

155. 155. The method of any one of claims 145-154, wherein the filtered protein preparation comprises less than about 1 ppm galectin-1 (as measured by LCMS).

156. 156. The method of any one of claims 145-155, wherein the filtered protein preparation comprises less than about 1 ppm peroxiredoxin-1 (as measured by LCMS).

157. 157. The method of any one of claims 145-156, wherein the filtered protein preparation comprises less than about 1 ppm cornifin alpha (as measured by LCMS).

158. 144. The method of any one of claims 141 to 143, wherein the host cell protein content in the drug substance preparation is less than 10 ppm (as measured by LCMS).

159. 159. The method of any one of claims 141-143 and 158, wherein the drug substance preparation comprises one, a combination, or all of the following host cell proteins: polyubiquitin, lysosomal protection protein, glutathione S-transferase Y1, 40S ribosomal protein S28, thioredoxin isoform X1, basement membrane-specific heparan sulfate proteoglycan core protein isoform X1, tubulointerstitial nephritis antigen-like protein, actin-partial cytoplasmic 2 isoform X2, galectin-1, peroxiredoxin-1, and cornifin alpha.

160. 160. The method of claim 159, wherein the drug substance preparation contains less than about 1 ppm polyubiquitin (as measured by LCMS).

161. 161. The method of claim 158 or 160, wherein the drug substance preparation contains less than about 1 ppm of lysosomal protective protein (as measured by LCMS).

162. 162. The method of any one of claims 158-161, wherein the drug substance preparation comprises less than about 1 ppm glutathione S-transferase Y1 (as measured by LCMS).

163. 163. The method of any one of claims 158-162, wherein the drug substance comprises less than about 1 ppm glutathione S-transferase Y1 (as measured by LCMS).

164. 164. The method of any one of claims 158-163, wherein the drug substance preparation comprises less than about 1 ppm 40S ribosomal protein S28 (as measured by LCMS).

165. 165. The method of any one of claims 158-164, wherein the drug substance preparation comprises less than about 1 ppm thioredoxin isoform X1 (as measured by LCMS).

166. 166. The method of any one of claims 158-165, wherein the drug substance preparation comprises less than about 1 ppm basement membrane-specific heparan sulfate proteoglycan core protein isoform X1 (as measured by LCMS).

167. 167. The method of any one of claims 158-166, wherein the drug substance preparation comprises less than about 1 ppm tubulointerstitial nephritis antigen-like protein (as measured by LCMS).

168. 168. The method of any one of claims 158-167, wherein the drug substance preparation comprises less than about 1 ppm actin-partial cytoplasmic 2 isoform X2 (as measured by LCMS).

169. 169. The method of any one of claims 158-168, wherein the drug substance preparation comprises less than about 1 ppm Galectin-1 (as measured by LCMS).

170. 170. The method of any one of claims 158-169, wherein the drug substance preparation comprises less than about 1 ppm peroxiredoxin-1 (as measured by LCMS).

171. 171. The method of any one of claims 158-170, wherein the drug substance preparation comprises less than about 1 ppm cornifin alpha (as measured by LCMS).

172. 172. A composition produced by the method of any one of claims 96 to 171.

173. 1. A pharmaceutical composition comprising an antibody that binds to human N3pGlu Aβ (anti-N3pGlu Aβ antibody), wherein the anti-N3pGlu Aβ antibody is prepared by a process comprising purifying the anti-N3pGlu antibody from mammalian host cells, and the total content of host cell proteins (HCPs) in the composition is less than about 100 ppm (as measured by LCMS).

174. 174. The pharmaceutical composition of claim 173, wherein the mammalian cell is a CHO cell.

175. The pharmaceutical composition of claim 173 or 174, wherein the anti-N3pGlu Aβ antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a bispecific antibody, or an antibody fragment.

176. The pharmaceutical composition of claim 175, wherein the anti-N3pGlu Aβ antibody is an IgG1 antibody.

177. The anti-N3pGlu Aβ antibody comprises a heavy chain (HC) and a light chain (LC), the light chain comprises a light chain variable region (LCVR), the heavy chain comprises a heavy chain variable region (HCVR), the LCVR comprises amino acid sequences LCDR1, LCDR2, and LCDR3, the HCVR comprises amino acid sequences HCDR1, HCDR2, and HCDR3, and LCDR1 is KSSQSLLYSRGKTYLN (SEQ ID NO: 17). wherein LCDR2 is AVSKLDS (SEQ ID NO: 18), LCDR3 is VQGTHYPFT (SEQ ID NO: 19), HCDR1 is GYDFTRYYIN (SEQ ID NO: 20), HCDR2 is WINPGSGNTKYNEKFKG (SEQ ID NO: 21), and HCDR3 is EGITVY (SEQ ID NO: 22).

