US20120301429A1 - Dac hyp compositions and methods - Google Patents

Dac hyp compositions and methods Download PDF

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Publication number
US20120301429A1
US20120301429A1 US13/481,081 US201213481081A US2012301429A1 US 20120301429 A1 US20120301429 A1 US 20120301429A1 US 201213481081 A US201213481081 A US 201213481081A US 2012301429 A1 US2012301429 A1 US 2012301429A1
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Prior art keywords
daclizumab
dac hyp
composition
dac
cell
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Inventor
Taymar E. Hartman
Paul W. Sauer
John E. Burky
Mark C. Wesson
Ping Y. Huang
Thomas J. Robinson
Braeden Partridge
J. Yun Tso
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AbbVie Biotherapeutics Inc
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AbbVie Biotherapeutics Inc
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Assigned to ABBOTT BIOTHERAPEUTICS CORP. reassignment ABBOTT BIOTHERAPEUTICS CORP. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BURKY, JOHN E, Partridge, Braeden, SAUER, PAUL W, ROBINSON, THOMAS J, WESSON, MARK C, HUANG, PING Y, HARTMAN, TAYMAR E, TSO, J. YUN
Publication of US20120301429A1 publication Critical patent/US20120301429A1/en
Assigned to ABBVIE BIOTHERAPEUTICS INC. reassignment ABBVIE BIOTHERAPEUTICS INC. CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: ABBOTT BIOTHERAPEUTICS CORP
Priority to US14/601,909 priority patent/US9676860B2/en
Assigned to ABBVIE BIOTHERAPEUTICS INC reassignment ABBVIE BIOTHERAPEUTICS INC CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: ABBOTT BIOTHERAPEUTICS CORP
Priority to US14/671,653 priority patent/US9340619B2/en
Priority to US14/735,062 priority patent/US9260528B2/en
Priority to US15/587,127 priority patent/US9815903B2/en
Priority to US15/724,443 priority patent/US20180127506A1/en
Priority to US16/779,336 priority patent/US20200157230A1/en
Abandoned legal-status Critical Current

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Definitions

  • Daclizumab is a humanized IgG 1 monoclonal antibody that binds to the alpha subunit (CD25 or Tac) of the human high-affinity interleukin-2 (IL-2) receptor, which is expressed on the surface of activated, but not resting, T- and B-lymphocytes.
  • IL-2 interleukin-2
  • DAC binds to CD25 with an approximate binding affinity (K D ) of 0.3 nM, and inhibits the proliferation of PHA blasts in a dose-dependent manner (Hakimi et al., 1993, J. Immunol. 151(2):1075-85).
  • K D approximate binding affinity
  • 15 nM DAC inhibits proliferation of IL-2-dependent cell line Kit225/K6 by 50% (Pilson et al., 1997, J. Immunol. 159(3):1543-56).
  • a version of DAC was previously marketed for the treatment of acute allograft rejection in renal transplant patients as an adjunct to an immunosuppressive regimen that includes cyclosporine and corticosteroids by Hoffman-La Roche, Inc. under the tradename ZENAPAXTM.
  • ZENAPAX was supplied as a concentrate for further dilution and intravenous administration.
  • Each vial of concentrate contained 5 mL of a solution containing 5 mg/mL DAC, 3.6 mg/mL sodium phosphate monobasic monohydrate, 11 mg/mL sodium phosphate dibasic heptahydrate, 4.6 mg/mL sodium chloride, 0.2 mg/mL polysorbate 80 and HCl and/or NaOH sufficient to adjust the pH to pH 6.9.
  • the recommended dose for both adult and pediatric patients was 1.0 mg/kg, prepared by diluting the calculated volume of 25 mg/5 mL ZENAPAX concentrate with 50 mL sterile 0.9% sodium chloride solution and administering intravenously via a peripheral or central vein over a 15-minute period.
  • DAC has also shown efficacy in the treatment of uveitis (Nussenblatt et al., 2004, FOCIS 2004 meeting; July 18-23, Montreal, QC. Abstract 4688; Nussenblatt et al., 2003, J. Autoimmun. 21:283-93) and multiple sclerosis (see, e.g., Bielekova et al., 2004, Proc. Nat'l. Acad. Sci. USA 101(23):8705-8708; Rose et al., 2007, Neurology 69:785-789; U.S. Pat. No. 7,258,859), and is currently the subject of ongoing clinical trials for the treatment of multiple sclerosis.
  • DAC has proven to be safe and effective, high concentration, liquid formulations that have long shelf lives and that can be conveniently administered without further formulation or manipulation, as well as new daclizumab molecules that have improved properties, such as enhanced safety, as compared to ZENAPAX DAC, would be desirable.
  • daclizumab is a humanized IgG 1 antibody that specifically binds the alpha subunit (also referred to as CD25 or Tac) of the human interleukin-2 receptor (IL-2R), which is an important mediator of lymphocyte activation.
  • IL-2R human interleukin-2 receptor
  • a version of daclizumab previously marketed by Hoffman-La Roche under the tradename ZENAPAXTM has demonstrated safety and efficacy in the treatment of renal allograft rejection when used as an adjunct to an immunosuppressive regimen including cyclosporine and corticosteroids (see, e.g., the European Medicines Agency (“EMEA”) market authorization for ZENAPAX), and has also demonstrated efficacy in the treatment of multiple sclerosis (see, e.g., Bielekova et al., 2004, Proc. Nat'l. Acad. Sci. USA 101(23):8705-8708; Rose et al., 2007, Neurology 69:785-789; U.S. Pat. No. 7,258,859).
  • EMEA European Medicines Agency
  • ZENAPAX DAC is expressed in GS-NS0 (murine myeloma) cells and purified using a process that involves Q-Sepharose chromatography, S-Sepharose chromatography, diafiltration, Q-Sepharose II chromatography, ultrafiltration, S-300 gel filtration chromatography and ultrafiltration. It has now been discovered that daclizumab expressed in an NS0 cell line that has been adapted to grow in serum-free, cholesterol-free and other animal product-free media and that is isolated by a different process has characteristics and properties that are different from, and in some instances, superior to, ZENAPAX daclizumab (“ZENAPAX DAC”).
  • This new daclizumab referred to herein as DAC HYP, has: a different isoform profile than ZENAPAX DAC (as determined via cation exchange chromatography); a different N-linked glycosylation profile than ZENAPAX DAC, even though both forms of daclizumab are expressed in NS0 cells; and less ADCC cytotoxicity than ZENAPAX DAC in biological assays.
  • the amino acid sequence of the mature V H chain of daclizumab begins at position 20 of the amino acid sequence shown in FIG. 2 (SEQ ID NO:4).
  • the N-terminal glutamine (Q) of the mature V H chain (in bold, underlined text in FIG. 2 ) can cyclize, forming pyroglutamate (pE).
  • the signal peptide sequence may truncate, leaving a valine-histidine-serine (VHS) sequence attached to the N-terminal glutamine residue of the mature V H chain.
  • VHS valine-histidine-serine
  • the various N-terminal isoforms of daclizumab 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 (0K, 1K or 2K), resulting in a complex isoform profile.
  • the cation exchange chromatogram of DAC HYP is characterized by a pE/Q isoform peak and a Q/VHS isoform peak. While not intending to be bound by any theory, it is believed that these unique pE/Q and Q/VHS isoforms may be influenced by the leader sequence used to express DAC HYP.
  • the present disclosure provides daclizumab compositions in which the pE/Q isoform ranges from 3%-17%, from 3%-15%, from 5%-15%, more preferably from 5%-12% or 7%-12% of the N-terminal isoforms, and/or in which the Q/VHS isoform ranges from 1%-15%, more preferably 3%-12% of the N-terminal isoforms, as determined by cation exchange chromatography.
  • the daclizumab composition is characterized by a cation exchange chromatography profile that is substantially similar to FIG. 18 or the DAC HYP profile of FIG. 23 .
  • Daclizumab has N-linked oligosaccharides attached to heavy chain residue Asn 296.
  • N-linked oligosaccharides are released using amidase PNGaseF and analyzed via HPLC, DAC HYP exhibits a glycosylation profile different from ZENAPAX DAC, despite the fact that both are recombinantly produced in NS0 cell lines. Indeed, the glycosylation profile of DAC HYP is unusually homogeneous. Referring to the upper panel of FIG.
  • the glycosylation profile of ZENAPAX DAC is characterized by peaks representing oligosaccharides G0-GlcNAc, G0, G1, Man5, G2, Man6, Man7 and sialylated oligosaccharide.
  • the lower panel of FIG. 21 shows that the glycosylation profile of DAC HYP is characterized by two main peaks corresponding to G0-GlcNAc glycoforms and G0 glycoforms and a minor peak corresponding to a G1 glycoform.
  • the G0-GlcNAc glycoforms can range from about 5% to about 20% of the AUC, more typically about 7.2% to 14.6% of the AUC.
  • the G0 glycoforms can range from 70% to 99.2% of the AUC, more typically from 80.9% to 99.2% of the AUC.
  • the G1 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.
  • Immunogenicity and high levels of effector function can be problematic for chronically administered drugs.
  • rapid clearance rates can reduce drug availability.
  • differences in glycosylation patterns of therapeutic antibodies can give rise to differences in immunogenicity.
  • Antibodies having highly homogeneous glycosylation patterns like DAC HYP may provide beneficial immunogenicity profiles, ADCC levels, and clearance rates.
  • biologics having more homogeneous glycosylation patterns reduce batch to batch variation and can improve consistency and stability.
  • the present disclosure provides daclizumab compositions that are characterized by a homogeneous N-linked glycosylation profile.
  • the daclizumab composition is characterized by an N-linked glycosylation profile that includes approximately 5-20% of the total AUC of G0-GlcNAc glycoforms, and in some embodiments approximately 5%-18% or approximately 7-15% (e.g., 7.2%-14.6% or 6.9%-14.7%) of the total AUC of G0-GlcNAC glycoforms (and in some specific embodiments 7.3% of the total AUC of G0-GlcNAc glycoforms), and approximately 70%-99.2% of the total AUC of G0 glycoforms, and in some embodiments approximately 75%-90%, approximately 75-92%, or approximately 81-88% of the total AUC of G0 glycoforms (and in some specific embodiments 86% of the total AUC of G0 glycoforms), as measured by HPLC.
  • the G1 peak is less than about 10% of the total AUC, less than about 5%, less than about 4% or less than about 3% of the total AUC and, in certain embodiments, ranges from about 1% to about 4% (e.g., 1.4% to 3.8%) or about 1% to about 3%.
  • the Man5 glycoforms are preferably about 3% of the total AUC or less.
  • the daclizumab composition is characterized by an HPLC N-linked glycoform profile substantially similar to a profile illustrated in FIG. 19 .
  • a daclizumab composition of the disclosure is characterized by the sum total of two or more glycoform peaks.
  • the daclizumab compositions of the disclosure are characterized by (a) two main peaks corresponding to G0-GlcNAc glycoforms and G0 glycoforms which together range from about 75% to about 100%, from about 80% to about 100%, or about 85% to about 100% of the total AUC and/or (b) peaks corresponding to Man5, Man6, and Man7 glycoforms which together are about 6% of the total AUC or less and/or (c) peaks corresponding to Man6 and Man7 glycoforms which together are about 2% of the total AUC or less.
  • the percentage of G0-GlcNAc G0, G1, and/or Man5 can be present in the amounts described in the preceding paragraph.
  • DAC HYP exhibits significantly less ADCC cytotoxicity than ZENAPAX DAC, which is likely due, at least in part, to differences in their non-fucosylated mannose glycosylation levels (see FIG. 21 ). As shown in FIG. 22A and FIG. 22B , DAC HYP exhibits at least 25% less ADCC cytotoxicity than ZENAPAX DAC as measured in a cellular assay.
  • the reduced ADCC cytotoxicity of DAC HYP may be beneficial for indications involving chronic administration where cell death is not desirable, for example, for the treatment of multiple sclerosis or uveitis.
  • DAC HYP therapy may be safer than therapy with ZENAPAXTM.
  • the disclosure provides daclizumab compositions that are characterized by exhibiting ADCC cytotoxicity of less than about 30%, 25%, 20%, 15%, 10%, 5%, or even lower, at a concentration of 1 ⁇ g/mL as measured in an in vitro assay using an effector to target cell ratio of 25:1, 40:1, 50:1 or 60:1, for example when using Kit225/K6 as a target cell and/or when using PBMC effector cells from 3 or more, 6 or more, 10 or more, or 50 or more healthy donors.
  • the disclosure provides daclizumab compositions that are characterized by exhibiting ADCC cytotoxicity ranging from 5-30%, from 10-30%, from 15-30%, from 15-30%, from 5-25%, from 10-25%, from 20-30%, from 15-25%, from 15-35%, or from 20-35% at a concentration of 1 ⁇ g/mL as measured in an in vitro assay using an effector to target cell ratio of 25:1, 40:1, 50:1 or 60:1, for example when using Kit225/K6 as a target cell and/or when using PBMC effector cells from 3 or more, 6 or more, 10 or more, or 50 or more healthy donors.
  • the lower levels of ADCC cytotoxicity observed with DAC HYP as compared to ZENAPAX DAC are surprising given that DAC HYP is an IgG 1 immunoglobulin and does not contain framework mutations known to reduce ADCC cytotoxicity.
  • the safety profile of DAC HYP as compared to ZENAPAX DAC may be further improved by the use of a high yield serum free process, that permits the production of a highly pure product free of bovine serum albumin (BSA). Accordingly, the present disclosure provides a daclizumab composition that is free of BSA and/or is the product of a cell culture process in which BSA is not present.
  • BSA bovine serum albumin
  • Daclizumab compositions characterized by one or more of the properties discussed above can be conveniently obtained via recombinant expression in mammalian cells. While not intending to be bound by any particular theory of operation, it is believed that one or more of the unique characteristics and/or properties discussed above may be due, at least in part, to the use of a high productivity recombinant expression system. This can be achieved by any method, such as by gene amplification using the DHFR, or using a selectable marker gene under the control a weak promoter, preferably in combination with a strong promoter driving the expression of the protein of interest (preferably a secreted protein).
