1. CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application no. 62/371,704, filed August 5, 2016, the contents of all of which are incorporated herein in their entireties by reference thereto.

2. REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

[0002] The Sequence Listing concurrently submitted herewith in a computer readable form (CRF) via EFS-Web as file name 386003_325WO.txt is incorporated herein by reference in its entirety. The electronic copy of the Sequence Listing was created on August 4, 2017, and has a file size of 17 Kbytes.

3. BACKGROUND

[0003] Daclizumab is a humanized IgGi monoclonal antibody that binds to the alpha subunit (CD25 or Tac) of the human high-affinity interleukin-2 (IL-2) receptor expressed on the surface of T- and B- lymphocytes.

[0004] Daclizumab was previously marketed under the trade name ZENAPAX ^® for the treatment of acute allograft rejection in renal transplant patients as an adjunct to an immunosuppressive regimen that includes cyclosporine and corticosteroids. Daclizumab has also been studied as a treatment for uveitis (Nussenblatt, 1999, Proc. Nat'l. Acad. USA 96:7462-7466), multiple sclerosis (see, e.g., U.S. Patent Nos. 7,575,742 and 8,298,525, and Bielekova et al, 2009, Arch Neurol. 66(4):483-9), ocular inflammation (Bhat et al, 2009, Graefes Arch. Clin. Exp. Ophthalmol. 247:687-692) and human T- cell leukemia virus-1 associated T-cell leukemia (Berkowitz et al, 2010, J Clin Oncol. 2010 ASCO Annual Meeting Proceedings 28 (May 20 Supplement):8043). Daclizumab as an add-on to interferon beta treatment was examined in the CHOICE study, a phase 2, randomized, double-blind, placebo- controlled trial of 230 patients with relapsing multiple sclerosis. (Wynn et al, 2010, Lancet 9(4):381- 90).

[0005] ZINBRYTA ^® (daclizumab) injection has been approved by the U.S. Food and Drug Administration for the treatment of adult patients with relapsing forms of multiple sclerosis.

ZINBRYTA ^® injection is supplied in a single-dose prefilled 1 mL syringe containing 150 mg/mL daclizumab. ZINBRYTA ^® contains a form of daclizumab referred to as Daclizumab High-Yield Process (DAC HYP). DAC HYP can be produced according to the process described in U.S. Patent Publication 2012/0301429. Among other characteristics, DAC HYP differs from daclizumab in ZENAPAX in its N-linked glycosylation profile, lower antigen-dependent cellular cytotoxicity ("ADCC") activity, and isoform profile on cation exchange chromatography. See U.S. Patent Publication 2012/0301429. DAC HYP was the subject of the SELECT and DECIDE studies.

SELECT was a large scale Phase lib, randomized, double-blind, placebo-controlled trial for safety and efficacy of two different dosage levels for treatment of relapsing-remitting multiple sclerosis (Gold et al, 2013, Lancet, 381 : 2167-75). DECIDE was a Phase III, global, randomized, double- blind, multicenter study comparing DAC HYP to interferon β-la for treatment of relapsing-remitting multiple sclerosis.

[0006] Daclizumab, e.g., DAC HYP, formulated as a high-concentration stable liquid composition are desirable for subcutaneous administration, but such high concentration of antibodies can have increased formation of aggregates, which is undesirable for injectable formulations.

4. SUMMARY

[0007] It has been discovered that in preparations of daclizumab (DAC) antibody, particularly those prepared at high concentrations such as ZINBRYTA ^® , the presence of acidic DAC isoforms correlates with an increased level of aggregates in the DAC antibody compositions. Moreover, analysis of these aggregates indicated that they are composed primarily of acidic DAC antibody species. Identification of process conditions which limit formation of acidic DAC isoforms and steps for removing acidic DAC isoforms yield compositions of DAC antibody having low levels of acidic DAC isoforms and reduction in levels of aggregates. Accordingly, the present disclosure provides compositions of DAC antibody, particularly DAC HYP, characterized by low levels of acidic DAC isoform content, and process conditions and isolation steps for preparing such compositions of DAC antibody.

[0008] In one aspect, present disclosure provides compositions of DAC antibody having a low level of total acidic DAC isoform content. In some embodiments, the compositions of DAC antibody have a total acidic DAC isoform content of about or less than about 20%, about or less than about 15%, or about or less than about 12%. The composition of DAC antibody having a low level of acidic DAC isoform content also has a level of aggregate which is less than about 2% of the total DAC antibody content upon long term storage at a temperature of about 2°C to 8°C. The composition of DAC antibody having a low level of acidic DAC isoform content also has a level of aggregate which is lower at stressed stability conditions as compared to DAC compositions prepared without taking steps to reduce the levels of acidic DAC isoforms.

[0009] The compositions of DAC antibody having the desired storage stable characteristics, e.g. , low level of acidic DAC isoform content and level of aggregates, can be prepared by using DAC antibody produced under cell culture conditions in which the pH is maintained at 6.90 or above, particularly at a H of about 6.9 to 7.2. In some embodiments, a method of producing a DAC antibody preparation having a low level of acidic DAC isoform content comprises: culturing a host cell capable of expressing a DAC antibody in a culture medium under conditions suitable for expressing the DAC antibody, wherein the culture medium is maintained at a pH in a range of about 6.9 to about 7.2 for substantially the entire period of time of culturing the cells.

[0010] In another aspect, reductions in the level of acidic DAC isoform content are achieved by including one or more steps for removing acidic DAC isoforms from the main DAC antibody of interest in the process used for preparing isolated DAC antibody. In some embodiments, a process for isolation of a DAC antibody includes at least a step using separation on an ion exchange medium to remove acidic DAC isoform species. The ion exchange medium can be an anion exchange medium or a cation exchange medium. In some embodiments, a process for preparing isolated DAC antibody of interest includes at least a separation step on an anion exchange medium to remove acidic DAC isoform species from the main DAC antibody of interest. In some embodiments, a process for preparing isolated DAC antibody of interest includes at least a separation step on a cation exchange medium for removal of acidic DAC isoform species from the main DAC antibody of interest. In some embodiments, removal of the acidic DAC isoforms uses a combination of separation on an anion exchange medium and separation on a cation exchange medium.

[0011] The compositions of DAC antibody produced by the processes and methods herein have a low level of acidic DAC isoform content, and corresponding improvements in storage-stable properties, particularly the level of aggregates. Accordingly, the present disclosure provides compositions of DAC antibody having low levels of acidic DAC isoforms prepared by the processes and methods described herein. In some embodiments, the compositions of DAC antibody having low levels of acidic DAC isoforms are prepared by the cell culturing process described in the present disclosure, and isolation of the DAC antibody by using a process which includes a step for removing acidic DAC isoform species, such as by ion exchange chromatography..

[0012] In a further aspect, the compositions of DAC antibody are formulated as a pharmaceutical composition with one or more pharmaceutically acceptable excipients. In some embodiments, the compositions of DAC antibody are aqueous formulations for parenteral administration, including among others, intravenous, intramuscular, and subcutaneous administration. In some embodiments, the DAC antibody is formulated at concentrations which provide therapeutically effective doses of DAC antibody when administered subcutaneously, such as from about 135 mg/mL to about 165 mg/mL. In some embodiments, the pharmaceutical formulations comprise DAC HYP. An exemplary formulation of DAC antibody, particularly DAC HYP, having a low level of acidic DAC isoform content comprises: 20-60 mM succinate, 0.02% - 0.04% polysorbate 80, 75-150 mM sodium chloride, 100 mg/mL or more of DAC antibody, (particularly 135 mg/mL to about 165 mg/mL), and pH of 5.5 to 6.5.

[0013] In another aspect, the composition of DAC antibody having the storage stable characteristics described herein is administered in a therapeutically effective amount to treat a disease or disorder involving CD25 activity. In some embodiments, the DAC antibody is administered to treat inflammatory diseases and disorders, such as neuro-inflammatory and ocular inflammatory diseases and disorders. Exemplary diseases and disorders indicated for treatment with DAC antibody include multiple sclerosis, neuromyelitis optica (NMO), uveitis, birdshot chorioretinopathy, age-related macular degeneration, and cicatrizing conjunctivitis. In particular, DAC antibody is indicated for treating forms of: relapsing multiple sclerosis, such as relapsing remitting multiple sclerosis;

progressive multiple sclerosis, including primary and secondary progressive multiple sclerosis; and relapsing progressive multiple sclerosis, such as relapsing secondary progressive multiple sclerosis. The composition of DAC antibody with storage-stable characteristics can be used as monotherapy, or in combination with another therapeutic agent. In various embodiments, a therapeutically effective amount of DAC antibody can be administered parenterally, including intravenously, intramuscularly, intraperitoneally or subcutaneously to a subject in need thereof. Pharmaceutical compositions of DAC HYP at concentrations of 135 mg/mL to 165 mg/mL are particularly suitable for subcutaneous administration.

[0014] Further provided are kits containing a DAC antibody having low levels of acidic DAC isoform content. The kit can comprise one or more unit doses of DAC antibody. Unit doses can be provided in vials, prefilled syringes or injection pens, for examples self-administration by the subject.

[0015] The features and advantages of the antibody compositions and processes for preparing the compositions will become further apparent from the following detailed description of embodiments thereof.

5. BRIEF DESCRIPTION OF THE TABLES AND FIGURES

[0016] FIG. 1 provides DAC-HYP light chain cDNA (SEQ ID NO: 1) and translated amino acid (SEQ ID NO:2) sequences. The bold, underlined aspartate (D) residue is the first amino acid in the properly processed mature protein; the amino acid sequence upstream of this residue corresponds to the signal sequence.

[0017] FIG. 2 provides DAC-HYP heavy chain cDNA (SEQ ID NO:3) and translated amino acid sequences (SEQ ID NO:4). The bold, underlined glutamine (Q) residue is the first amino acid in the properly processed mature protein; the amino acid sequence upstream of this residue corresponds to the signal sequence. [0018] FIG. 3 plots cell culture pH setpoint versus percentage acidic-pEpE content for DAC HYP formulated bulk. DAC HYP was isolated by Protein-A affinity chromatography.

[0019] FIG. 4 shows the stability trend for formulated bulk produced from different pH cell culture setpoints for DAC HYP.

[0020] FIG. 5 shows a chromatogram of DAC HYP preparation on Q-Sepharose Fast Flow for separating acidic DAC species from the main DAC HYP antibody.

[0021] FIG. 6 shows an overlay of CEX chromatograms for Q-Sepharose Fast Flow column flowthrough product and fraction retained by the Q-Sepharose Fast Flow column.

[0022] FIG. 7 shows a comparison of aggregation stability profiles of DAC HYP with and without the removal of acidic species using Q-Sepharose column. Circle plot is without removal and square plot is with removal of acidic species.

[0023] FIG. 8 shows chromatogram of DAC HYP following separation on an analytical CEX for determining acidic-pEpE content.

[0024] FIG. 9 shows a size exclusion chromatography of DAC HYP for determining level of aggregates.

6. DETAILED DESCRIPTION

[0025] The present disclosure provides daclizumab (DAC) antibody preparations having reduced levels of acidic DAC isoform content and improved storage-stable characteristics, particularly in regards to the formation of aggregates during storage. The present disclosure further provides processes for preparing such storage stable formulations of DAC antibody.

[0026] The foregoing general description, including the drawings, and the following detailed description are exemplary and explanatory only and are not restrictive of this disclosure.

[0027] The section headings used herein are for organizational purposes only and not to be construed as limiting the subject matter described.

6.1. Antibody Compositions and Process for Preparing Storage Stable Antibody Formulations

[0028] The present disclosure relates to compositions of daclizumab (DAC) having reduced and/or low levels of acidic DAC isoforms. "Daclizumab" or "DAC" or "DAC antibody" refers to an antibody with the light chain amino acid sequence represented by SEQ ID NO:2 (FIG. 1) and the heavy chain sequence amino acid sequence represented by SEQ ID NO:4 (FIG. 2). As noted above, it has been surprisingly found that the presence of acidic DAC isoforms correlate with an increase in the formation of aggregates in DAC antibody formulations, such as during purification and storage. In particular, formation of acidic species during antibody production in cell culture and their presence in intermediate process steps prior to removal by chromatography appears to correlate with aggregate formation. Aggregates in antibody formulations can affect antibody efficacy and result in increased immunogenicity of an administered antibody, particularly when prepared at high doses for subcutaneous administration. As further described herein, use of process conditions that significantly reduce levels of acidic DAC isoforms result in antibody preparations having low level of aggregates and reduced rates of aggregate formation, particularly at high antibody concentrations and under long term storage conditions. In particular and without being bound by any theory of operation, reducing formation of acidic species in cell culture and/or removing acidic species in the intermediate purification steps may uncouple the relationship between presence of acidic species and accelerated aggregate formation in the subsequent DAC antibody preparation such that the acidic species remaining in the subsequent DAC antibody preparation can increase but the preparation itself does not display a corresponding accelerated increase in aggregate formation.

[0029] Acidic DAC isoform species are DAC antibody species which are more acidic than the main DAC antibody species of interest. Without being bound by theory, acidic DAC isoforms may include charge variants, structure variants, glycosylation variants, and/or fragmentation variants. The acidic DAC isoforms can also be defined by their chromatographic behavior on an ion exchange medium. In some embodiments, acidic DAC isoform comprise a chromatographic species that elutes later than the main DAC antibody species on an anion exchange medium under chromatographic conditions which result in separation of the acidic DAC isoform from the main DAC antibody species. An example of chromatographic characteristic of acidic DAC HYP isoforms on an anion exchange medium Q- Sepharose is shown in FIGURE 5. The level of acidic DAC isoforms in a composition of DAC antibody, also referred to as acidic DAC isoform content, can be assessed by measuring the level of acidic-pEpE species following separation on a weak cation exchange chromatography medium (CEX) as described in Example 4 and as illustrated in FIGURE 8. The major antibody species in a preparation of DAC HYP antibody when analyzed by weak cation exchange chromatography are as follows:

Pyroglutamate on 1

heavy chain and

pEQ 0 C-terminal lysine on pEQ

glutamine on the

other

OK 0 C-terminal lysine on pEVHS

Pyroglutamate on 1 IK 1 C-terminal lysine on pEVHS heavy chain and

2K 2 C-terminal lysine on pEVHS pEVHS truncated signal

Acidic Acidic isoform on pEVHS peptide VHS on the

other Main Main isoform on pEVHS

Basic Basic isoform on pEVHS

Glutamine on 1

heavy chain and

QVHS truncated signal 0 C-terminal lysine QVHS

peptide VHS on the

other

OK 0 C-terminal lysine on VHSVHS

IK 1 C-terminal lysine on VHSVHS

Truncated signal

2K 2 C-terminal lysine on VHSVHS

VHSVHS peptide VHS on

Acidic Acidic isoform on VHSVHS both heavy chains

Main Main isoform on VHSVHS

Basic Basic isoform on VHSVHS

[0030] The levels of acidic-pEpE species is estimated to be representative of the total acidic DAC isoform content in a composition of DAC antibody. Thus, while reference to the level or percentage of acidic DAC isoform is in relation to the total acidic DAC isoforms in a DAC antibody composition, it is based on measurement of acidic-pEpE species as described herein.

[0031] Accordingly, in one aspect, the present disclosure provides compositions of DAC antibody having a low level of acidic DAC isoform content. In some embodiments, the composition comprises a DAC antibody, wherein the composition has an acidic DAC isoform content of about or less than about 20%. In some embodiments, the composition of the DAC antibody has an acidic DAC isoform content of less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, to about 2%. In some embodiments, the DAC antibody composition has an acidic DAC isoform content of about 3% to about 20%, about 4% to about 20%, about 5% to about 20%, about 6% to about 20%, about 7% to about 20%, about 8% to about 20%, about 9% to about 20%, about 10% to about 20%, about 11% to about 20%, about 12% to about 20%, about 13% to about 20%, about 14% to about 20%, or about 15% to about 20%. In some embodiments, the DAC antibody composition has an acidic DAC isoform content of about 3% to about 19%, about 4% to about 18%, about 5% to about 17%, about 6% to about 16%, about 7% to about 15%, about 8% to about 14%, or about 9% to about 13%. In some embodiments, the composition of DAC antibody is substantially free of acidic DAC isoform content. "Substantially free" in this context refers to compositions of DAC antibody where the acidic DAC isoform content is about or less than about 5%. In some embodiments, the composition of DAC antibody has the foregoing acidic DAC isoform content when measured as of completion of the final step in the purification of the DAC antibody, e.g., ultrafiltration/diafiltration as described below. In some embodiments, the foregoing acidic DAC isoform content is for the finished drug substance.