178. the LC of the anti-N3pGlu Aβ antibody comprises an LCVR, the HC of the anti-N3pGlu Aβ antibody comprises an HCVR, and the LCVR is DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSRGKTYLNWLLQKPGQSPQLLIYAVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHYPFTFGQGTKLEIK (SEQ ID NO: 13); 178. The pharmaceutical composition of claim 177, wherein the HCVR is QVQLVQSGAEVKKPGSSVKVSCKASGYDFTRYYINWVRQAPGQGLEWMGWINPGSGNTKYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCAREGITVYWGQGTTVTVSS (SEQ ID NO: 14).

179. the LC of the anti-N3pGlu Aβ antibody is DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSRGKTYLNWLLQKPGQSPQLLIYAVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 15); The HC of the Aβ antibody is QVQLVQSGAEVKKPGSSVKVSCKASGYDFTRYYINWVRQAPGQGLEWMGWINPGSGNTKYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCAREGITVYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL 179. The pharmaceutical composition of claim 177 or 178, wherein the compound is GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 16).

180. The pharmaceutical composition of claim 179, wherein the anti-N3pGlu Aβ antibody is donanemab.

181. 181. The pharmaceutical composition of any one of claims 173-180, wherein the composition comprises one, a combination, or all of the following host cell proteins: protein S100-A6, protein S100-A11, phospholipase B-like 2 protein, lysosomal protective protein, ubiquitin-40S ribosomal protein S27a, kallikrein-11, serine protease HTRA1 isoform XI, complement C1r small component, actin, aortic smooth muscle isoform XI, heat shock cognate 71 kDa protein, and peroxiredoxin-1.

182. 182. The pharmaceutical composition of claim 181, wherein the composition comprises less than about 5 ppm of protein S100-A6 (as measured by LCMS).

183. 183. The pharmaceutical composition of claim 181 or 182, wherein the composition comprises less than about 5 ppm of protein S100-A11 (as measured by LCMS).

184. 184. The pharmaceutical composition of any one of claims 181-183, wherein the composition comprises less than about 10 ppm phospholipase B-like 2 protein (as measured by LCMS).

185. 185. The pharmaceutical composition of any one of claims 181-184, wherein the composition comprises less than about 5 ppm of lysosomal protective protein (as measured by LCMS).

186. 186. The pharmaceutical composition of any one of claims 181-185, wherein the composition comprises less than about 5 ppm ubiquitin-40S ribosomal protein S27a (as measured by LCMS).

187. 187. The pharmaceutical composition of any one of claims 181-186, wherein the composition comprises less than about 5 ppm kallikrein-11 (as measured by LCMS).

188. 188. The pharmaceutical composition of any one of claims 181-187, wherein the composition comprises less than about 5 ppm of serine protease HTRA1 isoform X1 (as measured by LCMS).

189. 189. The pharmaceutical composition of any one of claims 181-188, wherein the composition comprises less than about 5 ppm of the complement C1r minor component (as measured by LCMS).

190. 190. The pharmaceutical composition of any one of claims 181-189, wherein said composition comprises less than about 5 ppm actin, aortic smooth muscle isoform X1 (as measured by LCMS).

191. 191. The pharmaceutical composition of any one of claims 181-190, wherein the composition comprises less than about 5 ppm actin, aortic smooth muscle isoform X1 (as measured by LCMS).

192. 192. The pharmaceutical composition of any one of claims 181-191, wherein the composition comprises less than about 5 ppm of heat shock cognate 71 kDa protein (as measured by LCMS).

193. 193. The pharmaceutical composition of any one of claims 181-192, wherein the composition comprises less than about 5 ppm peroxiredoxin-1 (as measured by LCMS).