  • the weak promoter driving the expression of a selectable marker is an SV40 promoter (Reddy et al., 1978, Science 200:494-502) in which the activity of one or both enhancer regions has been reduced or eliminated, such as by partial or complete deletion, optionally in combination with a strong promoter, such as the CMV IE promoter (Boshart et al., 1985, Cell 41(2):521-30), driving expression of the protein of interest.
  • the disclosure provides vectors useful for generating recombinant cell lines that stably express high levels of a daclizumab such as DAC HYP, in which expression of the selection marker is under the control of an SV40 promoter whose enhancer function has been reduced, such as by partial deletion of one or both enhancer sequences (designated dE-SV40).
  • a specific dE-SV40 promoter sequence that can be used to produce stable expression cell lines is at positions 6536-6735 of vector pHAT.IgG1.rg.dE (SEQ ID NO:5), illustrated in FIG. 3A-FIG . 3 D, and in FIG. 3E (SEQ ID NO: 12).
  • vectors useful for expressing a daclizumab such as DAC HYP will include one or more of the features exemplified by pHAT.IgG1.rg.dE (described in Section 7.1 below), such as a promoter.
  • 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.
  • 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 (at positions 0001-0623 and 3982-4604 of pHAT.IgG1.rg.dE (SEQ ID NO:5)). Additional features include transcriptional initiation sites (if absent from the promoter chosen), transcription termination sites, and origins of replication. Examples of such features are illustrated in Table 1, which outlines the components of pHAT.IgG1.rg.dE.
  • a specific embodiment useful for expressing both heavy and light chains of a daclizumab such as DAC HYP from a single exogenous nucleic acid, in NS0 cells utilizes a selection marker operable in mammalian cells, such as neomycin phosphotransferase (neo r ), hygromycin B phosphotransferase (hyg r ), hygromycin B phosphotransferase (Hph), puromycin-N-acetyltransferase (puro r ), blasticidin S deaminase (bsr r ), xanthine/guanine phosphoribosyl transferase (gpt), glutamine synthetase (GS) or Herpes simplex virus thymidine kinase (HSV-tk).
  • neomycin phosphotransferase neo r
  • the selectable marker in a vector of the disclosure is an E. coli guanine phosphoribosyl transferase selectable marker under the control of an enhancer-less SV40 promoter, the encoding sequence of which can be found at positions 6935-7793 of pHAT.IgG1.rg.dE (SEQ ID NO:5) shown in FIG. 3A-FIG . 3 D.
  • the disclosure provides host cells transfected with vectors useful for recombinantly producing daclizumab, such as for example, DAC HYP.
  • the host cell may be any mammalian cell, including, for example, Chinese Hamster Ovary (CHO) cells, NS0 murine myeloma cells, Sp2/0 cells, PER.C6 cells, Vero cells, BHK cells, HT1080 cells, COST cells, WI38 cells, CV-1/EBNA cells, L cells, 3T3 cells, HEPG2 cells, MDCK cells and 293 cells.
  • the vector Once transfected, the vector may integrate into the genome to yield a stable production cell line. Skilled artisans will appreciate that it is undesirable to include animal products in compositions designated for administration to humans.
  • host cells that do not require serum or other animal products for growth are preferred.
  • Host cells that require such animal products can be adapted to utilize serum-free and other animal product-free medium.
  • a method for adapting murine myeloma NS0 cells to grow in serum- and cholesterol-free medium is described in Hartman et al., 2007, Biotech. & Bioeng. 96(2):294-306 and Burky et al., 2007; Biotech. & Bioeng. 96(2):281-293.
  • a specific strain of NS0 cells adapted to grow in serum-free and cholesterol-free medium that has been stably transfected with a vector as described above that can be used to produce DAC HYP (clone 7A11-5H7-14-43, also referred to as Daclizumab dWCB IP072911) has been deposited with the American Type Culture Collection (ATCC), under accession no. ______.
  • the basal and feed media used to culture cells for recombinant protein production can affect the yield and quality of the expressed protein. Methods of optimizing these conditions are within the purview of a skilled artisan; exemplary conditions are set forth in the Exemplary Embodiments of the disclosure.
  • cells are adapted to grow in media free of cholesterol-, serum-, and other animal-sourced components; in such instances the basal and feed media preferably include defined chemicals that substitute for such components. It has also been discovered that media containing high levels of glucose, e.g., 10-35 g/L glucose, advantageously increase the cell culture productivity.
  • the basal medium has about 10-20 g/L, more preferably about 15 g/L, glucose and/or the feed medium has 22-35 g/L, more preferably around 28 g/L, glucose.
  • the feed medium can be added to the cells according to an escalating feed schedule, as is known in the art, over a period of 8-15 days, 9-13 days, or, most preferably, 10-13 days.
  • DAC HYP expressed in NS0 producer strain 7A11-5H7-14-43
  • the components of the growth and feed media, and other variables affecting expression and production have been optimized. Accordingly, the disclosure also provides optimized basal media, feed media, feeding schedules and other culturing methods and conditions useful for producing daclizumab in high yield and purity. These media and culturing parameters and methods are described in more detail in Section 7.3.
  • the stable, high concentration daclizumab drug formulations are generally prepared by exchanging a concentrated daclizumab formulation with exchange buffer having 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.
  • exchange buffer having 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.
  • the dilution buffer is the same as the exchange buffer, but includes about 0-10% (w/v) polysorbate 80, and is used in an amount such that the final, stable, high concentration daclizumab formulation has a calculated polysorbate 80 concentration (nominal concentration) in a range of 0.02-0.04%, in some embodiments about 0.03% (w/v).
  • a variety of different buffering agents and excipients can be included in the exchange and dilution buffers to achieve an osmolality and pH within the defined ranges.
  • a specific, non-limiting example of an exchange buffer suitable for formulating stable, high concentration liquid daclizumab and DAC HYP drug formulations contains about 40 mM succinate and about 100 mM NaCl and has a pH of about 6.0 at 25° C.
  • a specific, non-limiting example of a dilution buffer suitable for use with this exchange buffer contains about 40 mM succinate, about 100 mM NaCl and about 1% (w/v) polysorbate 80 and has a pH of about 6.0 at 25° C.
  • the pH of the final formulation can be adjusted with acid or base to yield an actual pH of about 6.0 at 25° C.
  • the stable, high concentration liquid daclizumab formulations are characterized by a low level of aggregation, typically containing at least 95% monomer and less than 3% aggregates, sometimes less than 1.5% aggregates, and more usually greater than 99% monomer and less than 0.8% aggregates, as measured by size exclusion chromatography.
  • a low level of aggregation typically containing at least 95% monomer and less than 3% aggregates, sometimes less than 1.5% aggregates, and more usually greater than 99% monomer and less than 0.8% aggregates, as measured by size exclusion chromatography.
  • Other purity characteristics of the high concentration liquid daclizumab drug formulations are described in more detail in Section 7.6.
  • the high concentration daclizumab drug formulations are also characterized by a long shelf life, being stable against greater than 5% degradation and formation of greater than 3% aggregates (as measured by SDS-PAGE and size exclusion chromatography, respectively) for periods of up to 54 months or longer, for example, for at least 5, years, when stored at 2-8′C, for periods of up to 9 months when stored under accelerated conditions (23-27° C./60 ⁇ 5% relative humidity) and for periods of up to 3 months when stored under stressed conditions (38-42° C./75 ⁇ 5% relative humidity).
  • the stable, high concentration liquid daclizumab formulations can be prepared by diluting an intermediate formulation with polysorbate dilution buffer to yield finished daclizumab drug formulation.
  • the disclosure provides polysorbate-free purified daclizumab (preferably DAC HYP) intermediate formulations containing at least about 150 mg/mL daclizumab, in some embodiments about 170-190 mg/mL daclizumab, that can be diluted with polysorbate dilution buffer to yield a stable, high concentration daclizumab liquid drug formulations as described herein.
  • the concentrated polysorbate-free intermediate formulations nominally contain about 155 mg/mL or about 180 mg/mL daclizumab (preferably DAC HYP), about 40 mM sodium citrate and about 100 mN NaCl, pH 6.0 at 25° C. In a specific embodiment, the concentrated polysorbate-free intermediate formulations nominally contain about 155 mg/mL or about 180 mg/mL daclizumab (preferably DAC HYP), about 40 mM sodium succinate and about 100 mN NaCl, pH 6.0 at 25° C.
  • the daclizumab compositions are characterized by a low level of aggregates, described further below.
  • concentrating daclizumab via ultrafiltration induces aggregates to form, which can result in a high concentration daclizumab drug formulation containing unacceptable (e.g., >3%) levels of aggregates. Accordingly, it is preferable to utilize a “polishing” step prior to concentrating the daclizumab drug substance to remove aggregates.
  • the level of acceptable aggregates prior to concentration will depend upon the concentration of the daclizumab drug substance to be concentrated, the desired concentration in the final daclizumab drug formulation, and the acceptable level of aggregates in the final daclizumab drug formulation.
  • the daclizumab composition to be concentrated should contain ⁇ 0.3% aggregates, preferably ⁇ 0.2% aggregates, and preferably even lower levels, e.g., about 0.1% aggregates.
  • a variety of known techniques can be used to obtain a starting daclizumab drug substance composition containing acceptable levels of aggregates for concentration into concentrated daclizumab intermediate and final drug formulations as described herein, including, for example, strong cation exchange chromatography and hydrophobic interaction chromatography.
  • weak cation exchange chromatography reduces levels of aggregates of daclizumab compositions containing in the range of 4-12 mg/mL daclizumab and up to 2.5% aggregates to extremely low levels, typically to about 0.1% aggregates.
  • the use of weak cationic exchange to remove aggregates is more environmentally friendly than hydrophobic interaction chromatography, which utilizes nitrogen containing solutions (such as ammonium sulfate solutions).
  • the disclosure provides methods of polishing daclizumab compositions to remove aggregates such that the resulting polished composition generally contains about 4 to 15 mg/mL daclizumab, where 0.3% or less (e.g., 0.2% or less or 0.1% or less) is in aggregate form, as measured by size exclusion chromatography.
  • the method generally involves passing a daclizumab composition containing about 4-10 mg/mL, typically about 8-9 mg/mL, and preferably about 8.5 mg/mL daclizumab and >0.5% aggregates over a weak cation exchange resin in a suitable buffer to adsorb to the daclizumab, and eluting the adsorbed daclizumab with an elution buffer.
  • Useful weak cation exchange resins include, but are not limited to, CM-650M (Tosoh Biosciences), CM-Sepharose, CM-HyperD.
  • the components of the equilibration, washing and elution buffers will depend upon the weak cation exchange resin used, and will be apparent to those of skill in the art.
  • CM-650M resin Tosoh Biosciences, part Number 101392
  • an equilibration and wash buffer containing about 20 mM sodium citrate, pH 4.5 and an elution buffer containing 20 mM sodium citrate and 75 mM sodium sulfate, pH 4.5 works well.
  • the flow rate used will depend upon the choice of resin and size of the column.
  • CM-650M resin having a bed height in the range of about 10-30 cm (e.g., 17-19 cm) and a flow rate in the range of about 50-200 cm/hr (e.g., 90-110 cm/hr, preferably about 100 cm/hr), works well with the chromatography can be carried out at room temperature, or at lower temperature, for examples temperatures ranging from 4°, 10°, 15°, 20° or 25° C. A typical useful temperature range is 18-25° C. (e.g., 18-22° C.).
  • the purification process for ZENAPAX DAC involves the following twelve steps:
  • the present disclosure also provides improved methods for isolating and/or purifying both daclizumab drug substance and high concentration drug formulations.
  • the process utilizes Protein A affinity chromatography in conjunction with strong anion exchange (Q-Sepharose) chromatography and weak cation exchange (CM-650M) chromatography, permitting continuous flow processing without dilution of process intermediate.
  • the improved method for obtaining purified daclizumab drug substance involves the following steps:
  • Crude daclizumab to be purified and optionally formulated via the above methods can be harvested from the cell culture using a variety of conventional means, e.g., microfiltration, centrifugation, and depth filtration directly from bioreactor.
  • crude daclizumab can be conveniently 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 be removed via centrifugation.
  • crude daclizumab is harvested by lowering the pH of the cell culture to approximately pH 5, chilling the culture to less than 15° C., for example 4° C., for 30-90 minutes, and centrifuging the resultant suspension to remove cells.
  • This process is generally applicable to any cell culture that secretes recombinant proteins into the culture medium, and is not specific to cultures producing daclizumab or therapeutic antibodies.
  • 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.
  • citric acid works well.
  • a concentrated citric acid solution e.g., a 0.5 M-2 M solution, can be used for adjusting the pH of the culture prior to harvesting.
  • DAC HYP The purification of DAC HYP is accomplished by use of three chromatography steps, virus inactivation, virus filtration and final ultra filtrations.
  • Protein A affinity chromatography is the first step in the purification process, which clears the majority of process related impurities. To enable the reuse of protein A affinity column, it must be regenerated and sanitized. It has been discovered that aqueous NaOH solution is effective in accomplishing both column regeneration and sanitization. However, the use of NaOH solutions can degrade the protein A resin, increasing overall production costs. It has also been discovered that sanitizing protein A affinity chromatography resins with a solution containing NaOH and benzyl alcohol yields good results and significantly increases the number of purification cycles.
  • the disclosure also provides a sanitization solution and method for regenerating and sanitizing protein A affinity columns and resins.
  • the buffer generally comprises about 100 to 500 mM sodium citrate, about 10 to 30 mM NaOH and about 0.5 to 3% (v/v) of benzyl alcohol, and has a pH in the range of about pH 10 to 13.