[0032] In some embodiments, the composition of the DAC antibody has an acidic DAC isoform content of about or less than about 15%. In some embodiments, the DAC antibody composition has an acidic DAC isoform content of about 3% to about 15%, about 4% to about 15%, about 5% to about 15%, about 6% to about 15%, about 7% to about 15%, about 8% to about 15%, about 9% to about 15%, about 10% to about 15%, or about 11% to about 15%. In some embodiments, the DAC antibody composition has an acidic DAC isoform content of about 3% to about 14%, about 4% to about 13%, about 5% to about 12%, or about 6% to about 11%. In some embodiments, the composition of DAC antibody has the foregoing acidic DAC isoform content when measured as of completion of the final step in the purification of the DAC antibody, e.g. , ultrafiltration/diafiltration as described below. In some embodiments, the foregoing acidic DAC isoform content is for the finished drug substance.

[0033] In some embodiments, the composition of the DAC antibody has an acidic DAC isoform content of about or less than about 12%. In some embodiments, the DAC antibody composition has an acidic DAC isoform content of about 3% to about 12%, about 4% to about 12%, about 5% to about 12%, about 6% to about 12%, about 7% to about 12% or about 8% to about 12%. In some embodiments, the DAC antibody composition has an acidic DAC isoform content of about 4% to about 11%, about 5% to about 10%, or about 6% to about 9%. In some embodiments, the composition of DAC antibody has the foregoing acidic DAC isoform content when measured as of completion of the final step in the purification of the DAC antibody, e.g., ultrafiltration/diafiltration as described below. In some embodiments, the foregoing acidic DAC isoform content is for the finished drug substance.

[0034] Compositions of DAC antibody having a level of acidic DAC isoform content as described above also have associated a lower level of aggregate and/or reduced rate of aggregate formation as compared to DAC compositions in which the acidic isoforms have not been reduced or removed. In some embodiments, the composition of DAC antibody having a level of acidic DAC isoform content described above also has a level of aggregate which is less than about 2% of the total DAC antibody content. In some embodiments, the level of aggregate is less than about 1.5%, less than about 1.4%, less than about 1.3%, less than about 1.2%, less than about 1.1%, less than about 1%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, less than about 0.5%, less than about 0.4%, less than about 0.3% to about 0.2% of the total DAC antibody content. In some embodiments, the level of aggregate is about or less than about 1% of the total DAC antibody content. In some embodiments, the composition of DAC antibody has about or less than about 15% of acidic DAC isoform content and a level of aggregate which is about or less than about 2% or 1% of the total DAC antibody content. In some embodiments, the composition of DAC antibody has about or less than about 12% of acidic DAC isoform content and a level of aggregate which is about or less than about 1% of the total DAC antibody content.

[0035] In some embodiments, the composition of DAC antibody having a level of acidic DAC content as described above also has a level of aggregate which is about or less than about 2% of the total DAC antibody content upon storage for at least 1 month, 2 months, 3 months, 6 months, 9 months, 12 months (1 year), 18 months, 24 months (2 years), or 36 months (3 years) at a temperature of about 2°C to 8°C in aqueous solution at a pH of about 5.8 to 6.2. In some embodiments, the level of aggregate is less than about 1.5%, less than about 1.4%, less than about 1.3%, less than about 1.2%, less than about 1.1%, less than about 1%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, less than about 0.5%, to about 0.2% of the total DAC antibody content upon storage for at least 1 month, 2 months, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, or 36 months at a temperature of about 2°C to 8°C in aqueous solution at a pH of about 5.8 to 6.2. In some embodiments, the composition of DAC antibody having a level of acidic DAC isoform content as described above has a level of aggregate which is about or less than about 2% of the total DAC antibody content upon storage for 36 months at about 2°C to 8°C.

[0036] In some embodiments, the composition of DAC antibody having a level of acidic DAC isoform content as described above also has a level of aggregate which is about or less than about 2.5 % of the total DAC antibody content upon storage for at least 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months at a temperature of about 23°C to 27°C in aqueous solution at a pH of about 5.8 to 6.2. In some embodiments, the level of aggregate is less than about 2%, less than about 1.5%, less than about 1.4%, less than about 1.3%, less than about 1.2%, less than about 1.1%, less than about 1%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, or less than about 0.5%, to about 0.2% of the total DAC antibody content upon storage for at least 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months at a temperature of about 23°C to 27°C in aqueous solution at a pH of about 5.8 to 6.2. In some embodiments, the composition of DAC antibody having a level of acidic DAC isoform content as described above also has a level of aggregate which is about or less than about 2 % of the total DAC antibody content upon storage for 6 months at about 23°C to 27°C. [0037] In some embodiments, the composition of DAC antibody having a level of acidic DAC isoform content as described above also has a level of aggregate which is about or less than about 3% of the total DAC antibody content upon storage for at least 1 month or 2 months at a temperature of about 37°C to 43°C in aqueous solution at a pH of about 5.8 to 6.2. In some embodiments, the level of aggregate is about or less than about 2.5%, less than about 2%, less than about 1.9%, less than about 1.8%, less than about 1.7%, less than about 1.6%, less than about 1.5% to about 0.2% of the total DAC antibody content upon storage for at least 1 month or 2 months at a temperature of about 37°C to 43°C in aqueous solution at a pH of about 5.8 to 6.2. In some embodiments, the composition of DAC antibody having a level of acidic DAC isoform content as described above also has a level of aggregate which is about or less than about 3% of the total DAC antibody content upon storage for 2 months at about 37°C to 43°C.

[0038] In some embodiments, the composition of DAC antibody having a of acidic DAC isoform content described above also has a level of aggregate which does not increase by more than 2.5 times upon storage for at least 1 month, 2 months, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, or 36 months at a temperature of about 2°C to 8°C in aqueous solution at pH of about 5.8 to 6.2. In some embodiments, the level of aggregate does not increase by more than 2 times, 1.75 times, 1.5 times, 1.25 times upon storage for at least 1 month, 2 months, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, or 36 months at a temperature of about 2°C to 8°C in aqueous solution at a pH of about 5.8 to 6.2. In some embodiments, the level of aggregate does not increase by more than 2.5 times after storage for 36 months at about 2°C to 8°C. In some embodiments, the composition of DAC antibody upon storage for 3 months to 6 month or 3 months to 9 months at about 2°C to 8°C has an acidic DAC isoform content of about or less than about 15% and a level of aggregate which is less than about 1.5%. In some embodiments, the composition of DAC antibody upon storage for 12 months or more up to 36 months at about 2°C to 8°C has an acidic DAC isoform content of about or less than about 15% and a level of aggregate which is less than about 2%.

[0039] In some embodiments, the composition of DAC antibody having a low level of acidic DAC isoform content also has a level of aggregate which does not increase by more than 3 times upon storage for at least 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months at a temperature of about 23°C to 27°C in aqueous solution at a pH of about 5.8 to 6.2. In some embodiments, the level of aggregate does not increase by more than 2.5 times, 2 times, 1.75 times, 1.5 times, or 1.25 times upon storage for at least 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months at a temperature of about 23°C to 27°C in aqueous solution at a pH of about 5.8 to 6.2. In some embodiments, the level of aggregate does not increase by more than 3 times after storage for 6 months at about 23°C to 27°C. In some embodiments, the composition of DAC antibody upon storage for 1 month at about 23°C to 27°C has an acidic DAC isoform content of about or less than about 12% or less and a level of aggregate which is about or less than about 1.5% of the total DAC antibody content. In some embodiments, the composition of DAC antibody upon storage for 3 months at about 23°C to 27°C has an acidic DAC isoform content of about or less than about 20% or less and a level of aggregate which is about or less than about 2% of the total DAC antibody content. In some embodiments, the composition of DAC antibody upon storage for 6 months at about 23°C to 27°C has an acidic DAC isoform content of about or less than about 25% or less and a level of aggregate which is about or less than about 2.5% of the total DAC antibody content.

[0040] In some embodiments, the composition of DAC antibody having a low level of acidic DAC isoform content also has a level of aggregate which does not increase by more than 4 times upon storage for at least 1 month or 2 months at a temperature of about 37°C to 43°C in aqueous solution at a pH of about 5.8 to 6.2. In some embodiments, the level of aggregate does not increase by more than 3 times, 2.5 times, 2 times, 1.75 times, 1.5 times, or 1.25 times upon storage for at least 1 month or 2 months at a temperature of about 37°C to 43°C in aqueous solution at a pH of about 5.8 to 6.2. In some embodiments, the level of aggregate does not increase by more than 4 times after storage for 2 months at about 37°C to 43°C. In some embodiments, the composition of DAC antibody upon storage for 1 month at about 37°C to 43°C has an acidic DAC isoform content of about or less than about 30% or less and a level of aggregate which is about or less than about 2.5% of the total DAC antibody content. In some embodiments, the composition of DAC antibody upon storage for 2 months at about 37°C to 43°C has an acidic DAC isoform content of about or less than about 45% or less and a level of aggregate which is about or less than about 3% of the total DAC antibody content.

[0041] In some embodiments of the foregoing storage stability conditions of the DAC antibody compositions, the aqueous solution comprises 20-60 mM succinate, 0.02%-0.04% w/v polysorbate 80, 75 mM-150 mM NaCl, 100 mg/mL or more DAC antibody, pH 5.5-6.5. In some embodiments, the DAC antibody is at a concentration of about 135-165 mg/mL. In some embodiments, the aqueous solution for determining storage stability characteristics comprises 40 mM succinate, 0.03% w/v polysorbate 80, 100 mM NaCl, 135-165 mg/mL of DAC antibody, pH 6.0.

[0042] In the embodiments herein, the DAC antibody has a light chain amino acid sequence represented by SEQ ID NO:2 (FIG. 1) and a heavy chain amino acid sequence represented by SEQ ID NO: 4 (FIG. 2). It is to be understood that a DAC antibody as used herein includes forms having the referenced light chain and heavy chain amino acid sequences but which can differ in other properties, including, among others, glycosylation profile, antibody isoform profile, and/or activity

characteristics (e.g., antibody dependent cytotoxicity, etc.). [0043] In some embodiments, the DAC antibody is DAC ZENAPAX® or a DAC antibody with a characteristic profile of DAC ZENAPAX®. The characteristics of DAC ZENAPAX® are described in U.S. patent publication no. 2012/0301429, incorporated herein by reference. The glycosylation profile of ZENAPAX ^® is characterized by peaks representing oligosaccharides GO-GlcNAc, GO, Gl, Man5, G2, Man6, Man7 and sialylated oligosaccharide. DAC ZENAPAX® also has a characteristic chromatographic isoform profile on cation exchange chromatography as provided in FIG. 23 of U.S. publication no. 2012/0301429, incorporated herein by reference.

[0044] In some embodiments, the DAC antibody is DAC Penzberg or a DAC antibody with a characteristic profile of DAC Penzberg. The glycosylation profile of DAC Penzberg has peaks representing oligosaccharides GO-GlcNAc, GO, and Gl but with a lower GO-GlcNAc peak as compared to DAC ZENAPAX®. DAC Penzberg also has a characteristic chromatographic isoform profile on cation exchange chromatography as provided in FIG. 23 of U.S. publication no.

2012/0301429, incorporated herein by reference.

[0045] In some embodiments, the DAC antibody is DAC HYP, produced by a high yield process described in U.S. patent publication no. 2012/0301429. The glycosylation profile of DAC HYP is characterized by two main peaks corresponding to GO-GlcNAc and GO and a minor peak

corresponding to a Gl glycoform. DAC HYP is also characterized by low levels of N-linked non- fucosylated mannose glycosyls, such as mannose-5 glycans (Man5). The GO-GlcNAc glycoforms can range from about 5% to about 20% of the total area under the curve (AUC), more typically about 7.2% to 14.6% of the AUC. The GO glycoforms can range from 70% to 99.2% of the AUC, more typically from 80.9% to 99.2% of the AUC. The Gl glycoform can range from 1% to 9% of the AUC, more typically from 1.4% to 3.8% of the AUC. Sialylated oligosaccharides are 1.0% of the total AUC or less.

[0046] The DAC HYP antibody can have an N-linked glycosylation profile in which the total AUC of the peaks in the profile corresponding to non-fucosylated mannose glycosyls is less than about 6% of the total AUC of all peaks in the profile. In particular embodiments, the non-fucosylated mannose glycosyls are Man5, Man 6, and Man7. In some embodiments, the Man5 peak constitutes about 3%, about 2%, about 1% or less of the total AUC of all peaks in the profile, the Man6 peak constitutes about 3%, about 2%, about 1% or less of the total AUC of all peaks in the profile, and/or the Man7 peak constitutes about 3%, about 2%, about 1% or less of the total AUC of all peaks in the profile.

[0047] DAC HYP also includes its various isoforms. In some embodiments, the DAC HYP can have the N-terminal glutamine (Q) of the mature VH chain (in bold, underlined text in FIG. 1) cyclized, forming pyroglutamate (pE). In some embodiments, the signal peptide sequence can be truncated, leaving a valine-histidine-serine (VHS) sequence attached to the N-terminal glutamine residue of the mature VH chain. Because each DAC HYP molecule contains two VH chains, the various N-terminal isoforms of DAC HYP can include forms containing: (1) two glutamine residues (Q/Q); (2) one glutamine residue and one VHS sequence (Q/VHS or VHS/Q); (3) two VHS sequences (VHS/VHS); (4) one glutamine residue and one pyroglutamate residue (Q/pE or pE/Q); (5) one pyroglutamate residue and one VHS sequence (pE/VHS or VHS/pE); and (6) two pyroglutamate residues (pE/pE). Different C-terminal isoforms also are possible, which contain either 0, 1 or 2 C-terminal lysine (K) residues (OK, IK or 2K), resulting in a complex isoform profile. DAC HYP has N-linked oligosaccharides attached to heavy chain residue Asn 296, molecules that can be analyzed by amidase PNGaseF release followed by HPLC analysis.

[0048] DAC HYP also exhibits significantly less ADCC cytotoxicity than ZENAPAX ^® . DAC HYP exhibits at least 25% less ADCC cytotoxicity than ZENAPAX ^® as measured in a cellular assay. In some embodiments, the DAC antibody prepared herein exhibits less than 30% ADCC average cytotoxicity as measured in an in vitro cellular assay using effector cells from at least 3 healthy human donors and KIT225/K6 cells as target cells, at a DAC antibody concentration of 1 μg/mL and an effector to target cell ratio of about 25: 1.

[0049] The compositions of DAC antibody, including DAC HYP, having a low level of acidic DAC isoform content described above can be prepared using steps and conditions that reduce the formation or presence of acidic DAC isoforms. These steps and conditions include specific culture conditions that limit formation of acidic DAC isoforms during cell growth and DAC expression and process steps which remove the acidic DAC species from the main DAC antibody species of interest.

6.1.1. Culture Conditions

[0050] With regards to culture conditions, it has been found that the pH used for growth of antibody expressing cells and the allowable range of pH variation of the culture medium during growth can significantly affect the level of acidic DAC isoform content in the DAC antibody preparation. In some embodiments, culturing antibody expressing cells at a pH above 6.9 results in DAC antibody preparations having lower levels of acidic DAC isoform content as compared to cultures grown below 6.9. Furthermore, in some embodiments, limiting the pH variation to prevent or minimize pH fluctuations that go below 6.9 and narrowing the variation within the set pH to reduce significant fluctuations in pH also reduces formation of acidic DAC isoforms during the culturing process.

[0051] Accordingly, in some embodiments, a method of producing a DAC antibody preparation having a low level of acidic DAC isoform content comprises: culturing a host cell capable of expressing a DAC antibody in a culture medium under conditions suitable for expressing the DAC antibody, wherein the culture medium is maintained at a pH in a range of about 6.9 to about 7.2 for substantially the entire period of time of culturing the cells. In various embodiments, the pH variation is maintained within ± 0.01, ±0.02 or ±0.05 pH units of the set pH, wherein the set pH is a value from about 6.9 to about 7.2. In some embodiments, the pH of the cell culture is chosen in the described range and the level of allowable variation set to provide a DAC antibody preparation following cell culture having the level of acidic DAC isoform content described below.