194. The anti-N3pGlu Aβ antibody comprises a heavy chain (HC) and a light chain (LC), the light chain comprises a light chain variable region (LCVR), the heavy chain comprises a heavy chain variable region (HCVR), the LCVR comprises amino acid sequences LCDR1, LCDR2, and LCDR3, the HCVR comprises amino acid sequences HCDR1, HCDR2, and HCDR3, LCDR1 is RASQSLGNWLA (SEQ ID NO: 27), and LCDR2 is YQASTLES (SEQ ID NO: 28). The pharmaceutical composition of any one of claims 173 to 176, wherein LCDR3 is QHYKGSFWT (SEQ ID NO: 29), HCDR1 is AASGFTFSSYPMS (SEQ ID NO: 30), HCDR2 is AISGSGGSTYYADSVKG (SEQ ID NO: 31), and HCDR3 is AREGGSGSYYNGFDY (SEQ ID NO: 32).

195. the LC of the anti-N3pGlu Aβ antibody comprises an LCVR, the HC of the anti-N3pGlu Aβ antibody comprises an HCVR, the LCVR is DIQMTQSPSTSASVGDRVTITCRASQSLGNWLAWYQQKPGKAPKLLIYQASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYKGSFWTFGQGTKVEIK (SEQ ID NO: 23), and the HCVR The pharmaceutical composition of claim 194, wherein is EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGSGSYYNGFDYWGQGTLVTVSS (SEQ ID NO: 24).

196. the LC of the anti-N3pGlu Aβ antibody is DIQMTQSPSTLSASVGDRVTITCRASQSLGNWLAWYQQKPGKAPKLLIYQASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYKGSFWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 25); The HC of the Aβ antibody is EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSA ISGSGGSTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGSGSYYNGFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA 196. The pharmaceutical composition of claim 194 or 195, wherein the pharmaceutical composition is PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 26).

197. 197. The pharmaceutical composition of any one of claims 194 to 196, wherein the total content of host cell proteins (HCPs) in the composition is less than about 10 ppm (as measured by LCMS).

198. 200. The pharmaceutical composition of claim 197, wherein the composition comprises one, a combination, or all of the following host cell proteins: polyubiquitin, lysosomal protection protein, glutathione S-transferase Y1, 40S ribosomal protein S28, thioredoxin isoform X1, basement membrane-specific heparan sulfate proteoglycan core protein isoform X1, tubulointerstitial nephritis antigen-like protein, actin-partial cytoplasmic 2 isoform X2, galectin-1, peroxiredoxin-1, and cornifin alpha.

199. 200. The pharmaceutical composition of claim 198, wherein the composition contains less than about 1 ppm polyubiquitin (as measured by LCMS).

200. 200. The pharmaceutical composition of claim 198 or 199, wherein the composition comprises less than about 1 ppm of lysosomal protective protein (as measured by LCMS).

201. 201. The pharmaceutical composition of any one of claims 198-200, wherein the composition comprises less than about 1 ppm of glutathione S-transferase Y1 (as measured by LCMS).

202. 202. The pharmaceutical composition of any one of claims 198-201, wherein the composition comprises less than about 1 ppm glutathione S-transferase Y1 (as measured by LCMS).

203. 203. The pharmaceutical composition of any one of claims 198-202, wherein the composition comprises less than about 1 ppm of 40S ribosomal protein S28 (as measured by LCMS).

204. 204. The pharmaceutical composition of any one of claims 198-203, wherein the composition comprises less than about 1 ppm thioredoxin isoform X1 (as measured by LCMS).

205. 205. The pharmaceutical composition of any one of claims 198-204, wherein the composition comprises less than about 1 ppm basement membrane-specific heparan sulfate proteoglycan core protein isoform X1 (as measured by LCMS).

206. 206. The pharmaceutical composition of any one of claims 198-205, wherein the composition comprises less than about 1 ppm tubulointerstitial nephritis antigen-like protein (as measured by LCMS).

207. 207. The pharmaceutical composition of any one of claims 198-206, wherein said composition comprises less than about 1 ppm actin-partial cytoplasmic 2 isoform X2 (as measured by LCMS).

208. 208. The pharmaceutical composition of any one of claims 198-207, wherein said composition comprises less than about 1 ppm galectin-1 (as measured by LCMS).

209. 209. The pharmaceutical composition of any one of claims 198-208, wherein the composition comprises less than about 1 ppm peroxiredoxin-1 (as measured by LCMS).

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