  • the buffer may also optionally include other components, such as, for example, salts and/or detergents. Both sodium citrate and benzyl alcohol are important for protecting protein A resin from being destroyed by NaOH and enhancing microbicidal activities.
  • the Protein A sanitization buffer contains about 200 mM sodium citrate, about 20 mM NaOH, and about 1% (v/v) benzyl alcohol.
  • sanitization solutions containing benzyl alcohol and sodium hydroxide have beneficial antimicrobial effects, and can be used to sanitize protein A columns in purification processes for any antibody.
  • the sanitization buffer can be used to sanitize Protein A chromatography resin in a batch-wise process, where the resin is washed with excess (e.g., 1.5-2 ⁇ volumes) of sanitization buffer followed by incubation for about 30-45 min. in excess (e.g., 1.5-2 ⁇ volumes) sanitization buffer, followed by equilibration with equilibration buffer or storage buffer.
  • the sanitization buffer can also be used to sanitize a prepared Protein A chromatography column by washing the column with excess (e.g., 1.5-2 ⁇ column volumes) sanitization buffer at a suitable flow rate (e.g., ranging from about 110-190 cm/hr, or 135-165 cm/hr), holding the column under conditions of zero flow for about 30-40 min, and then washing the column with equilibration buffer or storage buffer. Suitable equilibration and storage buffers are described in Section 0.
  • Sanitizing Protein A columns with the sanitization buffers described herein significantly increases the number of purifications for which a single batch of resin can be used. For example, whereas a single batch of Protein A resin typically lasts only about 30 purification cycles when sanitized with conventional NaOH buffers (e.g., 50 mM NaOH, 0.5 M NaCl), Protein A columns sanitized with the sanitization buffers described herein can be used for more than 100 purification cycles. While not intending to be bound by any theory of operation, it is believed that the sanitization buffers described herein in part protect the immobilized Protein A from NaOH-induced degradation, thereby increasing the useful life of the resin.
  • conventional NaOH buffers e.g., 50 mM NaOH, 0.5 M NaCl
  • the sanitization buffers described herein are especially beneficial when used to sanitize Protein A resins and columns utilizing unmodified immobilized Protein As, or Protein As that have not been engineered to be NaOH stable.
  • the disclosure further provides methods comprising using a protein A affinity resin for more than 30, more than 35 or more than 40 antibody purification runs, and in some instances up to 50 or up to 100 protein purification cycles, comprising conducting the purification runs and washing the resin with a sanitization solution as disclosed herein.
  • daclizumab specifically binds CD25 expressed on activated and not resting T and B lymphocytes, and blocks binding of IL-2 to CD25, thereby inhibiting formation of the high affinity IL-2 receptor complex, inhibiting proliferation of the activated T- and B-cells.
  • the DAC compositions and formulations described herein, and in particular the DAC HYP compositions and formulations likewise specifically bind CD25 and exhibit similar biological properties.
  • the DAC compositions and formulations described herein, and in particular DAC HYP are therefore useful in any of the assays and therapeutic methods described for daclizumab generally, and ZENAPAX specifically.
  • the present disclosure also provides methods of using the DAC compositions and formulations described herein, and in particular the DAC HYP compositions and high concentration stable liquid formulation, to inhibit proliferation of activated T- and B-cells, both in in vitro applications and in vivo as a therapeutic approach towards the treatment of diseases in which activated T- and/or B-cell proliferation play a role, such as the treatment and prevention of allograft rejection, the treatment of uveitis, and the treatment of multiple sclerosis.
  • the methods generally involve contacting an activated T- and/or B-cell with an amount of a daclizumab composition or formulation described herein sufficient to inhibit its proliferation.
  • the methods generally involve administering to a subject an amount of a daclizumab composition, for example a DAC HYP composition or a high concentration DAC formulation as described herein, to provide therapeutic benefit.
  • a daclizumab composition for example a DAC HYP composition or a high concentration DAC formulation as described herein.
  • the daclizumab compositions and formulations can be used to treat multiple sclerosis, either alone or in combination with other agents such as interferon beta.
  • the DAC compositions described herein can be administered subcutaneously to a patient from weekly to monthly (e.g., weekly, every two weeks, twice a month, every four weeks or monthly) in doses ranging from 75 mg to 300 mg (e.g., 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg or 300 mg) or ranging from 1 mg/kg to 4 mg/kg.
  • the compositions can be provided in prefilled syringes convenient for subcutaneous use, preferably at nominal daclizumab concentrations of 100 mg/mL ⁇ 10-15% or 150 mg/mL ⁇ 10-15%.
  • the concentrated DAC compositions can also be diluted for intravenous administration.
  • 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.
  • 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.
  • FIG. 3A-FIG . 3 D together provide the full nucleotide sequence for vector pHAT.IgG1.rg.dE (SEQ ID NO:5).
  • FIG. 3E provides a specific embodiment of a dESV40 promoter (SEQ ID NO:12) that can be used to select high yielding producer strains.
  • FIG. 4A-4B provide a schematic diagram of vector pHAT.IgG1.rg.dE ( FIG. 4A ), which is derived from pABX.gpt, a vector that can be adapted to express any heavy and light chain genes or even a non-antibody polypeptide ( FIG. 4B ).
  • FIG. 5 provides an exemplary production process for DAC HYP.
  • FIG. 6 demonstrates UV (280 nm), pH and conductivity monitoring of product fractions during protein A affinity chromatography.
  • FIG. 7 demonstrates UV (280 nm), pH and conductivity monitoring of product fractions during Q-sepharose chromatography.
  • FIG. 8 demonstrates UV (280 nm), pH and conductivity monitoring of product fractions CM cation exchange chromatography.
  • FIG. 9 provides a schematic illustration of the DAC HYP ultrafiltration system.
  • FIG. 10 provides a 0-60 minute DAC HYP peptide map chromatogram.
  • the reference profile is a 100 mg/ml DAC HYP preparation and Batch 1 and Batch 2 correspond to 150 mg/ml DAC HYP preparations.
  • FIG. 11 provides a 55-115 minute DAC HYP peptide map chromatogram.
  • the reference profile is a 100 mg/ml DAC HYP preparation and Batch 1 and Batch 2 correspond to 150 mg/ml DAC HYP preparations.
  • FIG. 12 provides a 110-170 minute DAC HYP peptide map chromatogram.
  • the reference profile is a 100 mg/ml DAC HYP preparation and Batch 1 and Batch 2 correspond to 150 mg/ml DAC HYP preparations.
  • FIG. 13 provides overlaid circular dichroism spectra of DAC HYP 150 mg/ml lots Batch 1 and Batch 2.
  • the reference is a 100 mg/ml preparation of DAC HYP.
  • FIG. 14A-FIG . 14 B provides overlaid zero-order ultraviolet spectra and overlaid second derivative ultraviolet spectra, respectively.
  • the reference profile is a 100 mg/ml DAC HYP preparation and Batch 1 and Batch 2 correspond to 150 mg/ml DAC HYP preparations. All three spectra are present in each of FIG. 14A and FIG. 14B , but appear as a single spectrum once overlaid on one another.
  • FIG. 15A-FIG . 15 B provide full scale and expanded scale size exclusion chromatograms, respectively.
  • the reference profile is a 100 mg/ml DAC HYP preparation and Batch 1 and Batch 2 correspond to 150 mg/ml DAC HYP preparations.
  • FIG. 16 is a plot of DAC HYP aggregation as a function of time.
  • FIG. 17 shows reduced and non-reduced SDS-PAGE (left and right panels, respectively).
  • the reference profile is a 100 mg/ml DAC HYP preparation and Batch 1 and Batch 2 correspond to 150 mg/ml DAC HYP preparations.
  • FIG. 18 shows cation exchange chromatograms of DAC HYP.
  • the reference profile is a 100 mg/ml DAC HYP preparation and Batch 1 and Batch 2 correspond to 150 mg/ml DAC HYP preparations.
  • the peak labels correspond to the different N- and C-terminal isoforms.
  • FIG. 19 shows HPLC chromatograms of N-linked oligosaccharides enzymatically cleaved from DAC HYP.
  • the reference profile is a 100 mg/ml DAC HYP preparation and Batch 1 and Batch 2 correspond to 150 mg/ml DAC HYP preparations.
  • FIG. 20 shows ADCC response curves of DAC HYP.
  • the reference profile is a 100 mg/ml DAC HYP preparation and Batch 1 and Batch 2 correspond to 150 mg/ml DAC HYP preparations.
  • FIG. 21 shows HPLC chromatograms for N-linked oligosaccharides released from DAC HYP (lower panel) and ZENAPAX DAC (upper panel) illustrating their different glycosylation profiles.
  • FIG. 22A-FIG . 22 B provide a comparison between the ADCC activity of two DAC HYP preparations (referred to as DAC HYP Batch 3 and DAC HYP Batch 4), DAC Penzburg, and ZENAPAX DAC using the variable effector-to-target cell ratio ADCC assay format ( FIG. 22A ) and the variable antibody concentration ADCC assay format ( FIG. 22B ).
  • FIG. 23 provides a comparison of the charge isoforms of DAC HYP, DAC Penzberg and ZENAPAX DAC.
  • the present disclosure provides, among other things, DAC compositions having specified properties, high concentration DAC formulations especially useful for certain modes of administration that are shelf stable at different temperatures, vectors and host cells useful for producing the DAC compositions, optimized culture broths and culturing conditions useful for producing the DAC compositions, methods for purifying the DAC compositions and high concentration formulations, and methods of using the DAC compositions and high concentration formulations, for example to inhibit proliferation of activated T- and/or B-cells and to treat and/or prevent activated T- and/or B-cell mediated diseases, such as, for example, multiple sclerosis.
  • Daclizumab as used herein refers to a humanized IgG 1 monoclonal antibody having the light (V L ) chain sequence illustrated in FIG. 1 (positions 21-233 of SEQ ID NO:2) and the heavy (V H ) chain sequence illustrated in FIG. 2 (positions 20 to 465 of SEQ ID NO:4).
  • the CDR sequences of DAC are as follows:
  • V L CDR#1 (SEQ ID NO: 6) S A S S S I S Y M H V L CDR#2: (SEQ ID NO: 7) T T S N L A S V L CDR#3: (SEQ ID NO: 8) H Q R S T Y P L T V H CDR#1: (SEQ ID NO: 9) S Y R M H V H CDR#2: (SEQ ID NO: 10) Y I N P S T G Y T E Y N Q K F K D V H CDR#3: (SEQ ID NO: 11) G G G V F D Y
  • DAC Penzberg Another version of DAC, produced at a facility in Penzberg, Germany, although never sold commercially, has been used in certain clinical trials. This version of DAC is referred to herein as “DAC Penzberg.”
  • the present disclosure concerns, in part, a new version of DAC having characteristics and properties that differ from, and in some instances that are superior to, the characteristics and properties of ZENAPAX DAC and DAC Penzberg. Accordingly, the present disclosure in part concerns DAC compositions that are new.
  • the new DAC compositions are characterized by one or more of the following features, as described more fully in the Summary section:
  • DAC compositions having one or more of these characteristics and/or properties are referred to herein as “DAC HYP” compositions.
  • DAC HYP DAC HYP compositions having one or more of these characteristics and/or properties.
  • DAC HYP compositions For purposes of exemplifying the various aspects and features of inventions described herein, a specific DAC HYP having all four of the above properties is described, as are specific compositions and methods for its production and purification. However, it is to be understood that a DAC HYP composition need not have all of the above four characteristics to fall within the scope of the disclosure.
  • DAC HYP has at least two of characteristics (1) through (4) above (e.g., at least a combination of (1) and (2); (1) and (3); (1) and (4); (2) and (3); (2) and (4); or (3) and (4)) or at least three of characteristics (1) through (4) above (e.g., at least a combination of (1), (2) and (3); (1), (2) and (4); (1), (3), and (4); and (2), (3), (4)).
  • Such DAC HYP compositions can also have ⁇ 3% aggregates, ⁇ 2% aggregates and even lower levels, e.g., ⁇ 1% aggregates, when formulated at concentrations of 100 mg ⁇ 10-15% or even 150 ⁇ 10-15%.
  • DAC HYP While certain aspects and embodiments of the inventions described herein are illustrated and exemplified with DAC HYP, skilled artisans will appreciate that they are not limited to DAC HYP, and are useful for daclizumab compositions generally, and also to IgG 2 , IgG 3 , and IgG 4 anti-CD25 antibodies having specific CD25 binding properties similar to DAC, and to anti-CD25 antibodies suitable for administration to humans that have not been humanized. These various different anti-CD25 antibodies are referred to herein as “DAC analogs.” Such DAC analogs may usually include the six DAC CDRs mentioned above, but may include other CDR sequences.
  • DAC HYP compositions can be confirmed using standard assays and methods. For example, N-terminal and C-terminal isoform profiles can be assessed using cation exchange chromatography with detection at 220 nm.
  • 100 ⁇ L of test sample (1 mg/mL antibody dissolved in Buffer A) is resolved at room temperature on a ProPac WCX-10 column (Dionex Coporation) equipped with a ProPac WCX-10G guard column (Dionex Corporation) using the following separation gradient (column is equilibrated with Buffer A):
  • N-linked glycosylation profiles can be assessed by cleaving the N-linked oligosaccharides with amidase PNGase F, derivatizing the oligosaccharides with a fluorescent label and analyzing the resultant mixture via normal phase HPLC with fluorescent detection.
  • anthranilic acid-derivatized, cleaved N-linked glycans are resolved at 50° C. on a 250 ⁇ 4.6 mm polymeric-amine bonded Asahipak Amino NH 2 P-504E column (5 ⁇ m particle size, Phenomenex, cat. No. CHO-2628) using the following elution gradient (using a sample injection volume of 100 ⁇ L; column is equilibrated with 85% Buffer A/15% Buffer B):
  • test sample (20 mg/mL antibody in elution buffer) can be resolved at room temperature on a 7.8 mm ⁇ 30 cm TSK G3000SWXL column (Tosoh Biosciences, part no. 601342) equipped with a 0.5 ⁇ m pre-column filter (Upchurch, part no. A-102 ⁇ ) using an isocratic gradient of elution buffer (200 mM KPO4, 150 mM KCl, pH 6.9) at a flow rate of 1 mL/min.