[0052] In some embodiments, the pH of the culture medium is maintained above pH 6.9 for substantially the entire period of time of culturing the cells. In some embodiments, the culture medium is maintained at a set pH in a range of about 7.0 to about 7.2, about 7.05 to about 7.2, about 7.1 to about 7.2, about 7.15 to about 7.20, or about 7.0 to about 7.1 for substantially the entire period of culturing the cells. In some embodiments, the set pH of the culture medium is maintained at a pH of about 6.9, about 7.0, about 7.01, about 7.02, about 7.03, about 7.04, about 7.05, about 7.06, about 7.07, about 7.08, about 7.09, about 7.10, about 7.11, about 7.12, about 7.13, about 7.14, about 7.15, about 7.16, about 7.17, about 7.18, about 7.19, or about pH 7.20 for substantially the entire period of culturing the cells.

[0053] In some embodiments, the pH of the culture medium is maintained within ± 0.01, ±0.02, or ±0.05 pH units of a set pH, wherein the set pH is a value from about 6.9 to about 7.2. Preferably, the pH variation is ± 0.01 of the set pH. In some embodiments, the pH of the culture medium is maintained at a pH of 7.05 ± 0.01, ± 0.02 or ± 0.05 for substantially the entire period of time of culturing the cells. In some embodiments, the pH of the culture medium is maintained at a pH of 7.10 ± 0.01, ±0.02, or ± 0.05 for substantially the entire period of time of culturing the cells. In some embodiments, the pH of the culture medium is maintained at a pH of 7.15 ± 0.01, ±0.02, or ± 0.05 for substantially the entire period of time of culturing the cells. In some embodiments, the pH of the culture medium is maintained at a pH of 7.20 ± 0.01, ±0.02, or ± 0.05 for substantially the entire period of time of culturing the cells.

[0054] In some embodiments, cell culturing is carried out under the culture conditions above for the seed phase or growth phase cultures, the production phase cultures, or both the growth and production phase cultures. In some embodiments, the growth phase or seed phase cultures refers to culture(s) used for seeding the production phase cultures. Methods of controlling bioreactor pH are known and those methods capable of controlling the pH to the desired pH and pH variation can be chosen. In some embodiments, the pH controller is a proportional-integral-derivative controller (PID controller) as part of the control system. The PID controller has the ability to control pH and other parameters using an algorithm performed by the computer system. The algorithm produces a calculation using three parameters: (1) the proportional value, which determines the reaction to the current error, (2) the integral value, which determines the reaction based on the sum of recent errors, and (3) the derivative value, which determines the reaction based on the rate at which the error has been changing. The weighted sum of these three actions is used to adjust the process via a control element such as the position of a control valve, allowing for fine process control. Such control of pH can be applied to any known cultivation method including, but not limited to, batch, fed-batch, chemostat and perfusion, and with various cell culture equipment including, but not limited to, shake flasks, spinner flasks, stirred bioreactors, airlift bioreactors, membrane bioreactors, reactors with cells retained on a solid support or immobilized/entrapped as in microporous beads, and any other configuration appropriate for optimal growth and productivity of the desired cell line. In some embodiments, an "on/off controller is used. In some embodiments, a PID controller is used. Exemplary PID controllers are commercially available, for example from manufacturers such as Sartorius, Emerson, Allan Bradley, and ABEC Inc.

[0055] In some embodiments, the culturing is carried out under the culture conditions above for a defined time to limit the level of acidic DAC isoform content to about or less than about 18%. In some embodiments, the cells are cultured for a period of time such that the total acidic DAC isoforms content following the period of culturing is less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, to about 2% of the total DAC antibody content. For assessing the level of acidic DAC isoform content from the cell culture, Protein- A is used to affinity isolate the DAC antibody, which is then analyzed by weak cation exchange chromatography.

[0056] In some embodiments, the culturing is for a defined period of time where the DAC antibody content following the period of culturing has a level of aggregate less than about 2% of the total DAC antibody content. In some embodiments, the cells are cultured for a period of time where the level of aggregate is less than about 1.5%, less than about 1.4%, less than about 1.3%, less than about 1.2%, less than about 1.1%, less than about 1%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, or less than about 0.5% of the total DAC antibody content. In some embodiments, the cells are cultured for a period of up to 8 days, up to 9 days, up to 10 days, up to 11 days, up to 12 days, up to 13 days, up to 14 days, or up to 15 days. In some embodiments, the cells are cultured for a period of 8-15 days, 9-13 days, or preferably 10-12 days. Generally, the cell cultures are grown at a temperature of about 34°C to about 39°C, preferably 37°C, with the appropriate levels of CO2, dissolved O2, and mixing as required for growth of the cells. See US patent publication no. 2012/0301429. [0057] In various embodiments, the DAC antibody is produced by recombinant expression in mammalian cells. Generally, the expression vectors comprise promoter elements to drive mRNA expression and selection markers for identification of stable transfectants (see, e.g., Reddy et al., 1978, Science 200:494-502; Boshart et al., 1985, Cell 41(2):521-30). In some embodiments, the vector is a high productivity recombinant expression vector, which may comprise a strong promoter for driving expression of the antibody coding sequence and a weak promoter for driving expression of the selection marker. The use of such promoter elements can allow for selection of stably chromosomally integrated vectors displaying high expression levels of the antibody. In some embodiments, the selection marker is under the control of a SV40 promoter whose activity has been reduced by deleting one or both of the enhancer elements, designated dE-SV40. An exemplary dE- SV40 promoter sequence that can be used to produce stable expression cell lines is at positions 6536- 6735 of vector pHAT.IgGl .rg.dE, as described in US 2012/0301429. Various embodiments of specific vectors that can be used to produce stable expression cell lines are also described in U.S. Application No. 61/565,419 filed Nov. 30, 2011 and International Application No. PCT/US11/62720 filed Nov. 30, 2011, incorporated herein by reference.

[0058] In some embodiments, vectors useful for expressing a DAC antibody such as DAC HYP can include one or more of the features exemplified by pHAT.IgGl.rg.dE, as described in

US2012/0301429. The two chains of daclizumab can be placed under separate transcriptional control but are preferably on the same vector, and their coding regions can be cDNA or genomic DNA containing introns and exons. As an alternative to separate transcriptional control, the two chains can be expressed as a single transcript or a single open reading frame, with their coding regions separated by an internal ribosome entry site or a self-cleaving intein sequence, in which case the heavy and light chain coding sequences are under the control of a single promoter. An exemplary promoter is the CMV 113 promoter and enhancer. Additional features include transcriptional initiation sites (if absent from the promoter chosen), transcription termination sites, and origins of replication. See US patent publication no. 20120301429.

[0059] The culture medium for growing host cells expressing DAC antibody can use any suitable culture medium formulations. In some embodiments, the initial growth of host cells uses a basal medium, in particular a protein free basal medium (e.g., PFBM and PFBM-2) at the pH described for reducing the level of acidic DAC isoform content. In some embodiments, culturing of host cells, particularly following growth in basal medium in the production phase can use a suitable feed medium. These include, among others, Protein Free Feed Medium (PFFM), preferably PFFM-3. Specific culture media for preparing DAC antibodies are described in U.S. patent publication no. 2012/0301429 and U.S. Patent No. 8465739, incorporated herein by reference. [0060] Following completion of culturing of cells, the expressed DAC antibody is harvested from the culture medium. The DAC antibody can be harvested using a variety of conventional techniques, such as by microfiltration, centrifugation (e.g., stacked disc centrifugation), and depth filtration directly from the culture medium. In some embodiments, DAC antibody can be harvested by lowering the pH of the cell culture to approximately pH 5 at a temperature of less than 15°C to flocculate the cells, which can then be removed by centrifugation. In an exemplary embodiment, crude DAC antibody is harvested by lowering the pH of the cell culture to approximately pH 5, chilling the culture to about 4°C for 30-90 minutes, centrifuging, and depth filtering the resultant suspension to remove cells. The pH of the culture can be adjusted using a variety of different acids, including weak or strong organic acids, or weak or strong inorganic acids, such as citric acid (e.g., 0.5-2.0 M citric acid).

[0061] In another aspect, the present disclosure provides a composition of DAC antibody produced by the cell culture process above. In some embodiments, the DAC antibody composition prepared by the foregoing cell culturing process has an acidic DAC isoform content of about or less than about 18%. In some embodiments, the DAC antibody composition prepared by the culturing process has an acidic DAC isoform content of less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, or less than about 3% to about 2%. In some embodiments, the DAC antibody composition prepared by the cell culturing process has an acidic DAC isoform content of about 2% to about 18%, about 3% to about 18%, about 4% to about 18%, about 5% to about 18%, about 6% to about 18%, about 7% to about 18%, about 8% to about 18%, about 9% to about 18%, about 10% to about 18%, about 11% to about 18%, or about 12% to about 18%. In some embodiments, the DAC antibody composition prepared by the cell culturing process has an acidic DAC isoform content of about 3% to about 17%, about 4% to about 16%, about 5% to about 15%, about 6% to about 14%, or about 7% to about 13 %.

[0062] In some embodiments, the DAC antibody composition prepared by culturing the host cell capable of expressing the DAC antibody in a culture medium maintained at a pH of about 6.90 ± 0.01, ±0.02 or ±0.05 for substantially the entire period of time of culturing the cells has an acidic DAC isoform content of about or less than about 18%. In some embodiments, the DAC antibody composition prepared by the culturing process has an acidic DAC isoform content of less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, or less than about 3% to about 2%. In some embodiments, the DAC antibody composition prepared by maintaining the cell culture medium at a pH of about 6.9 ±0.01, ±0.02 or ±0.05 for substantially the entire period of time of culturing the cells has an acidic DAC isoform content of about 2% to about 18%, about 3% to about 18%, about 3% to about 18%, about 4% to about 18%, about 5% to about 18%, about 6% to about 18%, about 7% to about 18%, about 8% to about 18%, about 9% to about 18%, about 10% to about 18%, about 11% to about 18% or about 12% to about 18%. In some embodiments, the DAC antibody composition prepared by maintaining the cell culture medium at a pH of about 6.9 ±0.01, ±0.02 or ±0.05 for substantially the entire period of time of culturing the cells has an acidic DAC isoform content of about 3% to about 17%, about 4% to about 16%, about 5% to about 15%, about 6% to about 14%, or about 7% to about 13%.

[0063] In some embodiments, the DAC antibody composition prepared by culturing the host cell capable of expressing the DAC antibody in a culture medium maintained at a pH of about 7.0 ± 0.01, ±0.02 or ±0.05 for substantially the entire period of time of culturing the cells has an acidic DAC isoform content of about or less than 17%. In some embodiments, the DAC antibody composition prepared by maintaining the cell culture medium at a pH of about 7.0 ± 0.01, ±0.02 or ±0.05 for substantially the entire period of time of culturing the cells has an acidic DAC isoform content of less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, or less than about 3% to about 2% of the total DAC antibody content. In some embodiments, the DAC antibody composition prepared by maintaining the cell culture medium at a pH of about 7.0 ± 0.01, ±0.02 or ±0.05 for substantially the entire period of time of culturing the cells has an acidic DAC isoform content of about 2% to about 17%, about 3% to about 17%, about 4% to about 17%, about 5% to about 17%, about 6% to about 17%, about 7% to about 17%, about 8% to about 17%, about 9% to about 17%, about 10% to about 17%, about 11% to about 17% or about 12% to about 17%. In some embodiments, the DAC antibody composition prepared by maintaining the cell culture medium at a pH of about 7.0 ± 0.01, ±0.02 or ±0.05 for substantially the entire period of time of culturing the cells has an acidic DAC isoform content of about 3% to about 16%, about 4% to about 15%, about 5% to about 14%, about 6% to about 13%, or about 7% to about 12%.

[0064] In some embodiments, the DAC antibody composition prepared by culturing the host cell capable of expressing the DAC antibody in a culture medium maintained at a pH of about 7.05 ± 0.01, ±0.02 or ±0.05 for substantially the entire period of time of culturing the cells has an acidic DAC isoform content of about or less than about 16%. In some embodiments, the DAC antibody composition prepared by maintaining the cell culture medium at a pH of about 7.05 ± 0.01, ±0.02 or ±0.05 for substantially the entire period of time of culturing the cells has an acidic DAC isoform content of less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, or less than about 3% to about 2% of the total DAC antibody content. In some embodiments, the DAC antibody composition prepared by maintaining the cell culture medium at a pH of about 7.05 ± 0.01, ±0.02 or ±0.05 for substantially the entire period of time of culturing the cells has an acidic DAC isoform content of about 2% to about 16%, about 3% to about 16%, about 4% to about 16%, about 5% to about 16%, about 6% to about 16%, about 7% to about 16%, about 8% to about 16%, about 9% to about 16%, about 10% to about 16%, about 11% to about 16% or about 12% to about 16%. In some embodiments, In some embodiments, the DAC antibody composition prepared by maintaining the cell culture medium at a pH of about 7.05 ± 0.01, ±0.02 or ±0.05 for substantially the entire period of time of culturing the cells has an acidic DAC isoform content of about 3% to about 15%, about 4% to about 14%, about 5% to about 13%, about 6% to about 12%, or about 7% to about 11%.

[0065] In some embodiments, the DAC antibody composition prepared by culturing the host cell capable of expressing the DAC antibody in a culture medium maintained at a pH of about 7.10 ± 0.01, ±0.02 or ±0.05 for substantially the entire period of time of culturing the cells has an acidic DAC isoform content of less than 16%. In some embodiments, the DAC antibody composition prepared by the culturing process has an acidic DAC isoform content of less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, or less than about 3% to about 2% of the total DAC antibody content, the DAC antibody composition prepared by maintaining the cell culture medium at a pH of about 7.10 ± 0.01, ±0.02 or ±0.05 for substantially the entire period of time of culturing the cells has an acidic DAC isoform content of about 2% to about 16%, about 3% to about 16%, about 4% to about 16%, about 5% to about 16%, about 6% to about 16%, about 7% to about 16%, about 8% to about 16%, about 9% to about 16%, about 10% to about 16%, about 11% to about 16% or about 12% to about 16%. In some embodiments, the DAC antibody composition prepared by maintaining the cell culture medium at a pH of about 7.10 ± 0.01, ±0.02 or ±0.05 for substantially the entire period of time of culturing the cells has an acidic DAC isoform content of about 3% to about 15%, about 4% to about 14%, about 5% to about 13%, about 6% to about 12%, or about 7% to about 11%.

[0066] In some embodiments, the DAC antibody composition prepared by culturing the host cell capable of expressing the DAC antibody in a culture medium maintained at a pH of about 7.20 ±0.01, ±0.02 or ±0.05 for substantially the entire period of time of culturing the cells has an acidic DAC isoform content of about or less than about 14%. In some embodiments, the DAC antibody composition prepared by maintaining the cell culture medium at a pH of about 7.20 ± 0.01, ±0.02 or ±0.05 for substantially the entire period of time of culturing the cells has an acidic DAC isoform content less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, or less than about 3% to about 2% of the total DAC antibody content. In some embodiments, the DAC antibody composition prepared by maintaining the cell culture medium at a pH of about 7.20 ± 0.01, ±0.02 or ±0.05 for substantially the entire period of time of culturing the cells has an acidic DAC isoform content of about 2% to about 14%, about 3% to about 14%, about 4% to about 14%, about 5% to about 14%, about 6% to about 14%, about 7% to about 14%, about 8% to about 14%, or about 9% to about 14%. In some embodiments, the DAC antibody composition prepared by maintaining the cell culture medium at a pH of about 7.20 ± 0.01, ±0.02 or ±0.05 for substantially the entire period of time of culturing the cells has an acidic DAC isoform content of about 4% to about 13%, about 5% to about 12% or about 6% to about 11%.