  • elution buffer 200 mM KPO4, 150 mM KCl, pH 6.9
  • the DAC HYP compositions and other DAC formulations described herein, such as the stable, high concentration liquid DAC formulations described herein, are useful for treating a variety of disorders and conditions thought to be mediated, at least in part, by activated T- and/or B-cells, including, for example, rejection of allograft transplants and multiple sclerosis.
  • Specific patient populations, formulations, modes of administration and dosage amounts and schedules useful for treating or preventing allograft rejection are described in U.S. Pat. No. 6,013,256, and are incorporated herein by reference.
  • Specific patient populations, formulations, modes of administration, dosage amounts and schedules useful for treating patients with multiple sclerosis are described in U.S. Pat. No. 7,258,859, and are incorporated herein by reference. All of these formulations, modes of administration, dosing amounts and schedules, as well as disclosed specific patient populations and combination therapies, are equally suited to the DAC HYP compositions and, where applicable, the high concentration DAC formulations, described herein.
  • the DAC HYP compositions and formulations described herein are administered in amounts that provide therapeutic benefit.
  • Therapeutic benefit includes, but is not limited, treatment of the underlying disorder.
  • Therapeutic benefit may also include improving or ameliorating symptoms or side effects of a particular disease as assessed using standard diagnostic and other tests.
  • various means of assessing therapeutic benefit including, for example, the use of magnetic resonance imaging to assess brain lesions and/or assessing progression to disability are described in U.S. Pat. No. 7,258,859, incorporated herein by reference. All of these various tests can be used to assess therapeutic benefit in the context of patients suffering from multiple sclerosis.
  • the stable high concentration DAC formulations are particularly useful for subcutaneous administration in the treatment of chronic diseases such as multiple sclerosis.
  • the formulations can conveniently be administered as a single bolus subcutaneous injection or diluted for intravenous administration.
  • the formulations can be administered subcutaneously to a patient from weekly to monthly (e.g., weekly, every two weeks, twice a month, every four weeks or monthly) in doses ranging from 75 mg to 300 mg (e.g., 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg or 300 mg) or ranging from 1 mg/kg to 4 mg/kg.
  • the compositions can be provided in prefilled syringes convenient for subcutaneous use.
  • the diluted formulations can be administered intravenously at suitable dosages at the same frequencies as for subcutaneous administration.
  • the cell culture used to produce a DAC HYP does not include an antifoam emulsion, whereas the cell culture DAC HYP at 150 mg/mL uses a low concentration Dow Corning Antifoam C in the 10,000 L bioreactor to minimize foaming.
  • the CM-650M column (see Section 7.4.5) is sanitized with a buffer of 0.5 M NaOH, 0.5 M sodium sulfate when producing a DAC HYP formulation having a final antibody concentration of 100 mg/mL; the sodium sulfate is omitted from the sanitization buffer when producing a DAC HYP formulation having a final antibody concentration of 150 mg/mL.
  • a one step ultrafiltration/diafiltration is used at the end of the downstream process immediately prior to addition of polysorbate 80 and dilution of the drug substance to final volume (see Section 7.4.7), whereas for making the drug substance at a concentration to 150 mg/mL, a two step UF/DF is used.
  • the examples below show comparative analyses among various lots of DAC HYP at 100 mg/mL and at 150 mg/mL. In several studies, batch of DAC HYP at 150 mg/mL were compared against a lot of DAC HYP 100 mg/mL manufactured at the 10,000 L scale, referred to below as Reference Standard lot RS0801.
  • the hybridoma producing anti-Tac a murine IgG 2a monoclonal antibody, was generated by fusing the murine myeloma cell line NS-1 with spleenocytes from a mouse immunized with a human T-cell line developed from a T-cell leukemia patient (Uchiyama et al., 1981, J. Immunol. 126(4):1393-7).
  • Anti-Tac was selected for its reactivity with activated T-cells, but not with resting T-cells or B-cells.
  • Anti-Tac was later shown to react with the alpha subunit of human IL-2 receptor (Leonard et al., 1982, Nature 300(5889):267-9).
  • the amino acid sequences for the light and heavy chain variable regions of the murine anti-Tac were determined from the respective cDNA (Queen et al., 1989, Proc. Nat'l Acad. Sci. USA 86(24):10029-33). The binding affinity of the mouse anti-Tac was retained in the humanized form as described in Queen et al.
  • the complementarity determining regions (CDRs) of the murine anti-Tac were first grafted onto the acceptor framework of human antibody Eu. With the aid of a three-dimensional model, key mouse framework residues critical for the conformation of the CDRs and thus the binding affinity were identified and substituted for the human counterpart in the acceptor frameworks. In addition, atypical amino acids in the acceptor frameworks were replaced with the human consensus residues of the corresponding positions to eliminate potential immunogenicity.
  • DAC HYP V L and V H genes were constructed as mini-exons by annealing and extension of overlapping oligonucleotides as described in Queen et al. (1989).
  • the resultant V L and V H genes were cloned into a single expression vector, as outlined in Cole et al. (1997, J. Immunol. 159(7):3613-21) and Kostelny et al. (2001, Int. J. Cancer 93(4):556-65), to construct pHAT.IgG1.rg.dE (see FIG. 3 and FIG. 4A ).
  • Plasmid pHAT.IgG1.rg.dE contains the genes for both the IgG 1 heavy and kappa light chains of daclizumab, each under control of the human cytomegalovirus (CMV) promoter.
  • the plasmid contains the E. coli guanine phosphoribosyl transferase (gpt) gene as a selectable marker.
  • the genetic components in pHAT.IgG1.rg.dE are described in Table 1 below.
  • the dESV40 promoter spans positions of 6536-6735 of pHAT.IgG1.rg.dE (6536-6562 is 27 residues of 72 bp enhancer A; 6566-6629 are the three 21-bp repeats. 6536-6735 are the reverse complement of 5172-1 and 1-133 in GenBank: J02400.1 (Simian Virus 40 Complete Genome)).
  • the nucleotide sequences of DAC HYP light and heavy chain genes in the expression vector were confirmed by DNA sequencing.
  • Mouse myeloma cell line NS0 was obtained from European Collection of Cell Cultures (ECACC catalog #85110503, Salisbury, Wiltshire, UK). A vial of these NS0 cells was thawed into DMEM supplemented with 10% FBS. Cells were maintained in a humidified incubator at 37° C. and 7.5% CO 2 . The cells were subsequently cultured in basal medium SFM-3 supplemented with 1 mg/mL BSA. SFM-3 is a 1:1 mixture of DMEM and Ham's F-12 supplemented with 10 mg/mL insulin and 10 ⁇ g/mL transferrin.
  • the NS0 cells were adapted to SFM-3 without supplements, by gradually reducing the amount of FBS present in the culture medium until it was eliminated, and then finally removing BSA in a single step.
  • the resulting host cell line was passaged 15-20 times in SFM-3 and a frozen bank was prepared.
  • the SFM-3 adapted cells were transfected with pHAT.IgG1.rg.dE (linearized with FspI enzyme (New England Biolabs, cat. no. R0135L, lot 43)) by electroporation. Briefly, 30-40 ⁇ g of pHAT.IgG1.rg.dE was added to 1 ⁇ 10 7 exponentially growing adapted NS0 cells and pulsed twice at 1.5 kV, 25 ⁇ F using a Gene Pulser instrument (BioRad, Richmond, Calif.). Following electroporation, cells were plated in DMEM ⁇ 10% FBS in five 96-well plates at 20,000 cells/well, a density that favored a single colony per well after mycophenolic acid (“MPA”) selection.
  • MPA mycophenolic acid
  • transfectants that had stably integrated the vector were selected in the presence of mycophenolic acid.
  • NS0 stable transfectant that produced a high level of DAC HYP
  • FACS fluorescence activated cell sorting
  • the final production cell line (7A11-5H7-14-43, also referred to as Daclizumab dWCB IP072911) was chosen.
  • a seed bank of the final production cell line was then prepared by freezing 1 ⁇ 10 7 cells per vial in 1 mL of 90% FBS/10% DMSO (Sigma, St. Louis, Mo.).
  • Cells are thawed from a single cell bank vial and expanded in progressively larger volumes within T-flasks, roller bottles, spinner flasks, and bioreactors until the production scale is achieved.
  • the cell culture fluid is clarified by centrifugation and depth filtration, and transferred to a harvest hold tank.
  • the production culture duration is approximately 10 days.
  • Cell culture and recovery can be carried out in a variety of different cell culture facilities using standard equipment, as is known in the art.
  • cells are thawed from a single cell bank vial and expanded in progressively larger volumes within shaker flasks and bioreactors until the production scale is achieved.
  • the cell culture fluid is clarified by centrifugation and depth filtration, and transferred to a harvest hold tank.
  • the production culture duration is approximately 10 days.
  • Custom Powder for making PFBM-2 can be ordered from Invitrogen by requesting Hybridoma-SFM media powder prepared without NaCl, phenol red, transferrin, and insulin, including a quantity of EDTA iron (III) sodium salt that, when reconstituted, yields a concentration of 5 mg/L, and that has quantities of the remaining components adjusted such that, when reconstituted, their concentrations are the same as reconstituted Hybridoma-SFM.
  • Prepared PFBM-2 medium contains the following components: 8 g/L Custom Powder; 2.45 g/L sodium bicarbonate; 3.15 g/L NaCl; and 16.5 g/L D-glucose monohydrate (15 g/L glucose).
  • the cells are expanded by serial passage into roller bottles or spinner flasks every two days thereafter.
  • T-flasks, roller bottles, and spinner flasks are placed in an incubator operating under a temperature set point of 37° C. under an atmosphere of 7.5% CO 2 for T-flasks and roller bottles and 5% CO 2 for spinner flasks.
  • the spinner flasks are supplemented with 5% CO 2 either by overlay into the headspace or by sparge into the culture, depending on the cell culture volume, and impeller speed is controlled at constant revolutions per minute (RPM).
  • RPM revolutions per minute
  • the target seeding density at all inoculum expansion passages is approximately 2.5 ⁇ 10 5 viable cells/mL.
  • inoculum preparation can be carried out according to methods known in the art, using a variety of standard culture vessels, volumes, and conditions. For example, production batches can be initiated by thawing a single cell bank vial. Cells can be transferred to a shaker flask containing a chemically-defined medium, Protein Free Basal Medium-2 (PFBM-2).
  • PFBM-2 Protein Free Basal Medium-2
  • Custom Powder for making PFBM-2 can be ordered from Invitrogen by requesting Hybridoma-SFM media powder prepared without NaCl, phenol red, transferrin, and insulin, including a quantity of EDTA iron (III) sodium salt that, when reconstituted, yields a concentration of 5 mg/L, and that has quantities of the remaining components adjusted such that, when reconstituted, their concentrations are the same as reconstituted Hybridoma-SFM.
  • Prepared PFBM-2 medium contains the following components: 8 g/L Custom Powder; 2.45 g/L sodium bicarbonate; 3.15 g/L NaCl; and 16.5 g/L D-glucose monohydrate (15 g/L glucose).
  • cupric sulfate heptahydrate can be added, e.g., at a concentration of 0.04 mg/L.
  • the cells are expanded by serial passage into shaker flasks every two days thereafter. Shaker flasks are placed in an incubator operating under a temperature set point of 37° C. under an atmosphere of 7.5% CO 2 .
  • the shaker flasks are agitated at constant revolutions per minute (RPM) on a shaker platform in the incubators.
  • RPM revolutions per minute
  • the target seeding density at all inoculum expansion passages is approximately 2.2-2.5 ⁇ 10 5 viable cells/mL.
  • the first of several, typically three or four, stainless steel stirred-tank seed bioreactors is inoculated.
  • the seed bioreactors Prior to use, the seed bioreactors are cleaned-in-place, steamed-in-place, and loaded with the appropriate volume of PFBM-2 culture medium.
  • the pH and dissolved oxygen probes are calibrated prior to the bioreactor being steamed-in-place.
  • the first seed bioreactor is inoculated with a sufficient number of cells to target an initial cell density of 2.0-2.5 ⁇ 10 5 viable cells/mL.
  • Sequential transfer to the larger volume is performed following approximately two days of growth in each reactor and target initial cell densities Of 2.0-2.5 ⁇ 10 5 viable cells/mL.
  • Culture pH is maintained by addition of either CO 2 gas or 1 M sodium carbonate (Na 2 CO 3 ) via automatic control.
  • the target operating conditions of the seed and production bioreactors include a temperature set point of 37° C., pH 7.0 and 30% dissolved oxygen (as a percentage of air saturation).
  • the 100 L, 300 L and 1,000 L bioreactors are agitated at 100 rpm, 80 rpm and 70 rpm, respectively.
  • the target operating conditions of the seed and production bioreactors include a temperature set point of 37° C., a pH of 7.0 with CO 2 sparge and base addition control and 30% dissolved oxygen (as a percentage of air saturation).
  • the larger volume bioreactors can be agitated at speeds of 100 rpm, 80 rpm, 70 rpm, or 40 rpm.
  • the inoculum is transferred into a stainless steel stirred-tank production bioreactor.
  • the production bioreactor has a working volume of approximately 10,000 L. Prior to use, the bioreactor is cleaned-in-place, steamed-in-place, and loaded with approximately 4,000 L of PFBM-2 medium. The pH and dissolved oxygen probes are calibrated prior to the bioreactor being steamed-in-place.
  • the inoculum is grown in a 3750 L seed bioreactor before transfer to a stainless steel stirred-tank production bioreactor with a working volume of approximately 15,000 L, which is cleaned-in-place, steamed-in-place, and loaded with approximately 4,000-7,000 L of PFBM-2 medium prior to use.
  • the target seeding density of the production bioreactor is in the range of 2.0-2.5 ⁇ 10 5 viable cells/mL.
  • a chemically-defined Protein Free Feed Medium concentrate (PFFM-3) (a chemically-defined concentrated feed medium made by reconstituting PFFM3 subcomponents 1 and 2, L-glutamine, D-glucose, sodium phosphate dibasic heptahydrate, L-tyrosine, folic acid, hydrochloric acid, and sodium hydroxide) is added during culture.
  • PFFM3 contains the components shown in Table 4:
  • PFFM3 Medium Components Component Concentration PFFM3 Subcomponent 1 (amino acids) 20.4 g/L prepared PFFM3 Subcomponent 2 (vitamins and trace elements) 4.93 g/L prepared L-Glutamine 11.0 g/L prepared D-Glucose 28.0 g/L prepared L-Tyrosine, disodium salt 1.32 g/L prepared Folic Acid 0.083 g/L prepared Na 2 HPO 4 •7H 2 O 1.74 g/L prepared Sodium Hydroxide Varies, pH control Glacial Hydrochloric Acid Varies, pH control WFI water
  • PFFM3 Subcomponent 1 contains the components shown in Table 5 below:
  • PFFM3 Subcomponent 2 contains components shown in Table 6 below:
  • Culture pH is maintained at approximately pH 7.0, preferably between pH 7.0 and pH 7.1, by automatic control of CO 2 gas and 1 M sodium carbonate (Na 2 CO 3 ) addition. Dissolved oxygen content is allowed to drop to approximately 30% of air saturation.
  • An oxygen/air mixture is sparged into the culture to achieve a constant total gas flow rate and dissolved oxygen is controlled by adjusting the ratio of air to oxygen gases as needed and by increasing agitation speed after reaching a maximum oxygen to air ratio. In another example, agitation is adjusted to maintain a constant power/volume ratio.
  • a simethicone-based antifoam emulsion is added to the bioreactor on an as needed basis based on foam level.
  • Bioreactor culture is harvested approximately 10 days post-inoculation. Prior to harvest, the bioreactor contents are sampled as unprocessed bulk.
  • the production bioreactor is first chilled to ⁇ 15° C., then adjusted to a pH of 5.0 ⁇ 0.1 using 0.5 M or 1 M or 2 M citric acid, and held for a period of approximately 30-90 or 45-60 minutes to flocculate the cells and cell debris prior to transfer to the harvest vessel.
  • the pH-adjusted harvest is then clarified by continuous centrifugation operated under predefined parameters for bowl speed and flow rate as defined in batch record documentation.
  • centrate is filtered through a depth filter followed by a 0.22 ⁇ m membrane filter and collected in a pre-sterilized tank.
  • 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.
  • the DAC HYP purification and formulation process was designed to improve efficiency relative to the ZENAPAX production process and to ensure consistent clearance of product- and process-related impurities.
  • the following subsections describe the purification process.
  • the purification is based on three chromatography techniques (Protein A affinity chromatography, Q Sepharose anion exchange chromatography, and CM-650(M) cation exchange chromatography) in combination with low pH viral inactivation, viral filtration, ultrafiltration/diafiltration, and formulation steps. All of the steps take place in enclosed equipment.
  • An outline of the purification process for DAC HYP is presented in FIG. 5 and described below.
  • the Protein A affinity chromatography step is the first purification step in the sequence of downstream operations. This step occurs in one or more cycles depending on the size of the column, typically two or three cycles for the column described in Table 8A (i.e., the cell-free harvest is portioned into two aliquots, and then each aliquot is loaded and eluted separately on the Protein A column).
  • Recombinant Protein A affinity chromatography resin specifically binds IgG, separating antibody from other components of the cell culture harvest.
  • the equilibration buffer is 20 mM sodium citrate, 150 mM sodium chloride, pH 7.0.
  • the column is loaded to a capacity of no greater than 35 grams antibody (protein) per liter of the packed resin.
  • 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 absorbance of the effluent at a wavelength of 280 nm is monitored and used to guide the collection of the product fraction (see FIG. 6 ).
  • a sanitization buffer containing sodium hydroxide and benzyl alcohol advantageously kills a wide range of microbial organisms while minimally affecting the quality of the protein A resin.
  • various sanitization solutions were spiked with various microorganisms and incubated over a period of time. At different intervals of incubation time, portions of the spiked sanitization solutions were neutralized and the microorganism titers were measured and compared to control. The microbicidal activities are expressed in the log reduction of the microorganisms over a period of time.
  • Table 8B shows the reduction of microorganism titers as function of contact time with sanitization buffer 20 mM sodium hydroxide, 200 mM sodium citrate and 1% benzyl alcohol:
  • Table 8 C shows the reduction of microorganisms by different sanitization solutions:
  • aeruginosa >3.6 >4.6 - Stenotrophomonas altophilia 6.0
  • Candida albicans 3.1 >5.5 0 >4.8 Aspergillus niger 0.01 0.8 0 >4.7
  • A 50 mM NaOH, 0.5 M NaCl (60 min)
  • B 20 mM NaOH, 200 mM sodium citrate, 1% benzyl alcohol (60 min)
  • C 200 mM sodium citrate, 0.5% benzyl alcohol (48 hrs)
  • D 2% benzyl alcohol (24 hrs)
  • This step is designed to inactivate low pH-sensitive endogenous virus-like particles and viruses.
  • the Protein A eluate from each Protein A cycle is eluted into a collection tank, where 0.5 M HCl is added until a pH 3.5 ⁇ 0.1 is reached.
  • the product is transferred to a hold tank where the pH is verified by another pH meter.
  • the low pH hold step is tightly controlled at pH 3.5 ⁇ 0.1 or ⁇ 0.2 (e.g., pH 3.35-3.64) for 30-120 minutes, or 30-240 minutes.
  • the viral inactivated eluate is neutralized to a pH of 7.8 ⁇ 0.1 or ⁇ 0.3 (e.g., pH 8.05-8.34) using 1 M Tris base, and then transferred through a 0.22 ⁇ m filter into a product pool tank.
  • a summary of the low pH viral inactivation conditions is set forth in the Table 9:
  • the Q Sepharose anion exchange chromatography step is used to reduce product- and process-related impurities (e.g., nucleic acids, host cell proteins, product aggregates, leached Protein A ligand, etc.) and to provide additional viral clearance capacity to the purification process.
  • product- and process-related impurities e.g., nucleic acids, host cell proteins, product aggregates, leached Protein A ligand, etc.
  • the conductivity and pH of the load are chosen in a manner such that the antibody flows through the column and negatively-charged impurities, such as host cell proteins and cellular DNA, bind to the positively-charged resin.
  • the anion exchange column is equilibrated with an equilibration buffer of 20 mM Tris, 20 mM sodium chloride, pH 7.8.
  • the pH-adjusted product from the low pH hold step is loaded onto the column to a capacity of no greater than 60 grams of antibody (protein), or no greater than 30-60 grams of antibody (protein), per liter of packed resin.
  • unbound antibody and impurities are removed from the column with the equilibration buffer.
  • Collection of the product is guided by monitoring the absorbance of the effluent at a wavelength of 280 nm (see FIG. 7 ).
  • the sanitization flow rate is 100 cm/hr and the hold time is 60 min.
  • This chromatography step is the last step used in the process to reduce trace levels of process- and product-related impurities. In addition to reducing aggregates and cleavage fragments of the antibody, this step also reduces process-related impurities such as host cell nucleic acids and proteins, and leached Protein A.
  • the column is equilibrated with an equilibration buffer of 20 mM sodium citrate, pH 4.5.
  • the anion exchange eluate pool is adjusted to a pH of 4.5 ⁇ 0.1 or ⁇ 0.2 (e.g., 4.35-4.64) using 0.5 M citric acid and loaded onto the column to a target loading capacity of no greater than 25 or 30 grams of antibody (protein) per liter of packed resin.
  • the column is washed with the equilibration buffer to remove any unbound, or loosely-bound, impurities from the resin.
  • the bound antibody is then eluted from the column in a step elution mode with an elution buffer of 20 mM sodium citrate, 75 mM sodium sulfate, pH 4.5. Peak collection is guided by monitoring the absorbance of the effluent at a wavelength of 280 nm (see FIG. 8 ).
  • the purpose of the nanofiltration step is to provide additional viral clearance capacity to the purification process.
  • the removal of viruses and virus-like particles at this step occurs through a size-exclusion mechanism.
  • the pores of the filter are designed such that the antibody passes through the filter whereas the virus-like particles and viruses are retained on the upstream side of the filter.
  • the cation exchange eluate that has been filtered through a 0.22 ⁇ m or 0.1 ⁇ m filter is passed through a small virus-retaining nanofilter, followed by a filter flush with DAC HYP formulation buffer without polysorbate 80 (40 mM succinate, 100 mM sodium chloride, pH 6.0).
  • the buffer flush step is applied to recover antibody that remains in the line and filter housing.
  • This process step is designed to concentrate the product and exchange the buffer in the product to the DAC HYP formulation buffer without polysorbate 80. It is operated in a tangential flow mode using a 30 kDa nominal molecular weight cutoff membrane. Two ultrafiltration/diafiltration stages are used to produce 150 mg/mL formulation due to the expected product volume at the final concentration and relative hold-up volume of each UF system.
  • the first stage is processed using a large UF system (see FIG. 9 ).
  • the nanofiltration filtrate is first concentrated to approximately 30 mg/mL and then diafiltered into exchange buffer (formulation buffer without polysorbate 80).
  • the diafiltered antibody solution is further concentrated to approximately 100 mg/mL, then recovered at a concentration of approximately 55 mg/mL from the UF/DF system and transferred through a 0.22 ⁇ m filter.
  • the diafiltered antibody solution can also be recovered at a concentration of approximately 20-60 mg/mL.
  • Table 13 A summary of the parameters of the first stage is set forth in Table 13:
  • the second stage is processed using a smaller UF system, but with the same 30 kDa cutoff membrane.
  • the DAC HYP (typically 55 mg/mL) solution is concentrated to approximately 180 mg/mL, recovered from the UF system, and then transferred through a 0.22 ⁇ m filter.
  • the UF system is rinsed with formulation buffer without polysorbate 80 and transferred through the 0.22 ⁇ m filter obtaining the purified drug substance at approximately 170 mg/mL or approximately 150-170 mg/mL.
  • Table 14 A summary of the parameters of the second stage is set forth in Table 14:
  • the final process step is the dilution of the purified drug substance to a final target concentration of 150 or 100 mg/L ⁇ 10%, i.e., a final target concentration of 150 ⁇ 15 mg/mL (in the case of the 150 mg/mL formulation) or 100 ⁇ 10 mg/mL (in the case of the 100 mg/mL formulation) in buffer containing an appropriate concentration of polysorbate 80.
  • the formulation is performed in stages.
  • the formulation buffer without polysorbate 80 (40 mM sodium succinate, 100 mM sodium chloride, pH 6.0) is added to the purified drug substance to reach the 90% target volume of formulated drug substance. Then, a calculated amount of polysorbate 80 dilution buffer (40 mM succinate, 100 mM sodium chloride, 1% polysorbate 80, pH 6.0) is added to reach the target concentration of 0.03% (w/v) polysorbate 80 in the final formulation.
  • the product volume is adjusted, using the formulation buffer (made of succinate and succinic acid for the 150 mg/mL formulation, and succinate and HCl for the 100 mg/mL formulation) without polysorbate 80, to achieve a final antibody concentration of 150 ⁇ 15 mg/mL (preferably 150 ⁇ 8 mg/mL).
  • the 100 mg/mL drug product is formulated in a similar manner to a final concentration 100 ⁇ 10 mg/ml, (preferably 100 ⁇ 5 mg/mL).
  • the formulated drag substance is filtered through a 0.22 ⁇ m filter into a BioProcess ContainerTM (BPC®) bag (or equivalent) which is placed inside a semi-rigid cylindrical support.
  • BPC® BioProcess ContainerTM
  • the support encloses the BPC with a lid and provides a protective barrier between the flexible bag and the external environment.
  • the formulated drug substance is stored at 2-8° C. in an access-controlled cooler for drug product fill/finish operations.
  • DAC HYP is glycosylated at amino acid 296 of both heavy chain subunits, with the major oligosaccharide form existing as a core fucosylated biantennary structure lacking terminal galactose.
  • N-terminus of DAC HYP heavy chain exists as three major forms: 1) N-terminal glutamine (predicted from the DNA sequence), 2) N-terminal pyroglutamate (from the cyclization of N-terminal glutamine), and 3) N-terminal valine, histidine and serine residues in addition to the predicted N-terminal glutamine residue (results from incomplete cleavage of the signal peptide).
  • the C-terminus of DAC HYP heavy chain exists with and without the C-terminal lysine residue.
  • the major form lacks the C-terminal lysine residue, resulting in a C-terminal glycine.
  • DAC HYP has a calculated molecular weight of 144 kDa based on the primary amino acid composition defined by the nucleotide sequence.
  • the corresponding molecular weight of the reduced heavy chain is 48.9 kDa and the reduced light chain is 23.2 kDa; these weights do not account for carbohydrate content or post-translational modifications.
  • DAC HYP binding is highly specific for CD25, which is expressed on activated but not on resting T and B lymphocytes. DAC HYP binding to CD25 on these activated cells blocks the binding of IL-2 to CD25 and subsequent formation of the high affinity IL-2 receptor complex. Consequently, IL-2-induced proliferation of the activated cells is blocked.
  • the observed and potential therapeutic efficacy of DAC HYP is believed to rest in large part on its inhibitory effect on the proliferation of activated autoreactive T-cells. However, DAC HYP might also exert a therapeutic effect through its blocking effect on other CD25-bearing cell types such as eosinophils.
  • DAC HYP drug substance is assessed by visually examining the color and clarity of the solution in direct light against a black background and white background without magnification. The solution is also evaluated for the presence of visible particles. The typical appearance of various lots of DAC HYP drug product is described in Table 17.