[0067] In some embodiments, the composition of DAC antibody prepared from the culturing process has a level of aggregate which is less than about 2%, less than about 1.5%, less than about 1.4%, less than about 1.3%, less than about 1.2%, less than about 1.1%, less than about 1%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, or less than about 0.5% to about 0.2% of the total DAC antibody content. In some embodiments, the composition of DAC antibody produced by the culturing process has a level of aggregate which is less than about 1% of the total DAC antibody content.

[0068] In some embodiments, the composition of DAC antibody prepared from the culturing process has a level of aggregate which is less than about 2%, less than about 1.5%, less than about 1.4%, less than about 1.3%, less than about 1.2%, less than about 1.1%, less than about 1%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, or less than about 0.5% to about 0.2% of the total DAC antibody content when stored for about 7 days, about 14 days, about 21 days or about 30 days at 2°C to 8°C. In some embodiments, the storage medium can be the culture medium, Protein-A elution buffer or in succinate buffer, as further provided herein.

[0069] In some embodiments, the composition of DAC antibody prepared from the culturing process has an acidic DAC isoform content of about or less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or less than about 0.5%, and a level of aggregate which is less than about 2%, less than about 1.5%, less than about 1.4%, less than about 1.3%, less than about 1.2%, less than about 1.1%, less than about 1%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, or less than about 0.5% to about 0.2% of the total DAC antibody content. In some embodiments, the composition of DAC antibody produced by the culturing process has an acidic DAC isoform content of about or less than about 18% and a level of aggregate which is less than about 2% of the total DAC antibody content. In some embodiments, the composition of DAC antibody produced by the culturing process has an acidic DAC isoform content of about or less than about 16% and a level of aggregate which is less than about 1.5% of the total DAC antibody content. In some embodiments, the composition of DAC antibody produced by the culturing process has an acidic DAC isoform content of about or less than about 14% and a level of aggregate which is about or less than about 1% of the total DAC antibody content.

6.1.2. Removal of Acidic DAC Isoforms and DAC Antibody Purification

[0070] The DAC antibody harvested from the culture medium is subjected to purification steps, including among others, affinity purification, in particular Protein A or Protein G selections; ion exchange chromatography, such as on anion exchange mediums, cation exchange mediums, and combinations thereof; size exclusion chromatography; hydrophobic interaction chromatography; multimodal chromatography; differential solubility; and ultrafiltration/diafiltration. The various purification steps can be practiced in combinations and under appropriate conditions to result in a DAC antibody preparation having a level of acidic DAC isoform content described herein.

[0071] In some embodiments, a process for isolation of a DAC antibody includes one or more steps for removing or separating out acidic DAC isoforms from the main DAC antibody of interest. Various protein isolation techniques can be used to remove the acidic DAC isoforms, for example, separation on an ion exchange medium, multimodal chromatographic medium, and isoelectric focusing (e.g. , preparative IEF, recycling IEF, etc.).

[0072] In some embodiments, a process for isolation of a DAC antibody for therapeutic applications includes at least a step using separation on an ion exchange medium to remove acidic DAC isoform species to provide consistent storage stable aqueous formulations of DAC antibody that have reductions in aggregate formation as compared to DAC formulations in which the acidic DAC isoforms have not been removed. The ion-exchange medium can be an anion exchange medium or a cation exchange medium.

[0073] In some embodiments, a method for isolating a DAC antibody comprises: (a) contacting a DAC antibody composition having acidic DAC isoforms with an ion exchange medium; and (b) separating the main DAC antibody from the acidic DAC isoforms on the ion exchange medium. Collecting the main DAC antibody fraction separated from the acidic DAC isoforms provides an isolated DAC antibody preparation having a reduced and/or low level of acidic DAC isoform content, as assessed by determining acidic DAC isoform content. In performing the separation, the DAC antibody preparation can be contacted with the ion exchange medium by using any of a variety of techniques, e.g. , batch purification or chromatography. In various embodiments, separation on the ion-exchange medium can be carried out in bind-elute mode or flow-through mode. In the bind-elute mode, the DAC antibody preparation containing acidic DAC isoforms is contacted with the ion exchange medium at an appropriate ionic strength and pH where at least the main DAC antibody of interest is retained on the ion exchange medium. The acidic DAC isoforms may or may not be bound to the ion exchange medium. The ion exchange medium loaded with the DAC antibody preparation is washed to remove impurities, and the acidic DAC isoforms separated from the main DAC antibody by using an elution solution of increasing the ionic strength (i.e. , conductivity) and/or a change in pH to differentially elute the acidic DAC isoforms and the main DAC antibody. Collecting the fraction containing the main DAC antibody of interest provides a composition of isolated DAC antibody having reduced and/or low level of acidic DAC isoform content. The change in conductivity and/or pH can be a continuous gradient (e.g. , linear or exponential) or discontinuous (e.g. , step gradient). In the flowthrough mode, the ion exchange medium is loaded with the DAC antibody at an appropriate pH and conductivity where the main DAC antibody of interest is not retained on the ion exchange medium and the acidic DAC isoforms are retained on the ion exchange medium. Collection of the flowthrough fraction provides a composition of DAC antibody having a reduced (e.g. , low) level of acidic DAC isoform content.

[0074] In various embodiments, the separation conditions can be assessed from the pi of the main DAC antibody of interest and/or the pi of the acidic DAC isoforms, which can be measured by, for example, by IEF. A theoretical pi for the DAC antibody itself can be estimated based on the amino acid sequence. The pi of the main DAC antibody of interest and the acidic DAC isoforms can be used to estimate the pH range in which the different DAC antibody species are likely to be bound or not bound to the ion exchange medium, as exemplified below.

[0075] In some embodiments, a process for isolation of the DAC antibody includes separation on an anion exchange medium. Chromatography on an anion exchange medium can be used to separate the acidic DAC isoform from the main DAC antibody species of interest. Charged substituents for anion exchange mediums include, among others, diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and quaternary amine (Q) groups. In some embodiments, the anion exchange medium is a strong anion exchanger, such as those using ionic substituents quaternary aminoethyl (QAE) or quarternary amine (Q) groups. In some embodiments, the anion exchange medium is a weak anion exchanger, such as those using diethyaminoethyl (DEAE) groups. The charged substituents can be present on various mediums, such as cellulose; agarose, particularly crosslinked agarose; dextran, particularly crosslinked dextrans; polystyrene; polyacrylic; and polyphenolic resins. These mediums can be in various physical forms, such as particles or membranes. Exemplary anion exchange mediums include, among others, Poros 50PI and Poros 50HQ (cross-linked poly[styrene-divinylbenzene); Capto Q Impres and Capto DEAE (agarose matrix); Toyopearl QAE-550, Toyopearl DEAE-650, and

Toyopearl GigaCap Q-650 (polymeric); Fractogel® EMD TMAE Hicap (polymeric resin); Sartobind STIC® PA nano (salt-tolerant membrane with a primary amine ligand); Sartobind Q nano (anion exchange chromatographic membrane); CUNO BioCap; XOHC (depth-filter media based on cellulose, and mixed cellulose esters), and Q-Sepharose®. In a preferred embodiment, the anion exchange medium is Q-Sepharose®, which is based on crosslinked agarose. In some embodiments, the anion exchange chromatography is performed using Q-Sepharose Fast Flow (QSFF) resin.

[0076] In some embodiments, a method of isolating a DAC antibody comprises: (a) contacting a composition of DAC antibody containing acidic DAC isoforms with an anion exchange medium; and (b) separating the main DAC antibody from the acidic DAC isoforms on the anion exchange medium. The fraction containing the main DAC antibody separated from the acidic DAC isoform is collected to provide a composition of DAC antibody having a low level of acidic DAC isoform content, as assessed by determining the level of acidic DAC isoform content. In view of the type of anion exchange medium selected and the guidance provided herein, the person of skill in the art can select the appropriate chromatography parameters, such as pH, conductivity, and type of salt to effect separation of the acidic DAC isoforms from the main DAC antibody species of interest.

[0077] In some embodiments, the method of isolating a DAC antibody comprises: (a) contacting a composition of DAC antibody containing acidic DAC isoforms with an anion exchange medium under conditions in which the main DAC antibody and the acidic DAC isoforms are retained on the anion exchange medium; and (b) separating the main DAC antibody from the acidic DAC isoforms retained on the anion exchange medium. The separation can be effected by a continuous gradient of increasing conductivity and/or decrease in pH, or by use of step increases in conductivity and/or decreases in pH. Collecting the eluted fraction containing the main DAC antibody provides an isolated DAC antibody with a reduced and/or low level of acidic DAC isoform content. As noted above, this separation scheme on the anion exchange medium is referred to as bind and elute operation or mode.

[0078] In some embodiments, the method of isolating a DAC antibody comprises: (a) contacting a DAC antibody composition containing acidic DAC isoforms with an anion exchange medium under conditions in which the DAC antibody is not retained on the anion exchange medium and the acidic DAC isoforms are retained on the anion exchange medium; and (b) separating the main DAC antibody from the acidic DAC isoforms retained on the anion exchange medium. Collecting the flowthrough fraction containing the main DAC antibody provides a composition of DAC antibody with a reduced and/or low level of acidic DAC isoform content. As noted above, this separation scheme on the anion exchange medium is referred to as flowthrough operation or mode.

[0079] In some embodiments, the anion exchange medium is a quaternary amine anion exchange medium, particularly Q-Sepharose, more particularly Q-Sepharose Fast Flow (QSFF) medium. In some embodiments, the method for preparing isolated DAC antibody includes the step of chromatography on a Q-Sepharose column. In some embodiments, isolating the DAC antibody by chromatography on Q-Sepharose comprises: (a) loading a composition of DAC antibody containing acidic DAC isoforms on a column containing Q-Sepharose in a loading solution having a conductivity and a pH in a range in which the main DAC antibody is not retained on the Q-Sepharose and the acidic DAC isoforms are retained on the Q-Sepharose; and (b) eluting the unretained main DAC antibody with an eluting solution having a conductivity and a pH in a range in which the main DAC antibody is not retained on the Q-Sepharose and the acidic DAC isoforms are retained on the Q- Sepharose.

[0080] In some embodiments for separation on an anion exchanger, particularly for separation on a strong anion exchanger, e.g. , Q-Sepharose, in the flowthrough mode, the pH of the loading and eluting solution is about 0.3 to 1 pH unit below the pi of the DAC antibody, a pH where the main DAC antibody is likely net positively charged or uncharged, and the acidic DAC isoforms are likely net negatively charged.. In some embodiments, the loading and/or eluting solution is about 0.3 to 0.9 pH unit, 0.4 to 0.8 pH unit, or about 0.5 to 0.7 pH unit below the pi of the DAC antibody of interest. In some embodiments, the pH of the loading and/or eluting solution is about 0.3, 0.4, 0.5. 0.6, 0.7, 0.8, 0.9 or 1.0 pH unit below the pi of the DAC antibody of interest. The DAC HYP antibody has a theoretical pi of about 9.1 while the measured pi for DAC HYP is about 8.8. It is to be understood that a person of skill in the art given the guidance in the present disclosure and the knowledge in the art can use either the theoretical pi or measured pi as a starting point for determining the appropriate separation conditions to effect removal of the acidic DAC isoforms from the main DAC antibody. In some embodiments, the washing and eluting solution has a pH in the range of about 8.1 to 8.8, about 8.1 to 8.7, about 8.1 to 8.6, about 8.1 to 8.5, about 8.1 to 8.4, or about 8.1 to 8.3. Correspondingly, in some embodiments, the conductivity is in the range of about 1.5 to 4 mS/cm, i.e., in the range in which the acidic DAC isoforms are retained on the anion exchange medium and the main DAC antibody of interest is not retained on the anion exchange medium. In some embodiments, the conductivity is in the range of about 1.5 to 3.5 mS/cm, about 2.0 to 3.5 mS/cm, or about 2.5 to 3.5 mS/cm. In some embodiments, the conductivity is about 1.5, 2.0, 2.5, 3.0 or about 3.5 mS/cm. In some embodiments, the conductivity is >2 mS/cm.

[0081] In some embodiments, the loading solution has a conductivity in the range of about 1.5 to 4 mS/cm, about 1.5 to 3.5 mS/cm, about 2.0 to 3.5 mS/cm, or about 2.5 to 3.5 mS/cm and a pH in the range of about 8.1 to 8.3. In some embodiments, the eluting solution has a conductivity in the range of about 1.5 to 4 mS/cm, about 1.5 to 3.5 mS/cm, about 2.0 to 3.5 mS/cm, or about 2.5 to 3.5 mS/cm and a pH in the range of about 8.1 to 8.3. In some embodiments, the counterion is CI ^" , particularly in the form of NaCl. An exemplary embodiment of a loading/eluting solution for Q-Sepharose medium is 20 mM Tris, 20 mM NaCl, pH 8.1-8.3 (conductivity of about 2.0 to 3.5 mS/cm).

[0082] In some embodiments, a method of isolating a DAC antibody comprises: (a) contacting a composition of DAC antibody containing acidic DAC isoforms with a cation exchange medium; and (b) separating the main DAC antibody from the acidic DAC isoforms on the cation exchange medium. Collecting the main DAC antibody fraction separated from the acidic DAC isoforms provides a composition of DAC antibody having a reduced and/or low level of acidic DAC isoform content, as assessed by determining the level of acidic DAC isoform.

[0083] In some embodiments, the method of preparing isolated DAC antibody comprises: (a) contacting a composition of DAC antibody containing acidic DAC isoforms with a cation exchange medium under conditions in which the main DAC antibody and the acidic DAC isoforms are retained on the cation exchange medium; and (b) separating the main DAC antibody from the acidic DAC isoforms retained on the cation exchange medium. The separation can be effected by a continuous gradient of increasing conductivity and/or change in pH, or by use of step increases in conductivity and/or changes in pH. Collecting the eluted fraction containing the main DAC antibody provides an isolated DAC antibody with a reduced and/or low level of acidic DAC isoform content. As noted above, this separation scheme on the cation exchange medium is referred to as bind and elute operation or mode.

[0084] In some embodiments, the method of preparing isolated DAC antibody comprises: (a) contacting a composition of DAC antibody containing acidic DAC isoforms with a cation exchange medium under conditions in which the DAC antibody is retained on the cation exchange medium, and the acidic DAC isoforms are not retained on the cation exchanged medium; and (b) separating the main DAC antibody from the acidic DAC isoforms not retained on the cation exchange medium. Following removal of the acidic DAC isoform species, the bound main DAC antibody is eluted from the cation exchange medium with higher salt concentration and/or change in pH of the eluting solution. Collecting the eluted main DAC antibody fraction separated from the acidic DAC isoform species provides an isolated DAC antibody having a reduced and/or low level of acidic DAC isoform content. As noted above, this separation scheme on the cation exchange medium is referred to as flowthrough operation or mode.

[0085] In some embodiments, mixed mode or mixed modal or multimodal ("MM") chromatography may be used to remove acidic DAC species to provide DAC antibody having reduced and/or low level of acidic DAC isoform content. MM chromatography is a chromatographic strategy utilizing two or more different chromatographic mediums, where the different chromatographic mediums use different chemical basis for separation of analytes. Chromatographic mediums for MM chromatography include, among others, mixtures of the following: anion exchange medium, cation exchange medium, hydrophobic interaction medium, hydrophilic interaction medium, hydrogen bonding, pi-pi bonding, and metal affinity. In some embodiments, an MM chromatographic medium with at least an ion exchange medium, such as an anion exchange medium or a cation exchange medium is used in the MM chromatography.

[0086] Exemplary MM chromatography mediums include, among others, Capto Adhere, having a base matrix is a cross-linked agarose with a N-Benzyl-N-methyl ethanol amine group that exhibits multiple modes of interaction (e.g., ionic interaction, hydrogen bonding and hydrophobic interaction); and PPA-HyperCel and HEA-HyperCel, which have base matrices of high porosity cross-linked cellulose having phenylpropylamine and hexylamine groups. Other exemplary mixed mode chromatographic mediums include, but are not limited to, Nuvia C Prime, Toyo Pearl MX Trp 650M, Eshmuno® HCX, CaPure, and Ceramic Hydroxyapatite.

[0087] In various embodiments, the loading pH and conductivity, wash pH and conductivity, as well as elution pH and conductivity for MM chromatography can be modified to achieve the desired removal of acidic DAC isoforms. In some embodiments, additives such as polyethylene glycol, detergents, amino acids, sugars, and chaotropic agents can be added to enhance the performance of the separation or achieve efficient recovery or quality of the DAC antibody preparation.