  • the pH of DAC HYP is determined in accordance with the U.S. Pharmacopeia Protocol No. 791 .
  • the pH ranges of various lots of DAC HYP drug product are summarized in Table 17.
  • the concentration of DAC HYP is determined by UV spectroscopy. DAC HYP samples are diluted gravimetrically with buffer. The UV absorbance of each diluted sample solution is measured at 278 nm against a buffer blank. The protein concentration of the sample is calculated using the absorptivity coefficient for DAC HYP. The protein concentrations of various lots of DAC HYP drug product are summarized in Table 17.
  • DAC HYP 150 mg/mL lots were evaluated by N-terminal sequencing. The samples were analyzed using an automated Edman degradation sequencing instrument.
  • This sequence is a result of the native N-terminal heavy chain glutamine residue not undergoing cyclization to the pyroglutamate form.
  • the N-terminal sequencing results for the 150 mg/mL lots are consistent with the sequences predicted from the heavy and light chain coding sequences. Comparable results were obtained for the 100 mg/mL lots.
  • the molecular masses of the heavy chain and light chain of the DAC HYP 150 mg/mL lots and Reference Standard RS0801 were evaluated by liquid chromatography mass spectrometry (LC-MS) analysis. All lots were deglycosylated with the amidase PNGaseF, reduced with dithiothreitol, alkylated with iodoacetic acid, and separated by reversed phase chromatography. Theoretical heavy and light chain masses were calculated from the protein sequence. The observed masses of the samples were within 1 Da of the calculate masses, as shown in Table 18 below:
  • DAC HYP heavy chain As described in the preceding subsection, the two most prevalent forms of DAC HYP heavy chain are known to contain an N-terminal pyroglutamate (pE) residue or a valine, histidine, serine (VHS) sequence and lack C-terminal lysine.
  • pE N-terminal pyroglutamate
  • VHS valine, histidine, serine
  • the molecular weights obtained for the two predominant heavy chain variants and the light chain in the 150 mg/mL lots were comparable to those of Reference Standard RS0801 and consistent with the masses predicted from the protein sequences.
  • the heavy and light chain mass results confirm the presence of the expected light chain and heavy chain primary structures in the DAC HYP 150 mg/mL lots.
  • DAC HYP 150 mg/mL lots and Reference Standard RS0801 (DAC HYP produced from a 100 mg/mL drug substance lot manufactured at the 10,000 L scale) were evaluated using reversed phase HPLC peptide mapping. All lots were reduced with dithiothreitol, alkylated with iodoacetic acid, and enzymatically digested with trypsin. The resulting peptides were separated by reversed phase chromatography and detected by ultraviolet absorbance at 215 nm to generate peptide maps.
  • the peptide maps of the 150 mg/mL lots were compared to that of Reference Standard RS0801. Peptides corresponding to ninety eight percent of the expected heavy and light chain residues have previously been identified by mass spectrometry in the peptide map of Reference Standard RS0801. The residues that have not been accounted for in the peptide map are single amino acids or reside in very polar dipeptides, and are not expected to be retained by the reversed phase column. Masses consistent with pyroglutamate, glutamine, and the VHS sequence at the N-terminus of the heavy chain N-terminal peptide have been identified in the reference standard.
  • DAC HYP contains a consensus site for N-linked glycosylation in the Fc portion of the heavy chain at Asn296 and masses consistent with linked complex core biantennary oligosaccharide structures have been identified for the peptide containing the Asn296 residue.
  • FIG. 10 Peptide maps comparing the DAC HYP 150 mg/mL lots to Reference Standard RS0801 are shown in FIG. 10 (0 to 60 minutes), FIG. 11 (55 to 115 minutes), and FIG. 12 (110 to 170 minutes).
  • the peptide maps of the DAC HYP 150 mg/mL lots are comparable to those of the reference standard and confirm the presence of the expected primary structure in the 150 mg/mL lots.
  • DAC HYP 150 mg/mL lots and Reference Standard RS0801 were analyzed by far ultraviolet circular dichroism spectroscopy (far-UV CD) to evaluate secondary structure. Prior to analysis samples were diluted with water to a final protein concentration of 0.2 mg/mL. Spectra were acquired from 195 to 260 nm using a 0.1 cm cell and the signal obtained was converted to molar ellipticity after buffer subtraction.
  • the overlaid far-UV CD spectra of DAC HYP 150 mg/mL lots Batch 1 and Batch 2 and Reference Standard RS0801 are shown in FIG. 13 .
  • the spectra of all lots show a positive band at approximately 202 nm and a negative band at approximately 217 nm.
  • the negative band at 217 nm is characteristic of a ⁇ sheet conformation, the predominant conformation of IgG molecules.
  • the far-UV CD spectra of the lots were similar, which is supportive of a uniform secondary structure among the DAC HYP 150 mg/mL lots and Reference Standard RS0801.
  • DAC HYP 150 mg/mL lots and Reference Standard RS0801 were analyzed by ultraviolet (UV) spectroscopy to evaluate tertiary structure.
  • Prior to analysis samples were diluted with formulation buffer (40 mM succinate, 100 mM sodium chloride, 0.03% polysorbate 80, pH 6.0) to a final protein concentration of 0.5 mg/mL.
  • formulation buffer 40 mM succinate, 100 mM sodium chloride, 0.03% polysorbate 80, pH 6.0
  • Spectra were acquired from 250 to 350 nm using a 1 cm path length quartz cuvette and normalized to an absorbance of 1.0 at 280 nm.
  • the overlaid zero-order and second derivative UV spectra (calculated from the smoothed zero-order data) are shown in FIGS. 14A and 14B , respectively.
  • the zero-order and second derivative spectra of all lots evaluated were superimposable, which is supportive of a uniform tertiary structure among the DAC HYP 150 mg/mL lots Batch 1 and Batch 2 and Reference Standard RS0801.
  • Size exclusion chromatography was performed using a porous silica column with an aqueous mobile phase and ultraviolet absorbance detection at 280 nm.
  • 15 ⁇ L test sample (20 mg/mL antibody in elution buffer) was analyzed at room temperature on a 7.8 mm ⁇ 30 cm TSK G3000SW XL column (Tosoh Biosciences, part no. 601342) equipped with a 0.5 ⁇ m pre-column filter (Upchurch, part no. A-102X) using an isocratic gradient of elution buffer (200 mM KPO 4 , 150 mM KCl, pH 6.9) at a flow rate of 1 mL/min.
  • the 150 mg/mL lots and Reference Standard RS0801 were analyzed using SEC with multi-angle light scattering detection (SEC-MALS) to determine the molecular weight of the aggregate peak.
  • SEC-MALS multi-angle light scattering detection
  • the molecular weight obtained for the aggregate peak was approximately 300 kDa, which is consistent with antibody dimer.
  • the monomer and aggregates in DAC HYP 150 mg/mL and 100 mg/mL lots were characterized using sedimentation velocity analytical ultracentrifugation (SV-AUC).
  • SV-AUC sedimentation velocity analytical ultracentrifugation
  • Monomer was the major component observed in each of the lots.
  • the sedimentation coefficient of the monomer peak was highly consistent among the lots indicating that the conformation of the monomer is comparable between the 150 mg/mL and 100 mg/ml lots.
  • the monomer content of the 150 mg/mL lots was comparable to that of the 100 mg/mL lots.
  • the predominant aggregate species in each of the lots had a sedimentation coefficient consistent with antibody dimer. This is consistent with SEC-MALS results, which indicate that the SEC aggregate peak is composed primarily of antibody dimer (see preceding subsection). Low levels of two larger aggregate species that had sedimentation coefficients consistent with trimer and tetramer were also observed in each of the lots by AUC. The dimer, trimer, and tetramer content of the 150 mg/mL lots was comparable to that of the 100 mg/mL lots.
  • Purity was determined by SDS-PAGE using 4-20% (typically 14%) tris-glycine gels with Colloidal blue stain. Samples were analyzed under reducing conditions with a sample load of 10 ⁇ g. Purity was calculated by dividing the sum of the heavy chain and light chain band area by the total band area as measured by densitometry.
  • the 150 mg/mL lots are of high purity and comparable to the 100 mg/mL lots:
  • DAC HYP Purity of DAC HYP was assessed by both reduced and non-reduced gel electrophoresis. Precast 14% or 8-16% Tris-glycine gels were used for the analysis. Aliquots from the two batches of 150 mg/mL DAC HYP formulation were compared to a reference batch, as previously described. Reduced and non-reduced gels analyzing the purity of DAC HYP are shown in FIG. 17 . The band pattern of the 150 mg/mL lots was comparable to that of Reference Standard RS0801, with no new bands detected in the 150 mg/mL lots.
  • CEX cation exchange chromatography
  • the CEX chromatograms of the 150 mg/mL lots are consistent with those of Reference Standard RS0801, with no new charge isoforms detected in the 150 mg/mL lots.
  • the five major isoforms present in the CEX chromatograms are due to heterogeneity at the heavy chain N-terminus and include: 1) two pyroglutamate residues (pE/pE); 2) one pyroglutamate residue and one glutamine residue (pE/Q); 3) one pyroglutamate residue and one VHS sequence (truncated VHS signal peptide preceding the N-terminal glutamine residue of the mature heavy chain) (pE/VHS); 4) one glutamine residue and one VHS sequence (Q/VHS); 5) two VHS sequences (VHS/VHS).
  • C-terminal lysine (K) isoforms are also resolved and identified in FIG. 18 .
  • Each of the N-terminal isoforms described above may exist as different C-terminal isoforms (0, 1, or 2 K). Because of the complexity of the mixture, the C-terminal lysine isoforms are only clearly resolved and measurable for the major pE/pE and pE/VHS isoforms using the described method.
  • the oligosaccharide distributions of the DAC HYP 150 mg/mL and 100 mg/mL lots were evaluated by oligosaccharide mapping.
  • N-linked oligosaccharides were released enzymatically from heavy chain Asn296 using the amidase PNGaseF.
  • the oligosaccharides were subsequently derivatized with a fluorescent label (in this case anthranilic acid) and separated from the antibody via a nylon membrane.
  • the derivatized, cleaved N-linked glycans were resolved at 50° C. on a 250 ⁇ 4.6 mm polymeric-amine bonded Asahipak Amino NH 2 P-504E column (5 ⁇ m particle size, Phenomenex, cat. No. CHO-2628) with fluorescent detection, using the following elution gradient (using a sample injection volume of 100 ⁇ L; column is equilibrated with 85% Buffer A/15% Buffer B):
  • G0 Biantennary core structure with fucose attached to the N-linked N-acetyl glucosamine, one N-acetyl glucosamine on each branch of the core structure and no terminal galactose.
  • G1 Biantennary core structure with fucose attached to the N-linked N-acetyl glucosamine, one N-acetyl glucosamine on each branch of the core structure and terminal galactose on one branch of the core structure.
  • DAC HYP lots were evaluated for potential methionine oxidation, by monitoring oxidized and non-oxidized tryptic peptides present in the peptide maps.
  • the peak areas of the non-oxidized and oxidized forms of each methionine containing peptide were determined using the mass spectra extracted ion chromatograms.
  • the percent oxidized methionine was calculated by dividing the mass spectra peak area of the oxidized peptide by the sum of the peak areas of the oxidized and non-oxidized peptides.
  • Heavy chain Met251 and Met427 are the most labile and exhibits the greatest degree of oxidation. Among the lots tested concurrently to evaluate comparability, oxidation levels for Met251 and Met427 did not exceed 4.8% and 1.8%, respectively.
  • DAC HYP 150 mg/mL and 100 mg/mL lots were evaluated for binding to the alpha subunit of the IL-2 receptor (CD25) via ELISA as a measure of potency as part of release testing.
  • Microtiter plates were immobilized with soluble CD25 and incubated with varying amounts of DAC HYP.
  • Bound DAC HYP was detected using a horseradish peroxidase-conjugated goat anti-human IgG antibody in tandem with 3,3′,5,5′-tetra-methyl benzidine substrate. Resulting absorbance values were plotted against the log 10 of DAC HYP concentration using a 4-parameter fit and percent relative potency values were generated using parallel line analysis.
  • the binding potency results of the 150 mg/mL lots were comparable to those of the 100 mg/mL lots.
  • Goat anti-human IgG Fc antibody was immobilized on a chip surface to capture DAC HYP samples, after which soluble CD25 was injected at various concentrations in duplicate over captured DAC HYP using an automated method. Binding data were collected and corrected using a reference flow cell and buffer blank, and fit with BIA Evaluation software using a 1:1 Langmuir model to obtain equilibrium constants.
  • association constant (k a ), dissociation constant (k d ), and affinity constant (K D ) values of the 150 mg/mL lots were comparable to those of Reference Standard RS0801.
  • DAC HYP 150 mg/mL and 100 mg/mL lots were evaluated for functional potency as part of release testing.
  • the functional potency assay measures the inhibition of IL-2 induced T-cell proliferation by binding of DAC HYP to the alpha subunit of the IL-2 receptor (CD25).
  • varying amounts of DAC HYP were incubated with KIT-225 K6 cells (Hori et al., 1987, Blood 70:1069-1072) expressing the IL-2 receptor.
  • Inhibition of T-cell proliferation by DAC HYP was subsequently detected using alamar blue. Resulting fluorescence values were plotted against the log 10 of DAC HYP concentrations using a 4-parameter fit and percent relative potency values were generated using parallel line analysis.
  • DAC HYP 150 mg/mL formulations were evaluated relative to that of Reference Standard RS0801 100 mg/mL DAC HYP.
  • IL-2 receptor expressing KIT-225 K6 cells were labeled with 51 Cr, and subsequently incubated with DAC HYP.
  • Human effector cells PBMC
  • Fc receptor bearing monocytes interact with the DAC HYP Fc domain and subsequently cause target cell lysis.
  • the degree of cytotoxicity was determined by measuring the release of 51 Cr from target cells and was expressed as a percentage of maximum cell lysis.
  • ADCC results are summarized in Table 30 below:
  • the ADCC activity of the 150 mg/mL lots was comparable to that of Reference Standard RS0801.
  • Residual Protein A may be determined by an ELISA method, where standards, sample controls, a plate blank, and test samples are diluted with a denaturing buffer and placed into a boiling water bath to dissociate Protein A and denature and precipitate daclizumab. After boiling, standards, controls, and samples are cooled, centrifuged, and added to a micro-titer plate coated with polyclonal anti-Protein A capture antibody. Residual Protein A present in the samples is then detected using a biotinylated anti-Protein A antibody in tandem with streptavidin alkaline phosphatase and P-nitrophenyl phosphate (PNPP) substrate.
  • PNPP P-nitrophenyl phosphate
  • the plate is analyzed in a spectrophotometric plate reader and a log-log standard curve is generated, against which the concentration of Protein A is determined.
  • Test sample results are reported in parts per million (ppm) units. Parts per million results are calculated by dividing the ng/mL Protein A result by the antibody concentration of the test sample in mg/mL.
  • Detection of mouse DNA is determined at a contract laboratory using a quantitative polymerase chain reaction (Q-PCR) test method.
  • the sample is subjected to DNA extraction.
  • the sample extract is then analyzed by Q-PCR using mouse specific primers and probe to amplify a specific fragment of a repetitive element of mouse DNA. Amplification of the DNA results in a fluorescence signal that is detected.
  • the DNA in the sample is quantitatively measured by comparison to a standard curve generated using known amounts of mouse DNA. Results are expressed in picograms of DNA per milligram of antibody.
  • Table 17 The average DNA content in various lots of DAC HYP drug product are summarized in Table 17.
  • HCP Host Cell Proteins
  • Residual host cell proteins in the product are quantified using a commercially available kit.
  • An affinity purified goat polyclonal antibody to NS0 cell lysate is used for both the capture and detection of NS0 HCP.
  • the HCP standard is produced by collecting cell free harvest material from a mock production run. A standard curve is prepared using an HCP working standard and samples containing HCP are serially diluted to target the range of the standard curve. Standards, sample controls, and test samples are added to an anti-NS0 HCP polyclonal antibody coated plate. Host cell proteins are then detected with an anti-NS0 HCP polyclonal antibody conjugated to horseradish peroxidase (HRP) in tandem with 3,3′,5,5′-tetra-methyl benzidine (TMB) substrate. The plate is then analyzed in a spectrophotometric plate reader and a four parameter curve fit is generated to quantitate the amount of HCP in the samples.
  • HRP horseradish peroxidase
  • TMB 3,3′,
  • the results for the NS0 HCP ELISA assay are reported in parts per million (ppm) units. Parts per million results are calculated by dividing the ng/mL HCP result by the antibody concentration in mg/mL. The average HCP of various lots of DAC HYP drug product are summarized in Table 17.
  • Polysorbate 80 is quantitated using a spectrophotometric method that is based on the formation of a colored cobaltthiocyanate complex with polysorbate 80.
  • a standard curve is constructed using a series of polysorbate 80 standards.
  • the polysorbate 80 concentration in the sample is determined from the standard curve.
  • the ranges of polysorbate concentrations of various lots of DAC HYP drug product are summarized in Table 17.
  • Osmolality is measured using a vapor pressure depression osmometer. Prior to sample analysis the osmometer is calibrated using osmolality standards that bracket the expected osmolality of the sample. The osmolality ranges of various lots of DAC HYP drug product are summarized in Table 17.
  • the physicochemical and biological analyses conducted provide a comprehensive evaluation of DAC HYP 150 mg/mL and DAC HYP 100 mg/mL formulations. The physicochemical and biological characteristics of all lots tested to date are comparable.
  • the characteristics of DAC HYP are as follows:
  • N-linked glycan distribution of DAC HYP is as follows:
  • DAC HYP is biologically active, as confirmed in ELISA and surface plasmon resonance CD25 binding experiments, as well as functional to inhibit IL-2 induced T-cell proliferation. DAC HYP is also characterized by a low level of aggregation upon storage.
  • High concentration DAC HYP formulations are stable upon storage.
  • the following tables provide stability data for 150 mg/mL DAC HYP drug substance lots.
  • Table 31 below provides stability data following storage in 50 mL bags at the recommended conditions (2-8° C.).
  • Table 32 below provides accelerated stability data storage in 50 mL bags at 23-27° C.
  • Table 33 below provides stressed stability data.
  • DAC Penzberg is a 100 mg/ml subcutaneous formulation of daclizumab used in clinical trials by PDL BioPharma (see CHOICE study described in Section 7.9.1 below).
  • DAC HYP also has significantly lower levels of mannose glycosyls (e.g., Man5, Man6, Man7) and lower levels of sialylated glycosyls than ZENAPAX DAC (see, e.g., FIG. 21 ).
  • ADCC antibody dependent cell-mediated cytotoxicity
  • PBMC peripheral blood mononuclear cells
  • variable effector to target cell ratio format 51 Cr-labeled KIT225/K6 cells (12,500 cells/well) were pre-incubated with 1 ⁇ g/mL of antibody (final concentration) for 30 minutes at 4° C. in V-bottom 96-well plates in a volume of 100 ⁇ L of ADCC Assay Medium (containing 435 mL RPMI-1640; 5.0 mL L-glutamine; 50 mL heat inactivated fetal bovine serum; 500 ⁇ l 1000 ⁇ 2-mercaptoethanol; 5.0 mL of penicillin-streptomycin (100 ⁇ ); and 5.0 mL of HEPES (1 M stock) per 500 mL); control cells were incubated in ADCC Assay Medium in the absence of antibody for subsequent determination of antibody-independent 51 Cr release.
  • ADCC Assay Medium containing 435 mL RPMI-1640; 5.0 mL L-glutamine; 50 mL heat inactivated fetal bovine serum; 500 ⁇ l 1000 ⁇ 2-mercaptoethanol
  • the PBMC effectors were diluted serially in ADCC Assay Medium in a separate 96-well polypropylene plate, yielding cell concentrations per 100 ⁇ L of 6.25 ⁇ 10 5 cells, 3.13 ⁇ 10 5 cells, 1.56 ⁇ 10 5 cells, 7.81 ⁇ 10 4 cells, or 3.91 ⁇ 10 4 cells.
  • a volume of 100 ⁇ L per well of PBMC suspension was added to the plates containing 51 Cr-labeled KIT225/K6 and antibodies, yielding Effector to Target (E:T) ratios of 50:1, 25:1, 12.5:1, 6.25:1 and 3.13:1.
  • variable antibody concentration format 51 Cr-labeled KIT225/K6 cells (12,500 cells/well; targets) were pre-incubated with various doses of antibodies (5, 1, 0.2, 0.04, 0.008, and 0.0016 ⁇ g/mL) of mAbs (final concentration) for 30 minutes at 4° C. in V-bottom 96-well plates in a volume of 100 ⁇ L of ADCC Assay Medium. The control cells were incubated with ADCC Assay Medium alone (no mAb) for subsequent determination of antibody-independent 51 Cr release.
  • the PBMC effectors were diluted serially in ADCC Assay Medium, in a separate 96-well polypropylene plate to a concentration of 3.13 ⁇ 10 5 cells/100 ⁇ L.
  • a volume of 100 ⁇ L per well of PBMC suspension was added to the plates containing 51 Cr-labeled KIT225/K6+mAbs, yielding an Effector to Target (E:T) ratio of 25:1.
  • E:T Effector to Target
  • the assay plates were spun at 50 RCF for 2 minutes and incubated at 37° C. in a 7.5% CO 2 incubator for 4 hours.
  • FIG. 22A variable effector to target cell ratio format
  • FIG. 22B variable antibody concentration format
  • FIG. 23 A comparison of the charge isoform profiles (corresponding to the N terminal variants) of DAC HYP vs. ZENAPAX DAC vs. DAC Penzberg is shown in FIG. 23 .
  • the CHOICE trial was a phase 2, randomized, double-blind, placebo-controlled trial of daclizumab added to interferon beta therapy in 230 patients with relapsing MS.
  • the trial tested two dosing regimens of 100 mg/ml DAC Penzberg (see Section 7.8 above for a description of the product) administered as a subcutaneous injection: 1 mg/kg daclizumab administered every four weeks and 2 mg/kg daclizumab administered every two weeks. Results of the study showed that the addition of daclizumab, administered at 2 mg/kg every two weeks to interferon beta therapy, significantly reduced new or enlarged gadolinium-enhancing lesions at week 24, when compared to interferon beta therapy alone.
  • the CHOICE trial demonstrated that, in MS patients on a background of IFN ⁇ -a1 therapy, daclizumab was well-tolerated and caused a dose dependent reduction in new/enlarged gadolinium-enhancing (Gd+) lesions by 72% compared to IFN ⁇ -a1 alone.
  • Clinical efficacy was associated with a marked expansion of immunoregulatory CD56 bright natural killer (NK) cells.
  • a centralized Interactive Voice Response System was used for randomization across all sites.
  • a protocol-defined interim futility analysis was performed after 150 patients completed the Week 24 visit.
  • Eligibility criteria included patients 18-55 years of age with clinically definite relapsing remitting multiple sclerosis (according to 2005 McDonald criteria #1-4; see, Polman et al, 2005 Ann Neurol 58:840-846), a baseline Expanded Disability Status Scale (EDSS) of 0-0.50 (Kurtzke, 1983, Neurology 33(11):1444-52) and at least one MS relapse in the 12 months before randomization or one new Gd+ lesion on brain MRI performed within the 6 weeks prior to randomization, were randomized to receive either DAC HYP (150 mg or 300 mg) or placebo as a subcutaneous injection once every 4 weeks for 52 weeks. Patients with child-bearing potential needed to practice effective contraception.
  • DAC HYP 150 mg or 300 mg
  • placebo placebo as a subcutaneous injection once every 4 weeks for 52 weeks.
  • DAC HYP primary-progressive, secondary-progressive, or progressive-relapsing MS, a history of malignancy, severe allergic or anaphylactic reactions or known drug hypersensitivity, or other significant medical conditions that, in the opinion of the investigator, would preclude administration of DAC HYP. Additional exclusion criteria included previous treatment with DAC HYP or ZENAPAXTM, total lymphoid irradiation, cladribine, mitoxantrone, T-cell or T-cell receptor vaccination or any therapeutic mAb, except natalizumab or rituximab.
  • patients could not have received treatment with cyclophosphamide or rituximab within the previous year; natalizumab, cyclosporine, azathioprine, methotrexate, intravenous immunoglobulin, plasmapheresis or cytapheresis within the previous 6 months; or live virus vaccine, treatment with glatiramer acetate, IFN ⁇ , interferon-alpha, 3 months before randomization; or corticosteroids, 4-aminopyridine or related products within the previous 30 days.
  • the primary objective of this study was to determine whether DAC HYP monotherapy reduced MS relapses as defined by the annualized relapse rate (ARR) at Week 52. Relapses were defined as new or recurrent neurologic symptoms (not associated with fever or infection), lasting >24 hours, and accompanied by new neurological findings upon assessment by the examining neurologist. An Independent Neurology Evaluation Committee (INEC), consisting of three blinded MS neurologists, evaluated all suspected relapses to adjudicate whether the protocol definition of MS relapse was satisfied. Only INEC approved relapses were included in the primary analysis.
  • ARR annualized relapse rate
  • the secondary objectives were to determine whether DAC HYP was effective in reducing the number of cumulative new Gd+ lesions on brain MRI scans performed at Weeks 8, 12, 16, 20 and 24 in a subset of patients; reducing the number of new or newly-enlarging T2 hyperintense lesions at Week 52; reducing the proportion of relapsing patients between baseline and Week 52; and improving quality of life (QoL), as measured by the change from baseline in the 29-item Multiple Sclerosis Impact Scale (MSIS-29) (Hobart et al., 2001, Brain 124(Pt 5):962-73) physical impact score at Week 52.
  • MSIS-29 29-item Multiple Sclerosis Impact Scale
  • EQ-VAS EQ-Visual Analogue Scale
  • EQ-5D health survey EuroQol-a new facility for the measurement of health-related quality of life, 2011, Accessed 17.11.11, at http://www.euroqol.org/
  • 12-item short form health survey SF-12 Ware et al., 1996, Medical Care 34(3):220-33
  • MSIS-29 psychological scale at Week 52 Hobart et al., 2001, Brain 124(Pt 5):962-73.
  • Additional MRI endpoints were the number of Gd+ lesions at Week 52, the volume of total and new or newly enlarging T2 hyperintense lesions at Weeks 24 and 52, the volume of total and new T1 hypointense lesions “black holes” (defined as lesions that were iso/hypointense to gray matter and that did not enhance after gadolinium administration) at Weeks 24 and 52, and the percentage change in whole brain volume assessed by the SIENA method (Smith et al., 2001, J Comput Assist Tomogr 25(3):466-75).
  • Lymphocyte subsets were measured at multiple time points using a validated FACS assay.
  • CD56 bright NK cells were defined as CD3 ⁇ /CD16 + /CD56 bright lymphocytes.
  • Immunogenicity to DAC HYP was assessed using a standard ELISA to screen for anti-drug antibodies and a cellular assay was then used to test for neutralizing antibodies on all positive samples.
  • a sample size of approximately 600 patients was selected to have 90% power to detect a 50% reduction in the ARR between a DAC HYP treatment group and the placebo group, estimated from simulations assuming a negative binomial distribution with a 10% drop out rate, a 5% type 1 error rate and a two sided test.
  • the ARR in the placebo group was assumed to be 0.476, based on recently completed trials in RRMS subjects. All reported p-values are two-tailed.