[0088] In some embodiments, conditions for MM chromatography are chosen so that acidic DAC isoforms are not retained on the MM chromatography medium while the main DAC antibody of interest is retained on the chromatography medium. Under such conditions, the acidic DAC isoforms are present in the flowthrough and wash fractions while the main DAC antibody of interest can be eluted with an elution solution, thereby resulting in DAC antibody having a reduced and/or low level of acidic DAC isoform content. Guidance for use of MM chromatography for purification of antibody are provided in patent publications US20150132801 and WO2013/066707, incorporated herein by reference. In some embodiments, the protein load of the mixture comprising protein of interest is adjusted to a total protein load to the column of between about 50 and 750 g/L, or between about 75 and 500 g/L, or between about 100 and 300 g/L. In some embodiments, the protein concentration of the load protein mixture is adjusted to a protein concentration of the material loaded to the column of about 1 and 50 g/L, or between about 9 and 25 g/L.

[0089] In various embodiments, the solutions used for isolation of the DAC antibody using an ion exchange medium or MM chromatography medium contains a buffer for adjusting and/or maintaining the appropriate pH of the solution used in the isolation process. Useful buffers include, among others, arginine, formate, citrate, succinate, pyridine, piperazine, L-histidine, Bis-tris, Bis-tris propane, imidazole, N-Ethylmorpholine, TEA (triethanolamine), Tris, morpholine, N-Methyldiethanolamine, AMPD (2-amino-2-methyl-l,3-propanediol), diethanolamine, ethanolamine, AMP (2-amino-2- methyl-l-propaol), piperazine, 1,3-diaminopropane and piperidine. In addition, solutions with appropriate conductivity used for isolating the DAC antibody can be made by adjusting the concentration of salts having the appropriate counterions for the particular ion exchange medium or MM chromatography medium. In some embodiments, solutions with the appropriate conductivity are prepared by adjusting the concentration of NaCl, KC1, Na2S04, or other types of salts used generally for protein purification. Generally, conductivity of the solutions can be measured by standard procedures known in the art (e.g., conductivity meter).

[0090] In some embodiments, a process for isolating DAC antibody following harvesting from the cell culture includes at least the combination of anion exchange chromatography, particularly chromatography on Q-Sepharose FF to remove acidic DAC isoform species, and cation exchange chromatography, particularly a weak cation exchanger, more particularly CM-650 M Toyopearl. When the DAC purification process includes a combination of separation on an anion exchange medium and separation on a cation exchange medium, the step of separation on the anion exchange medium can be carried out before the step of separation on the cation exchange medium, or conversely, the separation on the cation exchange medium can be carried out before the separation on the anion exchange medium. In some embodiments, the separation on the ion exchange medium can be sequential, or alternatively, other purification steps can be carried out in between the separations on the ion exchange mediums.

[0091] In some embodiments, a process for isolating DAC antibody includes two or all of the following isolation steps: Protein-A affinity isolation, anion exchange chromatography, and cation exchange chromatography. Additional steps can be added to the forgoing, for example, among others, viral inactivation and/or virus removal, and filtration steps for removal of viruses and concentration of the antibody. In some embodiments, the process for isolating DAC antibody following harvesting from the cell culture includes at least the combination of Protein-A affinity isolation and anion exchange chromatography, particularly chromatography on a strong anion exchanger, such as Q- Sepharose FF, to remove acidic DAC isoform species. In some embodiments, the process for isolating DAC antibody following harvesting from the cell culture includes at least the combination of Protein- A affinity isolation and cation exchange chromatography, particularly chromatography on a weak cation exchange medium, such as CM-650 M. In some embodiments, the purification steps following isolation from the culture medium includes at least the combination of Protein-A affinity isolation; anion exchange chromatography, particularly chromatography on a strong anion exchanger, such as Q-Sepharose, to remove acidic DAC isoform species; and cation exchange chromatography, particularly chromatography on a weak cation exchange medium, such as CM-650 M. The method for separation on the anion exchange medium and the separation on the cation exchange medium can be carried out as described herein. In some embodiments, the Protein-A affinity isolation step is used to isolate the DAC antibody prepared from the cell cultures, prior to the separation on the ion exchange medium. Protein-A, for example using recombinant Protein-A, specifically binds IgG and allows separation of the DAC antibody from other non-binding components present in the DAC antibody composition harvested from the cell culture. Generally, Protein-A affinity isolation step comprises loading the DAC antibody preparation onto a Protein-A resin (e.g. , recombinant Protein-A;

MabSelect) in a chromatography apparatus under conditions in which the DAC antibody binds to the Protein-A. Materials that do not bind to Protein-A are then removed with a washing solution, which can be an equilibration solution. The DAC antibody bound to the Protein-A is then eluted off the Protein-A under elution conditions that remove the bound antibody from the Protein-A. Exemplary process steps and parameters for isolating and purifying DAC, including a Protein-A chromatography step, are described in U.S. publication 2012/0301429, incorporated herein by reference. For example, following equilibration of the Protein-A column with an equilibration buffer, a neutralized DAC antibody preparation from the cell culture is loaded onto the column in order to bind the antibody to the Protein-A medium. An exemplary equilibration buffer is: 20 mM sodium citrate, 150 mM sodium chloride, pH 7.0. Following loading, the column is washed with the equilibration buffer to remove the unbound and loosely bound impurities from the resin, as well as a pre-elution wash with a citrate buffer to adjust the citrate and sodium chloride concentration of the column. The citrate buffer is 10 mM sodium citrate pH 7.0. The bound antibody is then eluted from the column with a step change in pH using an elution buffer of 10 mM sodium citrate at pH 3.5. The eluent from the Protein-A medium can be monitored by various techniques, such as by absorbance at a wavelength of 280 nm.

[0092] In some embodiments, the DAC antibody produced under the cell culture conditions resulting in a reduced and/or low level of acidic DAC isoform content and harvested from the culture medium is isolated according to at least the following purification steps: (a) Protein-A affinity isolation;

(b) low pH viral inactivation;

(c) strong anion exchanger (e.g., Q-Sepharose Fast Flow) chromatography;

(d) weak cation exchanger (e.g., CM-650 M) chromatography; and

(e) ultrafiltration/diafiltration.

[0093] As noted above, the Protein-A step isolates the bulk DAC antibody, which includes any DAC isoforms species present in the cell culture medium that bind Protein-A. The separation on the strong anion exchanger, e.g. , Q-Sepharose, is carried out under conditions to remove acidic DAC isoforms.

[0094] The DAC antibody preparations are subjected to an ultrafiltration/diafiltration step, preferably using a filter membrane having a nominal molecular weight cutoff of about 30 kDa. The DAC antibody preparation is concentrated and diafiltered to exchange the buffer to an exchange buffer (e.g., formulation buffer without surfactant). In some embodiments, the exchange buffer can have an osmolality in the range of about 267-327 mOsm/kg (e.g., 270-310 mOsm/kg) and a pH in the range of about pH 5.8-6.2 at 25°C. (e.g., 5.9-6.1 at 25°C). The initial concentration of antibody can be about 30 mg/mL, and in some embodiments, subject to further concentration, e.g. , to about 100 mg/mL.

[0095] A further ultrafiltration step can use a filter membrane with the same molecular weight cutoff (e.g., 30 kDa) to obtain a more concentrated DAC antibody solution, e.g., 150-170 mg/mL, 150 mg/mL, 160 mg/mL or 170 mg/mL.

[0096] The concentrated DAC preparation from the filtration step can be processed by dilution to the desired concentration of the DAC antibody. In some embodiments, the dilution buffer is the same as the exchange buffer, but includes about 0.005%-10% (w/v) polysorbate 80, and is used in an amount such that the final, stable, high concentration DAC antibody formulation has a calculated polysorbate 80 concentration (nominal concentration) in a range of 0.02-0.04% w/v, preferably about 0.03% w/v. A variety of different buffering agents and excipients can be included in the dilution buffers to achieve an osmolality and pH within the defined ranges. An initial dilution can be done with a dilution buffer not containing a surfactant. The final dilution to the desired concentration can be done with a dilution buffer containing the surfactant. An exemplary dilution buffer for high concentration liquid DAC antibody (including DAC HYP drug formulations) contains about 40 mM succinate, 100 mM NaCl, about 1 % w/v polysorbate 80, pH of about 6.0 at 25° C, which is used to dilute the DAC antibody preparation from the filtration step to a final polysorbate concentration of 0.02% w/v to 0.04% w/v polysorbate 80.

[0097] While the steps above can be practiced in the described sequence, it is to be understood that some of the steps in the process can be performed in a different sequence. For example, in some embodiments, the step of low pH viral inactivation can be carried out at any position in the sequence of the process above following Protein-A affinity isolation. In addition, in some embodiments, the ion exchange chromatography steps can be in any sequence in between the steps of Protein-A affinity isolation and ultrafiltration/diafiltration. In some embodiments, a process for isolating a DAC antibody of interest includes a process sequence which has Protein-A affinity isolation in the first step of the process, an ultrafiltration/diafiltration in the last step of the process, and the steps of low pH viral inactivation, anion exchange chromatography, and cation exchange chromatography in any order between the Protein-A affinity isolation step and the ultrafiltration/diafiltration step. For example, a process for isolating a DAC antibody of interest can comprise the following sequence: Protein-A affinity isolation, low pH viral inactivation; weak cation exchanger (e.g., CM-650 M)

chromatography; strong anion exchanger (e.g. , Q-Sepharose Fast Flow) chromatography, and ultrafiltration/diafiltration. Another exemplary process includes the following sequence of steps: Protein-A affinity isolation, strong anion exchanger (e.g., Q-Sepharose Fast Flow) chromatography, low pH viral inactivation, weak cation exchanger (e.g., CM-650 M) chromatography, and ultrafiltration/diafiltration. It is to be understood that each and every such process sequence variation are encompassed within the present disclosure.

[0098] In some embodiments, the process for isolating a DAC antibody having low levels of acidic DAC isoform content and low levels of aggregate as described herein include the following steps:

(1) Seed reactor (e.g., 60L, 235L 950L, and 3750 Bioreactors), pH control >7.0;

(2) Production Bioreactor (cell culture conditions >7.0 to minimize acidic DAC isoform); and

(3) Clarification (e.g., disc stack centrifugation).

[0099] Step (1) involves thawing recombinant cells capable of expressing DAC antibody obtained from a cell bank and transferring the cells to a culture flask containing defined medium Protein Free Basal Medium-(e.g. , PFBM-2). The inoculum is prepared by expanding the cells under suitable operating conditions, e.g., 37°C with 5%-7.5% CO2. Culture flasks are seeded with the inoculum and expanded by sequential passage into increasing culture volumes (e.g., ~60 L, -235 L and -950 L) in a defined medium PFBM-2 to prepare the seed cultures. The seed cultures are then used to inoculate the production bioreactor containing a defined medium, e.g., PFBM-2 or PFFM-3. The culture pH is maintained at >7.05 ± 0.05 by automatic control. Air/02 is sparged into the production culture. The production cultures are run for about 9 to 12 days.

[0100] For harvesting the DAC antibody in step (3), the production culture is chilled to <15°C and its pH adjusted to pH 5.0 ±0.1 with 0.2 M citric acid, and held for a period of approximately 30-90 or 45- 60 minutes to flocculate the cells and cell debris. The pH-adjusted harvest is then clarified by continuous centrifugation operated under appropriate bowl speed and flow rate. The clarified culture solution is filtered through a depth filter followed by membrane filtration, e.g. , 0.22 um. The cell-free harvest is adjusted to an approximate pH of 6.4 using a 1-2 M Tris solution and stored at 2-8° C for further processing. In some instances, this pH adjustment occurs within 12 hours of the original bioreactor pH adjustment to pH 5.0.

[0101] Additional steps, such as Protein A affinity isolation; low pH viral inactivation; strong anion exchanger (e.g. , Q-Sepharose Fast Flow) chromatography; weak cation exchanger (e.g., CM-650 M) chromatography; and ultrafiltration/diafiltration can be carried out on the cell free harvest as described above and as provided in the Examples.

[0102] The processes and methods described above for isolating the DAC antibody can be performed at an appropriate temperature, generally at a temperature between 5°C to 30°C. In some embodiments, the processes and methods use to remove the acidic DAC isoforms can be carried out at a temperature of about 15°C to 30°C. For example, the separation of DAC antibody from acidic DAC isoforms of anion exchange medium can be carried out at a temperature of about 15°C to 30°C in view of studies indicating that temperatures around ambient temperature (e.g. , 20°C) result in lower level of aggregates as compared to separation conducted at lower temperatures. In some embodiments, the processes and methods used for removing or reducing the level of acidic DAC isoforms is carried out at a temperature of about 20°C to 30°C.

[0103] The DAC antibody prepared by the process herein can be characterized by one or more of the following analytical procedures: (i) mass spectroscopy, i.e. , for mass analysis, particularly following deglycosylation; (ii) peptide mapping, (iii) N terminal sequencing; (iv) UV spectroscopy; (v) circular dichroism; (vi) analytical ultracentrifugation; (vii) fluorescence spectroscopy; (viii) differential scanning calorimetry; (ix) binding to CD25, e.g. , ELISA; (x) binding affinity as determined by surface plasmon resonance; (xi) N-glycan analysis; (xii) isolelectric focusing, particularly imaged capillary isoelectric focusing; (xiii) analytical cation exchange chromatography; (xiv) SDS polyacrylamide gel electrophoresis (reducing and non-reducing); and (xv) analytical size exclusion chromatography (e.g. , for assessing level of aggregates). Each of these analytical procedures are described in US 2012/0301429, incorporated herein by reference. In various embodiments, the DAC antibody preparation can be characterized after one or more of the process steps, after purification and/or after a defined time under specified storage conditions.

[0104] The methods described herein for isolating DAC antibody with reduced levels of acidic DAC isoform content can be applied to any form of DAC antibody. In an exemplary embodiment, the process and methods described herein are applied to isolation of DAC HYP antibody. [0105] For assessing the storage stable characteristics and also for purposes of quality control, the DAC antibody preparations can be characterized following various storage conditions. In some embodiments, the DAC antibody preparations are stored at 2°C to 8°C and examined following at least 1 month, 2 months, 3 months, 6 months, 9 months, 12 months (1 year), 18 months, 24 months (2 years), or 36 months (3 years) of storage. In some embodiments, the DAC antibody preparations are stored at about 23°C to 27°C and examined following at least 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months of storage. In some embodiments, accelerated storage or stress storage conditions are used to assess storage stable characteristics of the DAC antibody preparations. In some embodiments, the DAC antibody preparations are stored at 37°C to 43°C and examined at least 1 month or 2 months of storage. Any one or more of the analytical procedures can be used to characterize the storage stable characteristics of the DAC antibody preparations. In a preferred embodiment, the DAC antibody preparations stored under the defined conditions are examined for the presence of acidic DAC isoform content and/or aggregates. In exemplary embodiments, the DAC antibody is stored in formulation buffer composed of 40 mM succinate, 0.03% polysorbate 80, 100 mM NaCl, and pH of about 6 for testing of storage stable characteristics.

[0106] In another aspect, the present disclosure provides an isolated DAC antibody prepared by the one or more or combination of the purification steps described above. In some embodiments, the present disclosure provides an isolated DAC antibody prepared by ion exchange chromatography, where the isolated DAC antibody has an acidic DAC isoform content of less than about 20%. In some embodiments, the composition of the DAC antibody prepared by ion exchange chromatography has an acidic DAC isoform content of less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%„ less than about 6%, less than about 5%, less than about 4%, less than about 3%, to about 2%. In some embodiments, the DAC antibody composition prepared by ion exchange chromatography has an acidic DAC isoform content of about 3% to about 20%, about 4% to about 20%, about 5% to about 20%, about 6% to about 20%, about 7% to about 20%, about 8% to about 20%, about 9% to about 20%, about 10% to about 20%, about 11% to about 20%, about 12% to about 20%, about 13% to about 20%, about 14% to about 20%, or about 15% to about 20%. In some embodiments, the DAC antibody composition prepared by ion exchange chromatography has an acidic DAC isoform content of about 3% to about 19%, about 4% to about 18%, about 5% to about 17%, about 6% to about 16%, about 7% to about 15%, about 8% to about 14%, or about 9% to about 13%. In some embodiments, the DAC antibody composition prepared by ion exchange chromatography is substantially free of acidic DAC isoform content. [0107] In some embodiments, the composition of the DAC antibody prepared by ion exchange chromatography has an acidic DAC isoform content of less than about 15%. In some embodiments, the DAC antibody composition prepared by ion exchange chromatography has an acidic DAC isoform content of about 3% to about 15%, about 4% to about 15%, about 5% to about 15%, about 6% to about 15%, about 7% to about 15%, about 8% to about 15%, about 9% to about 15%, about 10% to about 15%, or about 11% to about 15%. In some embodiments, the DAC antibody composition prepared by ion exchange chromatography has an acidic DAC isoform content of about 3% to about 14%, about 4% to about 13%, about 5% to about 12%, or about 6% to about 11%.