  • the primary analysis evaluated differences in the ARR between each DAC HYP group versus placebo. Relapses that occurred after rescue treatment with alternative MS medication were censored. The difference was evaluated using a negative binomial regression model adjusting for the number of relapses in the year before study entry, baseline EDSS (EDSS ⁇ 2.5 versus EDSS>2.5) and baseline age ( ⁇ 35 versus >35 years).
  • Statistical testing for efficacy endpoints utilized separate comparisons of the DAC HYP 300 mg group versus placebo and the DAC HYP 150 mg group versus placebo. A sequential closed testing procedure was used to control the overall Type I error rate due to multiple comparisons.
  • Efficacy analyses were evaluated in the intent-to-treat (ITT) population which included all patients who underwent randomization. However, 21 patients from a single study center were prospectively excluded from the ITT population prior to study completion due to evidence of incorrect dosing at the center, which was identified prior to study completion (all patients at the center were receiving active treatment). In a sensitivity analysis, all primary and secondary efficacy analyses were repeated using all randomized patients. All safety analyses were based on the safety population, which was defined as all patients who received at least one dose of study medication and who had at least one post randomization assessment.
  • a preplanned futility analysis was performed after 150 subjects completed the Week 24 visit, to provide an opportunity to stop if the hypothesized effects of DAC HYP were not evident. Since efficacy may change over the duration of the study there was no plan to stop the study early for evidence of superiority at the time of the futility analysis. Futility was assessed by estimating separately the conditional power for both the cumulative number of new Gd+ lesions between weeks 8 and 24 and the ARR endpoint for each dose group. The Safety Monitoring Committee reviewed the data at the time of the analysis and based on the overall consistency of the data and the assessment of risk benefit recommended to continue the study.
  • the proportion of relapsing patients was reduced in the DAC HYP 150 mg (19%) and 300 mg (20%) groups relapsed versus 36% in the placebo group (p ⁇ 0.001 for both comparisons) (Table 36).
  • DAC HYP reduced new MS lesion activity, as defined by MRI, in both the entire study population and a subset with monthly MRIs performed between weeks 8 to 24 (Table 36).
  • the point estimates of efficacy were marginally stronger in the 300 mg dose group compared to the 150 mg dose group even after adjustment for the potential baseline imbalances.
  • Longitudinal analysis demonstrated that Gd+ lesion activity was higher in the 150 mg dose group compared to the 300 mg dose group in the first few months of treatment but was similar by week 52. (Table 36).
  • Sensitivity analyses that included the 21 patients from the one excluded study site yielded similar results for all efficacy analyses.
  • a psoas abscess which had been previously undiagnosed, was found to involve a mesenteric artery and had resulted in local thrombosis and acute ischemic colitis.
  • CD56 bright NK cells expanded from a median of 0.6% of lymphocytes at baseline to 2.8% at Week 52.
  • patients treated with DAC HYP had a modest decrease in B-cell and total lymphocyte counts (Table 38). Both CD4 + and CD8 + T-cell counts decreased by approximately 7-10% at Week 52 in DAC HYP-treated patients and the CD4 + /CD8 + ratio remained constant during treatment.
  • LFT Liver function test
  • DAC HYP-treated patients 5 patients in the 150 mg dose group and 1 subject in the 300 mg dose group). In some patients these antibodies were transient, and at week 52 neutralizing antibodies to DAC HYP were present in only 1 subject from each DAC HYP dose group.
  • Antagonism of CD25 with monthly, subcutaneous DAC HYP monotherapy demonstrated robust and clinically meaningful effects over 1 year on MS disease activity, e.g., as measured by reduction in relapse rate, new MRI defined lesion activity and disability progression in a predominantly treatment na ⁇ ve population of MS patients.
  • a modified NS0 cell that has been adapted to grow in serum- and cholesterol-free media and that is engineered to express a recombinant protein, said cell being capable of achieving a volumetric productivity exceeding 100 mg/L/day recombinant protein in a culture of 100 L in a 10-day fed-batch process when grown in serum- and cholesterol-free media.
  • the modified NS0 cell of embodiment 1 which is capable of achieving a volumetric productivity exceeding 100 mg/L/day recombinant protein in a culture of 1,000 L in a 10-day fed-batch process when grown in media free of cholesterol and animal-derived components.
  • the modified NS0 cell of embodiment 1 which is capable of achieving a volumetric productivity exceeding 100 mg/L/day recombinant protein in a culture of 16,000 L in a 10-day fed-batch process when grown in media free of cholesterol and animal-derived components. 4.
  • the modified NS0 cell of embodiment 1 that is capable of achieving a volumetric productivity exceeding 200 mg/L/day recombinant protein in a culture of at least 100 L in a 13-day fed-batch process. 6.
  • the modified NS0 cell of embodiment 5 which is capable of achieving a volumetric productivity exceeding 200 mg/L/day recombinant protein in a culture of 1,000 L in a 13-day fed-batch process when grown in cholesterol-free media. 7.
  • the modified NS0 cell of embodiment 5 which is capable of achieving a volumetric productivity exceeding 100 mg/L/day recombinant protein in a culture of 16,000 L in a 10-day fed-batch process when grown in serum- and cholesterol-free media.
  • the modified NS0 cell of embodiment 1 which is stably transfected with a nucleic acid useful for expressing an anti-CD25 monoclonal antibody.
  • the modified NS0 cell of embodiment 8 in which the anti-CD25 monoclonal antibody comprises a VL chain corresponding in sequence to positions 21-233 of SEQ ID NO:2 and a VH chain corresponding in sequence to positions 20 to 465 of SEQ ID NO:4. 10.
  • the modified NS0 cell of embodiment 1 which was transformed with vector pAbX.gpt. 11.
  • the modified NS0 cell of embodiment 1 which was transformed with vector pHAT.IgG1.rg.dE. 12.
  • the modified NS0 cell of embodiment 1 which is designated as clone 7A11-5H7-14-43 (obtainable from ATCC under strain designation Daclizumab dWCB IP072911, accession no. ______).
  • a method of producing a recombinant protein comprising culturing the modified NS0 cell of any one of embodiments 1-12. 14.
  • the modified NS0 cell is cultured under conditions that result in the production of at least 100 mg/L/day recombinant protein in a 100 L, 1,000 L or 16,000 L culture in a 10-day fed-batch process, or at least 200 mg/L/day recombinant protein in a 100 L, 1,000 L or 16,000 L culture in a 13-day fed-batch process.
  • the method of embodiment 13 or embodiment 14 wherein the modified NS0 cell is cultured in the absence of serum and cholesterol.
  • the method of embodiment 15 wherein the modified NS0 cell is cultured in the absence of tropolone and hydrocortisone. 17.
  • a vector useful for recombinantly expressing a protein of interest comprising a weak promoter driving expression of a selectable marker operable in mammalian cells and a strong promoter driving expression of a protein of interest.
  • the vector of embodiment 23, wherein the protein of interest is a therapeutic antibody.
  • the vector of embodiment 24, wherein the therapeutic antibody is an anti-CD25 antibody.
  • the vector of embodiment 25, wherein the anti-CD25 antibody comprises the CDRs of daclizumab.
  • the vector of embodiment 26, wherein the anti-CD25 antibody is daclizumab. 28.
  • a method for obtaining a mammalian host cell that has a high volumetric productivity of a protein of interest comprising transfecting the cell with the vector of any one of embodiments 23-27, and selecting a cell that is capable of producing at least 100 mg/L/day protein of interest in a 100 L, 1,000 L or 16,000 L culture in a 10-day fed-batch process or at least 200 mg/L/day recombinant protein in a 100 L, 1,000 L or 16,000 L culture in a 13-day fed-batch process.
  • a composition comprising daclizumab, where the daclizumab is characterized by the presence of a pE/Q heavy chain N-linked isoform and/or a Q/VHS heavy chain N-terminal isoform.
  • composition of embodiment 29 in which the pE/Q heavy chain N-terminal isoform constitutes approximately 6-15% of the daclizumab. 31. The composition of embodiment 29 in which the pE/Q heavy chain N-terminal isoform constitutes approximately 7-12% of the daclizumab. 32. The composition of any one of embodiments 29-31 in which the Q/VHS heavy chain N-terminal isoform constitutes approximately 1-15% of the daclizumab. 33. The composition of embodiment 32 in which the Q/VHS heavy chain N-terminal isoform constitutes approximately 3-12% of the daclizumab. 34. The composition of embodiment 29 in which the heavy chain of daclizumab exists in the following N-terminal isoforms:
  • a composition comprising daclizumab, where the daclizumab is characterized by an N-linked glycosylation HPLC profile containing two main peaks, one corresponding to oligosaccharide G0 GlcNAc and one corresponding to oligosaccharide G0, where the combined AUC of these two peaks constitutes about 88-99.5% of the total AUC of all peaks.
  • composition of embodiment 39 in which the AUC of the G0-GlcNAc peak constitutes about 6-16% of the total AUC of all peaks and the AUC of the G0 peak constitutes about 78-90% of the total AUC of all peaks. 41.
  • composition of embodiment 38 in which the N-linked glycosylation profile contains a third peak corresponding to sialylated oligosaccharides, and the AUC of the sialylated oligosaccharide peak constitutes 1% or less of the total AUC of all peaks. 44.
  • composition of embodiment 38, in which the daclizumab has an N-linked glycosylation HPLC profile substantially similar to that of FIG. 19 or to that of the lower panel of FIG. 21 . 47.
  • the composition of embodiment 38, in which the daclizumab is DAC HYP. 48.
  • a composition comprising daclizumab which exhibits less than 35% ADCC average cytotoxicity as measured in an in vitro cellular assay using effector cells from at least 3 healthy donors and Kit 225 K6 cells as target cells, at a daclizumab concentration of 1 ⁇ g/mL and an effector to target cell ratio of about 25:1.
  • the composition of embodiment 48 wherein the daclizumab exhibits 10% to 30% ADCC average cytotoxicity in said assay. 50.
  • composition of embodiment 48 or embodiment 49 wherein said assay uses effector cells from at least 6 healthy donors.
  • 51. The composition of embodiment 48 or embodiment 49 wherein said assay uses effector cells from at least 10 healthy donors.
  • a composition useful for making a daclizumab drug formulation comprising about 150-190 mg/mL daclizumab and quantities of excipients such that dilution of the composition with a dilution buffer yields a diluted composition that contains about 85-165 mg/mL daclizumab and has an osmolality in the range of about 267-327 mOsm/kg and a pH in the range of about pH 5.8-6.2 at 25° C., and in which at least about 95% of the daclizumab is in monomer form, as measured by size exclusion chromatography. 54.
  • composition of embodiment 53 which contains quantities of excipients such that when diluted with a dilution buffer the diluted composition contains about 85-115 mg/mL daclizumab.
  • a composition comprising about 4 to 15 mg/mL daclizumab, where 0.1% or less of the daclizumab is in aggregate form. 57.
  • composition of embodiment 56 which is obtained by purifying a daclizumab composition comprising about 4 to 15 mg/ml daclizimab, where up to 2.5% of the daclizumab is in aggregate form, via column chromatography on a weak cation exchange resin.
  • composition of embodiment 59 where the chromatography is carried out in a cylindrical column using a resin bed having a height of about 10-30 cm or about 17-19 cm, and the daclizumab is eluted at a temperature in the range of about 4-22° C. or about 18-22° C., and a flow rate in the range of about 50-200 cm/hr or about 90-110 cm/hr. 61.
  • a composition suitable for administration to humans comprising about 85-165 mg/mL daclizumab; and about 0.02-0.04% (w/v) polysorbate 80, where the composition has an osmolality in the range of about 267-327 mOsm/kg and a pH in the range of about pH 5.8-6.2 at 25° C., and at least about 95% of the daclizumab is in monomer form, as measured by size exclusion chromatography.
  • the composition of embodiment 61 in which at least about 99% of the daclizumab is in monomer form, as measured by size exclusion chromatography.
  • the composition of embodiment 61 which comprises about 85-115 mg/mL daclizumab. 64.
  • composition of embodiment 63 which consists essentially of about 100 mg/mL daclizumab, about 40 mM sodium succinate, about 100 mM sodium chloride, and about 0.03% (w/v) polysorbate 80, and has a pH of about 6.0 at 25° C. 65.
  • the composition of embodiment 61 which comprises about 135-165 mg/mL daclizumab.
  • the composition of embodiment 65 which consists essentially of about 150 mg/mL daclizumab, about 40 mM sodium succinate, about 100 mM sodium chloride, and about 0.03% (w/v) polysorbate 80, and has a pH of about 6.0 at 25° C. 67.
  • composition of embodiment 65 which is obtained by a process comprising the steps of concentrating a daclizumab composition comprising about 4 to 15 mg/mL daclizumab via ultrafiltration in a suitable buffer to achieve a daclizumab concentration in the range of about 85-180 mg/mL and optionally diluting the concentrated composition with a dilution buffer.
  • a pharmaceutical composition suitable for subcutaneous administration comprising about 85-165 mg/mL daclizumab, where the percentage of daclizumab in aggregate form does not exceed about 3% following storage for a period of about 12 months at a temperature in the range of about 2-8° C. 69.
  • the pharmaceutical composition of embodiment 68 which comprises about 85-115 mg/mL daclizumab. 70.
  • the pharmaceutical composition of embodiment 68 which comprises about 135-165 mg/mL daclizumab. 71.
  • the pharmaceutical composition of embodiment 69 or embodiment 70 in which the percentage of daclizumab in aggregate form does not exceed about 2% following storage for a period of about 12 months at a temperature in the range of about 2-8° C. 72.
  • the pharmaceutical composition of embodiment 69 or embodiment 70 in which the percentage of daclizumab in aggregate form does not exceed about 3% following storage for a period of about 18 months at a temperature in the range of about 2-8° C. 73.
  • a process for harvesting a recombinant protein from a cell culture comprising the steps of:
  • ATCC American Type Tissue Collection

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US15/724,443 US20180127506A1 (en) 2011-05-27 2017-10-04 Dac hyp compositions and methods
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