[0108] In some embodiments, the composition of the DAC antibody prepared by ion exchange chromatography has an acidic DAC isoform content of less than about 12%. In some embodiments, the DAC antibody composition prepared by ion exchange chromatography has an acidic DAC isoform content of about 3% to about 12%, about 4% to about 12%, about 5% to about 12%, about 6% to about 12%, about 7% to about 12%, or about 8% to about 12%. In some embodiments, the DAC antibody composition prepared by ion exchange chromatography has an acidic DAC isoform content of about 4% to about 11%, about 5% to about 10%, or about 6% to about 9%.

[0109] In some embodiments, the isolated DAC antibody prepared by ion exchange chromatography and having a level of acidic DAC isoform content above also has a level of aggregate which is about or less than about 2%, less than about 1.5%, less than about 1.4%, less than about 1.3%, less than about 1.2%, less than about 1.1%, less than about 1% , less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, less than about 0.5%, less than about 0.4%, less than about 0.3%, to about 0.2% of the total DAC antibody content.

[0110] In some embodiments, the isolated DAC antibody prepared by ion exchange chromatography and having a level of acidic DAC isoform content above also has a level of aggregate which is about or less than about 2%, less than about 1.5%, less than about 1.4%, less than about 1.3%, less than about 1.2%, less than about 1.1%, less than about 1% , less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, less than about 0.5%, less than about 0.4%, less than about 0.3% to about 0.25% of the total DAC antibody content upon storage for at least 1 month, 2 months, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, or 36 months at about 2°C to 8°C in aqueous solution at pH of about 5.8 to 6.2. In some embodiments, the isolated DAC antibody prepared by ion exchange chromatography has a level of aggregate which is about or less than about 2% upon storage for 36 months at about 2°C to 8°C.

[0111] In some embodiments, the isolated DAC antibody prepared by ion exchange chromatography and having a level of acidic DAC isoform content above also has a level of aggregate which is about or less than about 2.5%, less than about 2%, less than about 1.5%, less than about 1.4%, less than about 1.3%, less than about 1.2%, less than about 1.1%, less than about 1% , less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, or less than about 0.5%, to about 0.25% of the total DAC antibody content upon storage for at least 1 month, 2 months, 3 months, 4 month, 5 months, or 6 months at about 23°C to 27°C in aqueous solution at pH of about 5.8 to 6.2. In some embodiments, the isolated DAC antibody prepared by ion exchange chromatography has a level of aggregate which is about or less than about 2.5% upon storage for 6 months at about 23°C to 27°C.

[0112] In some embodiments, the isolated DAC antibody prepared by ion exchange chromatography and having a level of acidic DAC isoform content above also has a level of aggregate which is about or less than about 3.0%, less than about 2.5%, less than about 2%, less than about 1.9%, less than about 1.8%, less than about 1.7%, less than about 1.6%, or less than about 1.5%, to about 0.25% of the total DAC antibody content upon storage for at least 1 month or 2 months at about 37°C to 47°C in aqueous solution at pH of about 5.8 to 6. In some embodiments, the isolated DAC antibody prepared by ion exchange chromatography has a level of aggregate which is about or less than about 3.0% upon storage for 2 months at about 37°C to 47°C.

[0113] In some embodiments, the isolated DAC antibody prepared by ion exchange chromatography and having a level of acidic DAC isoform content above also has a level of aggregate which does not increase by more than 2.5 times, 2 times, 1.75 times, 1.5 times, 1.25 times upon storage for at least 1 month, 2 months, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, or 36 months at about 2°C to 8°C in aqueous solution at pH of about 5.8 to 6.2. In some embodiments, the isolated DAC antibody prepared by ion exchange chromatography has a level of aggregate which does not increase by more than 2.5 times after storage for 36 months at about 2°C to 8°C.

[0114] In some embodiments, the isolated DAC antibody prepared by ion exchange chromatography and having a level of acidic DAC isoform content above also has a level of aggregate which does not increase by more than 3 times, 2.5 times, 2 times, 1.75 times, 1.5 times, 1.25 times upon storage for at least 1 month, 2 months, 3 months, 4 month, 5 months, or 6 months at about 23°C to 27°C in aqueous solution at pH of about 5.8 to 6.2. In some embodiments, the isolated DAC antibody prepared by ion exchange chromatography has a level of aggregate which does not increase by more than 3 times after 6 months of storage at about 23°C to 27°C.

[0115] In some embodiments, the isolated DAC antibody prepared by ion exchange chromatography and having a level of acidic DAC isoform content above also has a level of aggregate which does not increase by more than 4 times, 3 times, 2.5 times, 2 times, 1.75 times, 1.5 times, or 1.25 upon storage for at least 1 month or 2 months at about 37°C to 47°C in aqueous solution at pH of about 5.8 to 6.2. In some embodiments, the isolated DAC antibody prepared by ion exchange chromatography has a level of aggregate which does not increase by more than 4 times after 2 months of storage at about 37°C to 47°C.

[0116] In some embodiments, the isolated DAC antibody prepared by anion exchange

chromatography, particularly strong anion exchanger, e.g., Q-Sepharose FF, has an acidic DAC isoform content of less than about 20%. In some embodiments, the composition of the DAC antibody prepared by anion exchange chromatography has an acidic DAC isoform content of less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, to about 2%. In some embodiments, the DAC antibody composition prepared by anion exchange chromatography has an acidic DAC isoform content of about 3% to about 20%, about 4% to about 20%, about 5% to about 20%, about 6% to about 20%, about 7% to about 20%, about 8% to about 20%, about 9% to about 20%, about 10% to about 20%, about 11% to about 20%, about 12% to about 20%, about 13% to about 20%, about 14% to about 20%, or about 15% to about 20%. In some embodiments, the DAC antibody composition prepared by anion exchange chromatography has an acidic DAC isoform content of about 3% to about 19%, about 4% to about 18%, about 5% to about 17%, about 6% to about 16%, about 7% to about 15%, about 8% to about 14%, or about 9% to about 13%.

[0117] In some embodiments, the composition of the DAC antibody prepared by anion exchange chromatography has an acidic DAC isoform content of less than about 15%. In some embodiments, the DAC antibody composition prepared by anion exchange chromatography has an acidic DAC isoform content of about 3% to about 15%, about 4% to about 15%, about 5% to about 15%, about 6% to about 15%, about 7% to about 15%, about 8% to about 15%, about 9% to about 15%, about 10% to about 15%, or about 11% to about 15%. In some embodiments, the composition of DAC antibody prepared by anion exchange chromatography has an acidic DAC isoform content of about 3% to about 14%, about 4% to about 13%, about 5% to about 12%, or about 6% to about 11%.

[0118] In some embodiments, the composition of the DAC antibody prepared by anion exchange chromatography has an acidic DAC isoform content of less than about 12%. In some embodiments, the DAC antibody composition prepared by anion exchange chromatography has an acidic DAC isoform content of about 3% to about 12%, about 4% to about 12%, about 5% to about 12%, or about 6% to about 12%. In some embodiments, the DAC antibody composition prepared by anion exchange chromatography has an acidic DAC isoform content of about 4% to about 11%, about 5% to about 10%, or about 6% to about 9%. [0119] In some embodiments, the isolated DAC antibody prepared by anion exchange

chromatography and having a level of acidic DAC isoform content above also has a level of aggregate which is about or less than about 2%, less than about 1.5%, less than about 1.4%, less than about 1.3%, less than about 1.2%, less than about 1.1%, less than about 1%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, less than about 0.5%, less than about 0.4%, less than about 0.3%, to about 0.2% of the total DAC antibody content.

[0120] In some embodiments, the isolated DAC antibody prepared by anion exchange

chromatography and having a level of acidic DAC isoform content above also has a level of aggregate which is about or less than about 2%, less than about 1.5%, less than about 1.4%, less than about 1.3%, less than about 1.2%, less than about 1.1%, less than about 1% , less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, less than about 0.5%, less than about 0.4%, or less than about 0.3%, to about 0.2% of the total DAC antibody content upon storage for at least 1 month, 2 months, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, or 36 months at about 2°C to 8°C in aqueous solution at pH of about 5.8 to 6.2. In some embodiments, the isolated DAC antibody prepared by anion exchange chromatography has a level of aggregate which is about or less than about 2% after storage for 36 months at about 2°C to 8°C.

[0121] In some embodiments, the isolated DAC antibody prepared by anion exchange

chromatography and having a level of acidic DAC isoform content above also has a level of aggregate which is about or less than about 2.5%, less than about 2%, less than about 1.5%, less than about 1.4%, less than about 1.3%, less than about 1.2%, less than about 1.1%, less than about 1% , less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, or less than about 0.5% of the total DAC antibody content upon storage for at least 1 month, 2 months, 3 months, 4 month, 5 months, or 6 months at about 23°C to 27°C in aqueous solution at pH of about 5.8 to 6.2. In some embodiments, the isolated DAC antibody prepared by anion exchange chromatography has a level of aggregate which is about or less than about 2.5% after storage for 6 months at about 23°C to 27°C.

[0122] In some embodiments, the isolated DAC antibody prepared by anion exchange

chromatography and having a level of acidic DAC isoform content above also has a level of aggregate which is about or less than about 3.0%, less than about 2.5%, less than about 2%, less than about 1.5%, less than about 1.4%, less than about 1.3%, less than about 1.2%, less than about 1.1%, less than about 1% , less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, or less than about 0.5% of the total DAC antibody content upon storage for at least 1 month or 2 months at about 37°C to 47°C in aqueous solution at pH of about 5.8 to 6. In some embodiments, the isolated DAC antibody prepared by anion exchange chromatography has a level of aggregate which is about or less than about 3.0% upon storage for 2 months at about 37°C to 47°C. [0123] In some embodiments, the isolated DAC antibody prepared by anion exchange

chromatography and having a level of acidic DAC isoform content above also has a level of aggregate which does not increase by more than 2 times, 1.75 times, 1.5 times, 1.25 times upon storage for at least 1 month, 2 months, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, or 36 months at about 2°C to 8°C in aqueous solution at pH of about 5.8 to 6.2. In some embodiments, the isolated DAC antibody prepared by anion exchange chromatography has a level of aggregate which does not increase by more than 2 times upon storage for 36 months at about 2°C to 8°C.

[0124] In some embodiments, the isolated DAC antibody prepared by anion exchange

chromatography and having a level of acidic DAC isoform content above also has a level of aggregate which does not increase by more than 2.5 times, 2 times, 1.75 times, 1.5 times, 1.25 times upon storage for at least 1 month, 2 months, 3 months, 4 month, 5 months, or 6 months at about 23°C to 27°C in aqueous solution at pH of about 5.8 to 6.2. In some embodiments, the isolated DAC antibody prepared by anion exchange chromatography has a level of aggregate which does not increase by more than 2.5 times upon storage for 6 months at about 23°C to 27°C.

[0125] In some embodiments, the isolated DAC antibody prepared by anion exchange

chromatography and having a level of acidic DAC isoform content above also has a level of aggregate which does not increase by more than 4 times, 3 times, 2.5 times, 2 times, 1.75 times, 1.5 times, 1.25 times upon storage for at least 1 month or 2 months at about 37°C to 47°C in aqueous solution at pH of about 5.8 to 6.2. In some embodiments, the isolated DAC antibody prepared by anion exchange chromatography has a level of aggregate which does not increase by more than 4 times upon storage for 2 months at about 37°C to 47°C.

[0126] In some embodiments, the DAC antibody produced by the methods above and having the described characteristics are provided as storage stable aqueous solutions at DAC antibody concentrations of at least about 50 mg/mL, 100 mg/mL, 110 mg/mL, 120 mg/mL, 130 mg/mL, 135 mg/mL 140 mg/mL, 145 mg/mL 150 mg/mL, 155 mg/mL 160 mg/mL, 165 mg/mL 170 mg/mL, 175 mg/mL 180 mg/mL, 185 mg/mL 190 mg/mL, 195 mg/mL, 200 mg/mL, 220 mg/mL, 240 mg/mL, 260 mg/mL, 280 mg/mL, 300 mg/mL, 320 mg/mL 340 mg/mL, or 360 mg/mL. In some embodiments, the DAC composition produced by the methods above and having the described characteristics are provided as storage stable aqueous solutions at DAC concentrations of about 50 mg/mL to 360 mg/mL, 100 mg/mL to 300 mg/mL, 120 mg/mL to 220 mg/mL, or 135 mg/mL to 165 mg/mL. In some embodiments of the foregoing, the DAC antibody is DAC HYP. 6.2. Pharmaceutical Compositions

[0127] In another aspect, the DAC antibody having the characteristics described herein are provided as a pharmaceutical composition. In some embodiments, the antibody compositions are provided as a sterile, pharmaceutical composition with one or more pharmaceutically acceptable carriers. The pharmaceutical compositions can be provided in any suitable form, for example as a powder, lyophilate, or liquid formulation.

[0128] In some embodiments, the pharmaceutical compositions of the DAC antibody compositions can be prepared for storage as lyophilized formulations or aqueous solutions by mixing the antibody having the desired degree of purity with optional pharmaceutically-acceptable carriers, excipients or stabilizers typically employed in the art, all of which are referred to herein as "carriers," including, among others, buffering agents, stabilizing agents, preservatives, isotonicity agents, surfactants, antioxidants, and other additives. See Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Generally, such excipients and additives should be nontoxic to the recipients at the dosages and concentrations employed.

[0129] In some embodiments, the pharmaceutical composition includes a buffering agent to maintain the pH of the composition in the range suitable for administration. In some embodiments, the buffers can be present at concentration ranging from about 1 mM to about 100 mM, about 2 mM to about 80 mM, about 5 mM to about 60 mM, or about 10 mM to about 40 mM. Suitable buffering agents include organic and inorganic acids and salts thereof such as, for example, citrate (e.g. , monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), succinate (e.g., succinic acid monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid disodium succinate mixture, etc.), tartrate buffers (e.g. , tartaric acid- sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate (e.g. , fumaric acid-monosodium fumarate mixture, fumaric acid disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconate buffers (e.g. , gluconic acid-sodium glyconate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid- potassium glyuconate mixture, etc.), oxalate (e.g. , oxalic acid-sodium oxalate mixture, oxalic acid- sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate (e.g. , lactic acid- sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.) and acetate (e.g. , acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.). In some embodiments, the buffer is a phosphate, histidine, aspartate, trimethylamine, or combinations thereof. In some embodiments, the pH of the pharmaceutical composition is at about 5 to about 9, about 5 to about 8, about 5.5 to about 7.5, or about 6 to about 7. In some embodiments, the H of the pharmaceutical composition is at a pH of about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, or about 9.0.

[0130] In some embodiments, the pharmaceutical compositions include a surfactant, particularly a non-ionic surfactant. The surfactant can be added to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation-induced aggregation. Exemplary non-ionic surfactants suitable for the pharmaceutical formulations include, among others, polysorbates (20, 40, 60, 80, etc.), polyoxamers (184, 188 etc.), pluronic polyols, and polyoxy ethylene sorbitan monoethers (TWEEN ^® -20, TWEEN ^® -80, etc.). In some embodiments, the nonionic surfactant can be present in a range of about 0.001 % w/v to about 2% w/v, about 0.005% w/v to about 1.5% w/v, about 0.01% to about 1% w/v, about 0.02% w/v to about 0.5% w/v, or about 0.03% w/v to about 0.1% w/v. In some embodiments, the nonionic surfactant is present at about 0.001% w/v, about 0.005% w/v, about 0.01% w/v, about 0.02% w/v, about 0.03% w/v, about 0.04% w/v, about 0.06% w/v, about 0.08% w/v, about 0.1% w/v, about 0.2% w/v, about 0.3% w/v, about 0.4% w/v, about 0.5% w/v, about 1% w/v, about 1.5% w/v, or about 2% w/v. An exemplary nonionic surfactant is polysorbate 80, preferably at about 0.02% w/v to about 0.04% w/v, more preferably at about 0.03% w/v.

[0131] In some embodiments, the pharmaceutical composition may include a preservative, such as to retard microbial growth. Suitable preserves for use with the present disclosure include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides (e.g. , chloride, bromide, and iodide), hexamethonium chloride, and alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol. In some embodiments, preservatives can be present in amounts ranging from 0.2%-l% (w/v). In some embodiments, the pharmaceutical compositions of isolated DAC antibody, such as aqueous formulations of DAC HYP, do not include a preservative.

[0132] In some embodiments, the pharmaceutical composition includes an isotonicifier, also referred to as "stabilizers." Such stabilizers can be included in the compositions to provide appropriate isotonicity to liquid compositions. Exemplary stabilizers include, among others, polyhydric sugar alcohols, for example trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol. Typical stabilizers can be polyhydric sugar alcohols, such as those enumerated above; amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine; polyethylene glycol; amino acid polymers; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglycerol and sodium thiosulfate; proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; and hydrophilic polymers, such as polyvinylpyrrolidone. Stabilizers can be present in the range from 0.1 to 10,000 weights per part of weight active protein.

[0133] In some embodiments, the pharmaceutical compositions include salts, for example to adjust the ionic strength, isotonicity (osmolality), and/or reduce insolubility of the antibody. In some embodiments, useful salts include NaCl, KC1, MgCh, and CaC . In some embodiments, the salt can be present at about 1 mM to about 300 mM, about 10 mM to about 250 mM, about 20 mM to about 200 mM, or about 50 mM to about 150 mM. In some embodiments, the salt can be present at about 1 mM, about 10 mM, about 20 mM, about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, about 200 mM, about 250 mM, or about 300 mM.

[0134] In some embodiments, a storage stable DAC antibody pharmaceutical composition comprises: 20-60 mM succinate, 0.02% - 0.04% polysorbate 80, 75-150 mM NaCl, DAC antibody at a concentration of 50 mg/ml or more, and a pH of 5.5 to 6.5. In some embodiments, the storage stable pharmaceutical composition comprises:

20-60 mM succinate;

0.02% - 0.04% polysorbate 80;

75-150 mM NaCl, and

100 mg/ml or more of DAC antibody;

pH of 5.5 to 6.5.

[0135] In an exemplary embodiment, the storage stable pharmaceutical composition comprises:

40 mM succinate;

0.03% polysorbate 80;

100 mM NaCl;

135-165 mg/ml DAC antibody; and

pH of about 6.

[0136] In another exemplary embodiment, the storage stable pharmaceutical composition comprises:

40 mM succinate;

0.03% polysorbate 80,

100 mM sodium chloride;

150 mg/mL DAC antibody;

pH of about 6.0.

[0137] In some embodiments, the pharmaceutical compositions can be conveniently presented in unit dose forms containing a predetermined amount of antibody per dose. Such a unit dose can contain, by way of example and not limitation, DAC antibody in an amount of 0.1 mg to 0.5 g, about 20 mg to 500 mg, about 50 mg to 250 mg, or about 100 mg to 300 mg. In some embodiments, the unit dose contains the antibody in an amount of about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 250 mg or about 300 mg of DAC. In some embodiments, the unit doses can be supplied in single use vials or pre-filled syringes or auto-injectors, as further described below. In some embodiments, the DAC antibody in the pharmaceutical compositions above is a DAC HYP antibody.

6.3. Treatments with DAC Antibody and Administration

[0138] In another aspect, the DAC antibody having the characteristics described herein are used to treat disorders associated with CD25 activity. In some embodiments, the DAC antibody compositions described herein are used to treat a subject with an autoimmune disease, such as neuroinflammatory disorders, including multiple sclerosis and neuromyelitis optica (NMO); and ocular inflammatory disease, such as uveitis, birdshot chorioretinopathy, age-related macular degeneration, and cicatrizing conjunctivitis. See, e.g. , U.S. Pat. No. 7,258,859, incorporated herein by reference. In some embodiments, a subject with the specified disorder is administered a therapeutically effective amount of a DAC antibody described herein, particularly a DAC HYP antibody.

[0139] In some embodiments, the DAC antibody compositions, particularly DAC -HYP antibody compositions, are used to treat a subject with multiple sclerosis. In some embodiments, the subject selected for treatment with DAC antibody has relapsing form of multiple sclerosis (RMS). Clinical trials of DAC-HYP for treatment of multiple sclerosis are described in the SELECT trials and the CHOICE study. See, e.g., US 20120301429789; see also Wynn et al, 2010, Lancet Neurol. 9(4):381- 90. Patients with RMS experience discrete episodes of neurological dysfunction (referred to as relapses, exacerbations, or attacks), each lasting several days to several weeks, that occur intermittently over many years. In some embodiments, the subject for treatment with DAC antibody has relapsing remitting multiple sclerosis (RRMS). In some embodiments, the subject for treatment with DAC antibody has progressive multiple sclerosis. In some embodiments, the subject for treatment with DAC antibody has primary progressive multiple sclerosis. In some embodiments, the subject for treatment with DAC antibody has secondary progressive multiple sclerosis, particularly relapsing secondary progressive multiple sclerosis. In some embodiments, the subject for treatment with DAC antibody has relapsing progressive multiple sclerosis.

[0140] In some embodiments, the subject with multiple sclerosis selected for treatment with DAC antibody has either failed to respond to prior treatment with interferon beta or has discontinued prior treatment with interferon beta. In some embodiments, the subject with relapsing multiple sclerosis for treatment with DAC antibody has had at least one relapse while being treated with interferon beta. [0141] The DAC antibody compositions can be administered by a variety routes, taking into consideration the indication to be treated, the formulation, and dosage to be administered. In some embodiments, the DAC antibody of the disclosure can be administered parenterally, including intravenously, intramuscularly, intraperitoneally, or subcutaneously.

[0142] The dosage of a DAC antibody composition to be administered and the frequency of administration will vary according to the type of antibody preparation, the type and severity of the disease or disorder, the subject and physical condition of the subject, the therapeutic regimen (e.g., whether a combination therapeutic agent is used), and the selected route of administration. The DAC antibody compositions are administered in a therapeutically effective amount. In some embodiments, the effective dose of DAC antibody can range from about 0.1 to about 5 mg/kg of DAC antibody per single (e.g., bolus) administration, multiple administrations or continuous administration, or any effective range or value therein depending on the condition being treated, the route of administration and the age, weight and condition of the subject. In some embodiments, each dose of DAC antibody can range from about 0.1 mg/kg to about 4 mg/kg of body weight, 0.1 mg/kg to about 3 mg/kg of body weight, 0.1 mg/kg to about 2 mg/kg of body weight, 0.2 mg/kg to about 1.5 mg/kg of body weight, or about 0.5 mg/gm to about 1.0 mg/kg body weight. In some embodiments, the DAC antibody is administered at a dose of about 1 mg/kg body weight. In some embodiments, the DAC antibody can be administered to a subject in doses ranging from about 25 mg to about 300 mg, about 50 mg to about 200 mg, or about 75 to about 175 mg per dose. In some embodiments, the DAC antibody is administered as a dose of about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg or about 300 mg. The frequency of administration can be weekly, monthly, or bimonthly or any frequency of administration to provide effective treatment of the disease or condition, as determined by the skilled medical personnel. The duration of treatment is for a sufficient time to provide effective treatment of the disease or condition. In some embodiments, the treatment is for at least 4 weeks (about 1 month), 6 weeks, 8 weeks (about 2 months), 12 weeks (about 3 months), 16 weeks (about 4 months), or 24 weeks ( 8 months) or more. In some embodiments, the duration of treatment is for 1 year or more, 2 years or more, or indefinitely as necessary to treat the disease or condition. In some embodiments, the DAC antibody is administered intravenously at about 1 mg/kg once per week for a period of at least 4 weeks, 6 weeks, 12 weeks, or 24 weeks or longer.

[0143] The DAC antibody compositions and formulations described herein are particularly useful for subcutaneous administration. The DAC antibody formulations can be administered subcutaneously to a patient once a month (once every 4 weeks) in doses ranging from about 25 mg to about 300 mg, about 50 mg to about 200 mg, about 75 to about 175 mg per dose, preferably in the range of 100 mg to 300 mg. In some embodiments, the DAC antibody is administered subcutaneously as a dose of about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg or about 300 mg, particularly 150 mg or 300 mg per dose. In some embodiments, the duration of treatment by subcutaneous administration is for about 4 weeks, 8 weeks, 12 weeks, 16 weeks, or 24 weeks or more. In some embodiments, the duration of treatment with DAC antibody is for 1 year or more, 2 years or more, 3 years or more, 5 years or more, 6 years or more, or indefinitely as necessary to treat the disease or condition. In some embodiments, the DAC antibody can be administered subcutaneously at about once every two weeks when used in an induction phase of treatment. A subsequent maintenance phase can use administration once a month.

[0144] For the treatment of multiple sclerosis, particularly relapsing remitting multiple sclerosis, the DAC antibody is administered subcutaneously once per month (every 4 weeks) at 150 mg per dose (concentration of 150 mg/mL). The duration of treatment with DAC antibody for multiple sclerosis is 8 weeks or more, 12 weeks or more, 16 weeks or more, or 24 weeks or more. In some embodiments, the duration of treatment with DAC antibody is for 1 year or more, 2 years or more, 3 years or more, 5 years or more, 6 years or more, or indefinitely as necessary to treat the disease or condition.

[0145] In some embodiments, the DAC antibody can be used as monotherapy, or used in combination with other drug compounds or active pharmaceutical ingredients for treating the disease or disorder. For the treatment of multiple sclerosis, the DAC antibody, particularly DAC-HYP, can be administered as monotherapy.

[0146] In some embodiments, the DAC antibody composition is administered adjunctively with another (second) therapeutic agent. In some embodiments, the second therapeutic agent is interferon- β, particularly for treating multiple sclerosis. Other secondary therapeutic agents that can be used with DAC antibody include, among others, a steroid, for example, corticosteroids such as prednisolone or methyl prednisolone; glatiramir acetate (e.g., Copaxone®); immunosuppressive/immunomodulating agents, such as cyclosporine, fingolimod (Gilenya®); anti-metabolites, such as methotrexate, teriflunomide (Aubagio®); anti -neoplastic agents, such as mitoxantrone, azathioprine, cladribine, and cyclophosphamide; anti-CD20 antibodies, such as ocrelizumab; anti- a4-integrin antibodies, such as natalizumab (Tysabri®); anti-CD52 antibodies, such as alemtuzumab (Lemtrada®); and other agents for treating multiple sclerosis, such as riluzole and dimethyl fumatrate (Tecfidera®). The combination therapeutic agent can be administered concurrently, either simultaneously or successively. As used herein, the DAC antibody composition and the combination therapeutic agent are said to be administered successively if they are administered to the patient on the same day, for example during the same patient visit. Successive administration can occur 1, 2, 3, 4, 5, 6, 7 or 8 hours apart. In contrast, the DAC antibody composition and the combination therapeutic agent are said to be administered separately if they are administered to the patient on the different days, for example, the DAC antibody composition and the combination therapeutic agent can be administered at a 1-day, 2- day or 3-day, one-week, 2-week or monthly intervals. In the methods of the present disclosure, the DAC antibody composition can precede or follow administration of the other therapeutic agent.

6.4. Pharmaceutical Kits

[0147] Encompassed by the present disclosure are pharmaceutical devices or kits containing the compositions of isolated DAC antibody described herein. In some embodiments, the pharmaceutical kit comprises the DAC antibody composition disclosed herein, either in lyophilized form or as an aqueous solution. The kit can include, among others, a combination therapeutic; a device for administering the DAC antibody composition; or pharmaceutical grade water or buffer to resuspend the antibody if the antibody is in lyophilized form.

[0148] In some embodiments, the kit can contain one or more unit doses, two or more unit doses, three or more unit doses, four or more unit doses, five or more unit doses, eight or more unit doses, ten or more unit doses of the DAC antibody composition.

[0149] In some embodiments, the unit dose can be provided in a pre-filled syringe, or in a self- injectable syringe pen. In some embodiments, each prefilled syringe or self -injectable syringe pen contain a dose of 50 mg to 300 mg, 100 mg to 200 mg or 125 mg to 175 mg. In some embodiments, the prefilled syringe or self-injectable syringe pen contains a unit dose of about 50 mg, about 75 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg or about 300 mg of DAC antibody of the disclosure. In an exemplary embodiment, a formulation of DAC HYP is provided at a unit dose of about 150 mg. In some embodiments, the pre-filled syringes and auto-inject syringe pen are single use devices. In some embodiments, the pharmaceutical kit comprises 1 to 5, 1 to 4, or 1 to 3 injectable single use syringes or pens per kit.

7. EXAMPLES

7.1. Example 1 : pH Control and Production of Low Acidic DAC HYP

[0150] Vials of NS0 cells (a mouse myeloma cell line) that have been stably transfected with a DAC expression vector and capable of producing DAC HYP were thawed in a Precision Water Bath Model 180 and expanded every two days as indicated in the inoculum preparation batch records. Basal growth medium PFBM-2 was used in the inoculum expansion process, including the seed bioreactors. PFBM-2 was made by combining the basal powder (HPFM-8), sodium chloride, sodium bicarbonate, and glucose, pH adjusted (if sufficient mixing is provided to produce a slight dimple in the liquid level, pH adjustment is typically not required) and osmolality verified. [0151] The culture was expanded in various sizes of shake flasks to accumulate enough cell mass to inoculate production bioreactors. The target seeding density at each passage was 2.0x10 ⁵ cells/ml. Cultures were grown to a viable cell density of approximately 8.0-12.0 xlO ⁵ cells/ml in an incubator controlled to 37°C and 7.5% CO2 and agitated at 100 rpm using a Bellco Orbital Shaker platform.

[0152] The shake flasks were passaged within 48 hours (± 2hrs) after inoculation and sampled by aseptically removing approximately 1.5 ml of culture in a Biosafety Cabinet (BSC). The sample was then assayed for pH, pCC , and pC using the Radiometer ABL-5 blood gas analyzer (using approximately 85 μΐ,). The ABL-5 measurements were used to verify that the culture was growing under the correct conditions of pH and dissolved oxygen. The sample was also assayed using a Cedex to measure viable cell density (VCD) and viability.

[0153] Cell culture was passaged for 14-19 days prior to the inoculation of the production bioreactors.

[0154] Prior to inoculating the 2 L production bioreactors, the DAC cultures were grown in water jacketed Applikon 3 L glass N-l seed bioreactors. The operating conditions of the N-l seed bioreactors are listed in Table 1. A variable split ratio was used at the time of N-l seed bioreactor inoculation to target a starting density of approximately 2.3 xlO ⁵ cells/mL. The N-l seed bioreactors were sampled on Day 0 after inoculation and then again on Day 1 and Day 2. Each sample was assayed using a BGA for pH, pC02, and p02, for YSI glucose and lactate, and for Cedex VCD and viability. The BGA measurements were used to verify proper functioning of the pH probe (via the offline pH and pCC measurement) and the DO probe (via the offline p02 measurement).

Split ratio at inoculation Variable

Seed bioreactor stage duration 2 days ± 4 hours

[0155] After the sample was taken on Day 2, the contents of the N-l bioreactors were pumped into sterile bottles. The culture was then transferred into water jacketed Applikon 3 L glass production bioreactors.

[0156] The bioreactors were inoculated using a fixed split ratio of 1:4.93 seed:total volume. Prior to inoculating with 213mL of seed culture, each production bioreactor was filled with 837 mL of PFBM- 2 or PFBM-2 + 20X Cu basal medium and warmed overnight prior to inoculation with pH and DO controls enabled. The N-l seed bioreactors were grown to a density of approximately 9 xlO ⁵ to 12 x 10 ⁵ viable cells/mL in order to target a seeding density of the production bioreactor of approximately 2 x 10 ⁵ to 3 x 10 ⁵ viable cells/mL at the time of sampling. In addition to the cell culture inoculum, 0.48 mL of 3% Antifoam C solution was added into the inoculum bottle prior to inoculation (this equates to an Antifoam concentration of 8 ppm based on the expected harvest volume of 1800 mL).

[0157] The production bioreactor operating conditions shown in Table 2 and Table 3 and the feed schedule is shown in Table 4.

Table 2

Production Bioreactor operating procedures

Prior to Inoculation

Media held in bioreactor at 37°C for ~ 24 hours

pH, temperature and DO controls turned on

Gasflow and agitation turned on, no overlay

DO and pH probe standardization prior to inoculation

Sparging at 100 seem performed for 100% DO calibration with pH control turned on first pH recalibration decision made using Day 0 sample

Operation /sampling

pH recalibrated if offline and online differ by≥ 0.02

Bioreactor condenser not connected to water supply

Feeding: See Feed Schedule Table

Light Exposure Mitigation

Bioreactor aluminum foil wrapped and feed bottle held in a dark room

Table 3

Product ion Itior eaclor Operating Conditions

Parameter Parameter Value

Agitation (rpm) 325 (constant)

Bioreactor baffles (Yes/No) No

60/1 (~1 cm above shaft bottom) / Applikon

Impeller size (mm) / number /type

marine scoping Sparger Type Applikon drilled hole

Temperature (°C) 37.0

pH Setpoints 6.90, 7.00, 7.20

pH Deadband ±0.05

Dissolved Oxygen 30%

Dissolved Oxygen Control Strategy Ratio control, total gasflow constant

Total gasflow (seem) 12: Days 0 to 5; 9: Days 5 post feed to Day 10

Seed split ratio, basal medium (mL),

1:4.93, 837, 213

cells added (mL)

Initial working volume (L) 1.05

Daily sampling volume (mL) 10

Overlay gasflow (seem) No overlay

Carbon dioxide gas (rotameter set at 20 seem) / pH control strategy

liquid 1M Na ₂ C0 ₃ base

[0158] For each production bioreactor sample time point (sample taken once daily, or twice if necessary for pH verification), prior to removing a liquid sample for analysis, the bioreactor controller parameters of online pH, temperature, online dissolved oxygen percentage, air flow, oxygen flow, and agitation were recorded. For each sample time point, 5 mL of waste was removed from the sample line and then 5 mL of sample was collected. The sample was assayed for offline pH, pC02, and p02 using an ABL-5 BGA; assayed for glucose and lactate concentrations using the YSI 2700 analyzer; assayed for glutamine, glutamate, and ammonium ion concentration using a Nova Bioprofile lOOplus; and titer assayed using an Agilent protein A HPLC system beginning on Day 2 or 3.

[0159] At harvest the bioreactor contents were chilled to less than 15°C and then pH adjusted to 5.0 ± 0.1 using 1M Citric acid. Then the pH adjusted harvest was held for approximately 45 minutes to allow enough time for full flocculation, centrifuged at 7000 x g's, and then pumped through Millipore 0.027 m ² B1HC Pod filters at 110 L/m ² /hr. The Pods were then flushed with about 600 mL of deionized water to ensure full antibody recovery. Finally, the pH was titrated to pH 7.0 ±0.1 using 2 M Tris. This procedure was performed for each pool of a given bioreactor condition and then separately processed through purification to formulated bulk.

[0160] Cell culture pH was controlled around a range of set-points, detailed in Table 5.

7.1.1. Results

[0161] The formulated bulk DAC antibody was then assayed by CEX with the results shown in FIGURE 3. There is a clear trend in reduced percent acidic DAC isoform species in the formulated bulk as a function of pH setpoint in the production bioreactor. Similar results are observed for DAC antibody intermediate process when cell harvest samples are acidified, held, batch centrifuged, depth filtered, and then neutralized prior to protein A based isolation and analyzed for ACS by CEX.

[0162] After the harvest material was processed using the full DAC HYP downstream purification steps (without viral filtration), it was concentrated to 100 mg/mL subject to stability testing at 5°C for thirty days. The results, shown in FIGURE 4 indicate that there is a correlation between the cell culture production bioreactor pH setpoint and rate of aggregation in formulated bulk.

[0163] The pH 7.2 condition begins with the lowest level of aggregate and over 30 days exhibits the smallest increase, and does not go above 0.8%. The pH 7.0 condition begins at a lower initial aggregate level but tracking almost identically with the standard pH 7.0 condition aggregate trend. The pH 6.9 condition exhibited the highest absolute aggregate amount at 30 days as well as the greatest increase from its starting value.

[0164] Examining the data in FIGURE 3 and FIGURE 4, it can be seen that there is a link between higher acidic isoform content and higher amount of aggregate and rate of aggregation formation.

7.2. Example 2: Screening of Chromatographic Conditions for Removal of Acidic Species from DAC-HYP

[0165] Eight conditions were tested in a DOE (Design of Experiment) study for Q-Sepharose anion exchange removal of acidic species from DAC HYP preparations. A column with 1.1 cm (inner diameter) was packed with Q-Sepharose Fast Flow (QSFF) Resin (GE Healthcare, Piscataway, New Jersey) to 20.1 cm bed height (19.5 mL packed volume) with an ΑΚΤΑ Purifier Chromatography Workstation (GE Healthcare, Piscataway, NJ). All chromatography steps were performed in down- flow mode and at a flow rate of 100 cm/hr. Both the ΑΚΤΑ Purifier Chromatography Workstation, the QSFF column, buffers and load were set up in the cold room maintained at 2 - 8 ^° C. QSFF flow through collection criteria started at 0.10 AU (AU = absorbance unit) up trend and ended at 0.10 AU down on 280 nm absorbance trace measured over 0.2 cm path length.

[0166] A single buffer (20 mM Tris and 20 mM NaCl) was used for column equilibration and wash, with conductivity adjusted using NaCl.

[0167] The conditions tested are listed in Table 6. All runs had a load capacity of 30 mg/ml resin, pH values (measured at 2-8 °C) ranged from 8.1 - 8.5 with a center point of pH 8.3, and conductivity ranged from 2.0 - 3.0 mS/cm for the load and from 2.0 - 4.0 mS/cm for the wash buffer.

[0168] Q flow-through samples from each run were analyzed for product quality (primarily aggregates), process consistency (step yield), and acidic isoforms distribution (acidic pEpE). Results are show in Table 7.

7 92.9 0.4 13.7

8 94.1 0.7 16

'SEC-HPLC

² Charged Isoforms Distribution, CEX-HPLC.

[0169] It shows that acidic species in the Q-Sepharose column flow through product ranged from 11.1 to 16 depending on the process conditions. More acidic species were removed at higher pH and lower conductivity conditions, but with the drawback of a lower step yield. This set of experiment clearly showed that acidic species can be removed by Q column.

7.3. Example 3 : Preparation of Low Acidic Isoform Antibody Compositions for Stability Testing

[0170] DAC -HYP was purified using Q-Sepharose chromatography under conditions to reduce the amount of acidic species.

[0171] ΑΚΤΑ Purifier Chromatography Workstation (GE Healthcare, Piscataway, NJ) was used, with GE XK column 2.2 cm (inner diameter) packed with Q-Sepharose Fast Flow (QSFF) Resin (GE Healthcare, Piscataway, New Jersey). A bed height of 20 cm was used, and the column was equilibrated with equilibration buffer containing 20 mM Tris and sufficient amount of NaCl to reach 3 mS/cm conductivity and at pH 8.2 (pH measured at 20°C). Solution of DAC HYP, also at 3 mS/cm and pH 8.2, was then loaded on the column at 30 g/L of resin and at a flow rate of 100 cm/hr. When the flow through showed a UV measurement of higher than 60 mAU, the eluate was collected, and the column was washed with equilibration buffer until the UV measurement dropped below 60 mAU. The column was then cleaned with a solution of 0.5 M NaCl and sanitized with 0.5M NaOH. A typical chromatogram is shown in FIGURE 5, The product flow through peak is the UV absorbance solid blue line from 240 min to 320 min, The acidic species being retained by Q-Sepharose column and then eluted by 0.5 M NaCl cleaning solution is shown in the blue line UV peak at approximately 360 min. They were collected separately and analyzed for acidic species by analytical CEX method specified in Example 4. An overlay of the CEX chromatograms is shown in FIGURE 6, in which black line is flow through product and the red line is retained fraction by Q-Sepharose column. As shown in FIGURE 6, the acidic species are in the region between 10 to 16 min. The red line clearly showed significant amounts of acidic species in the retained fraction compared to the black line for flow through product.

[0172] The flow through product from this experiment was further purified by CEX and UF/DF and prepared at 150 mg/mL formulation according to the process described in U.S. Patent Publication 2012/0301429. At the same time, the same lot of DAC HYP was purified and formulated according to the process described in U.S. Patent Publication 2012/0301429 without optimizing Q column process conditions. These two formulated bulks were put on stability study at 2-8°C and the results are shown in FIGURE 7, which shows that the bulk with acidic species removed by Q column (black line) has significant lower starting aggregate at time zero, and the rate of aggregate increase during the first several weeks of storage at 2°C -8°C is also much lower as compared to bulk without acidic species removed (red line). This once again demonstrated the strong correlation of acidic species in the bulk and the aggregation of DAC HYP during storage.

7.4. Example 4: Acidic Isoform Assay

[0173] Quantitating the acidic isoform content as well as the content of other Daclizumab charge variants is carried out by separation by HPLC cation-exchange chromatograpy (CEX). This method applies to the analysis of Daclizumab process intermediates, drug substance and drug product. The chromatographic separation uses Propac WCX-10 column, 4.0 x 250 mm (Dionex, USA). ProPac WCX-10 is a weak cation exchanger having a carboxylate functional group on 10 μιη nonporous, polymeric beads and coated with a hydrophihc layer. Column is stored in 20 mM Sodium Phosphate with 0.1% azide, pH 6.5. The HPLC system is a Waters 2695 or equivalent.

[0174] Sample preparation. DAC HYP reference samples and samples for analysis are adjusted to about 1 mg/mL protein with Mobile Phase A (MP A) solution (20 mM MES, 2 mM oxalic Acid, pH 5.9). The volume of solution to be prepared is based on the number of injections, with each injection being about 50 uL of the 1 mg/mL Daclizumab solution). MPA is used for Blank Injections.

[0175] Analysis procedure. Prepared samples are analyzed on the CEX column by conditioning the column with a reference sample followed by blank samples. Actual samples are then analyzed. After a certain number of samples have been analyzed, the column is retreated with a reference sample before a second set of actual samples are examined. An exemplary regimen is provided is Table 8.

[0176] The chromatographic conditions and mobile phase characteristics are shown in Table 9 and Table 10, respectively: Table

Chromatographic Conditions

Column: ProPac WCX-10

Column Temperature: 50°C

Flow rate: 1 mL/min

Detection: 280 nm

Mobile Phase A (MP A) 20 mM MES, 2 mM oxalic acid, pH 5.9

Mobile Phase B (MPB) 20 mM MES, 240 mM NaCl, 2 mM oxalic acid, pH 5.9

[0177] Data analysis: The chromatograms are analyzed using Empower chromatography data software (Waters, USA). The parameters for integration of the chromatograms are provided in Table 11.

[0178] It is to be understood that the integration parameters can be modified to obtain integration of the chromatogram peaks.

[0179] Calculation of Acidic DAC isoform Content. The percentage of the acidic DAC isoform content is estimated from the acidic-pEpE content, also referred to as % Acidic pEpE, relative to the total pEpE content. The percentage of acidic-pEpE is determined by integrating the area under the curve of the peaks encompassing the acidic-pEpE species and the area under curve of the main pEpE and basic pEpE species and calculating the percentage of acidic-pEpE species based on the following formula:

% Acidic pEpE = 100% ^χ Acidic pEpE / (Acidic pEpE + Main pEpE + Basic pEpE), where: (a) Acidic pEpE = sum of peak areas of all peaks eluting prior to Main pEpE peak; (b) Main pEpE = peak area of Main pEpE peak, and (c) Basic pEpE = sum of peak areas of all peaks eluting after then Main pEpE peak and prior to the pEQ-OK peak. A chromatogram showing the relevant peaks for calculating the percentage of acidic-pEpE species is shown in FIGURE 8.

7.5. Example 5 : Aggregates Assay

[0180] The aggregate content and the percent main DAC HYP antibody product in the process intermediates, drug substance and drug product are determined by size exclusion chromatography (SEC). The chromatographic medium and system, and the chromatographic conditions for SEC are provided in Table 12 and Table 13, respectively.

[0181] If the columns are to be stored for more than 12 hours between assays, it is flushed and stored in 10 % methanol using a 0.5 mL/min flow rate for 3 hours.

Table 13

( hroiiialoarapln condit ions

Flow rate: 1 mL/min, isocratic

Injection load 300 ug

Sampling rate 1 pt/sec

Autosampler temperature 5°C +/- 4°C

Column temperature: 18 - 30°C

Run time 15 min

Mobile phase program isocratic

Needle wash: 10% methanol

Seal wash: 10% methanol

[0182] Injection protocol. The chromatographic column is conditioned by running mobile phase and reference sample prior to sample preparation and analysis. An exemplary injection protocol is shown in Table 14:

1 injection/sample

[0183] Reference sample is used to bracket samples every 12 injections or when closing out the run. If more than 12 samples are assayed, the injection protocol can be altered as long as samples are bracketed by reference injections and a minimum of 5 reference injections are completed per assay.

[0184] Sample preparation. Sample is diluted to 20 mg/mL with cold mobile phase solution (2-8°C). If the sample concentration is less than 20 mg/mL, an injection volume equivalent to approximately 300 μg (+/- 20 μg) is used. If sample concentration is less than 3.0 mg/mL, about 100 μL of the sample is injected. Samples are vortexed briefly to mix and immediately place in cooled autosampler prior to sample analysis.

[0185] Determining system suitability. For each individual reference injection, the chromatographic system suitability values are assessed for each of the following parameters via chromatography software or manually.

USP Theoretical Plates flsT)*

N = 16(Rt/W) ²

N = plate count

Rt = Retention time

W = Peak width at the baseline determined by tangents drawn to the % of peak height

% = tangent % entered by user (use 61%)

*System is suitable when N is greater than 3,000

USP Re

R = resolution

Rt = retention times

W1+W2 = sum of tangent peak widths at 50%

C - constant of 2.0

*System is suitable when R is greater than 1.6.

Retention time

Retention time of the main peak must be between 6.8 and 8.3 minutes

Main Peak Height

Peak height of Main Peak must be between 1,000,000 μΥ and 1,600,000 μΥ at 280 nm. [0186] Data analysis. Analysis of the chromatograms is performed using software for the chromatographic system. All peaks that are not present in the mobile phase injection are integrated. Exemplary parameters for integration of peaks are provided in Table 15.

[0187] For identifying the main peak, it is the largest peak, with a retention time of approximately 7.7 minutes. The aggregate includes all peaks that elute before the Main Peak (excluding mobile phase peaks). The clip includes all peaks that elute after the Main Peak (excluding mobile phase peaks).

[0188] Peak area determination. Total Peak Area is determined by summing all integrated peak areas in the chromatogram. The % Area Aggregate is determined by summing the areas of all peaks of greater molecular weight than main peak (i.e., peaks eluting before the main peak), dividing by the Total Peak Area, and then multiplying by 100. The % Area Main Peak is determined by dividing the Main Peak area by the Total Peak Area, and then multiplying by 100.

[0189] Acceptance Criteria. Main peak height must be between 1,000,000 μν and 1,600,000 μν at 280 nm. The foregoing acceptance criteria need not be met if the sample concentration is less than 3.0 mg/mL.

[0190] A SEC chromatogram of DAC HYP for determining aggregate level is shown in FIGURE 9. The SEC chromatogram shows the integration of the aggregate peak eluting at 6.635 min; the main peak eluting at 7.779 min; and the clip peak eluting at 9.617 min.

[0191] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes. [0192] While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the subject matter herein.