Selective reduction of cysteine residues in il-17 antibodies

Abstract

To provide a method for selectively reducing CysL97 in a preparation of IL-17 antibody recombinantly produced by a mammalian cell or antigen binding fragment thereof (e.g., a preparation of secukinumab antibody).SOLUTION: The method comprises the steps of: a) forming a reducing mixture by contacting the preparation to a reductant in a vessel where the reductant is a thiol-containing reductant having a standard oxidation-reduction potential, E0, of -0.20 V to -0.23 V at pH 7.0, as measured by thiol-disulfide exchange, and the molar ratio of reductant to antibody in the reducing mixture is between 46:1 to 118:1; and b) incubating the reducing mixture while maintaining a volumetric oxygen mass-transfer coefficient (kLa*) in the vessel to 0.37 h-1 or lower as calculated by adapting a dissolved oxygen curve to a saturation curve.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application was filed on December 22, 2014, which is incorporated by reference in its entirety. This application claims priority to U.S. Provisional Application No. 62 / 095,361, filed on 2006 / 02 / 13.

[0002] The present disclosure relates to IL-17 antibodies or their antigens that are recombinantly produced by mammalian cells. Selectively reducing CysL97 in a preparation of a binding fragment, e.g., a preparation of secukinumab It concerns the method. [Background technology]

[0003] Classical antibodies consist of two light chains (L) each with a molecular weight of about 25 kD and two heavy chains (H) each with a molecular weight of about 50 kD. It is composed of two heavy chains (H) with a molecular weight of D. The light and heavy chains are separated by disulfide bonds. The two LH units are connected by an inter-heavy chain bond (LSSH). The general formula for a classical antibody is L-SS- H(-SS-)2H-SS-L or simply H2L2 (HHLL). In addition to interchain disulfide bonds, there are also conserved intrachain disulfide bonds. Sulfide bonds are important for the stability and behavior (e.g., affinity) of antibodies. The disulfide bond is formed between two cysteine ​​residues (Cys-SH) at conserved sites in the antibody chain. ), and these residues spontaneously form disulfide bonds (Cys-SSC Disulfide bond formation is regulated by the redox potential of the environment and by the Depends on the presence of enzymes specialized for all-disulfide exchange. Internal disulfide bonds (Cys-SS-Cys) stabilizes the three-dimensional structure of the antibody.

[0004] Additional free cysteines (i.e., unpaired cysteines) involved in antigen recognition and binding There are unusual antibodies that contain the free cysteine. Modification of the free cysteine ​​on these antibodies results in This can negatively affect activity and stability and may lead to increased immunogenicity. As a result, processing of these antibodies can result in the final product being inactive, misfolded, or unusable. This can be difficult because the antibody may contain significant amounts of unusable antibody material. No. 2009 / 0280, the disclosure of which is incorporated herein by reference. The specification of No. 131 describes an IL-17 antibody, e.g., a light chain complementarity determining region (CDR) 3 loop A free cysteine ​​residue after the cis-proline in (L-CDR3) (i.e., SEQ ID NO: 10 corresponds to amino acid 97 (hereafter referred to as "CysL97") of the light chain variable region Secukinumab (SEQ ID NO: 6) having amino acid 8 of L-CDR3 In order to maintain full activity, other cysteine ​​residues are required. Oxidative disulfide pairing with other groups also prevents oxidation by exogenous compounds (e.g., other proteins). Formation of mixed disulfides with proteins, products of cellular metabolism [e.g., cysteine ​​or glutathione] derivatization with ethyl 2-propanediol and formation of a sulfoxide with oxygen) also Unfortunately, secukinumab cannot mask the unpaired cysteine ​​residues in mammalian cells. Produced using mammalian cells, which may result in unwanted cell-based CysL97 modifications. This.

[0005] The literature describes unfolded, insoluble aggregates (inclusion bodies) with mixed disulfides. Refolding of mammalian proteins expressed in bacterial cells to produce mammalian proteins To obtain mammalian proteins from bacteria, only inclusion body proteins have been reported. It is isolated, solubilized, and denatured with strong chaotropic and reducing agents. Disulfide adduct-forming agents, and a weakly oxidizing / reducing environment (pH 7-9) Complete denaturation and reduction of sulfide bonds was also performed using cellulose obtained from commercial sources or synthesized by yeast. It has been used to correctly refold recombinant plant proteins (U.S. Pat. No. 6,333,411). These methods use complete denaturation and refolding of proteins. The process is costly, corrosive, time consuming, and requires the production of It is not necessary for proteins that are

[0006] Reduction / oxidation cations to correct misfolding of non-naturally occurring Fc fusion proteins The use of coupling reagents is known (WO 02 / 68455). The Fc fusion proteins of PCT Publication No. 02 / 68455 are The Fc region probably contains interchain disulfides that are reduced and reoxidized by oxidative stress. However, we have not yet fully explained how to generate molecules with selectively reduced cysteine ​​residues. There is no teaching of this in the pamphlet. Furthermore, Fc fusion proteins are not simply antibodies. The structure and activity of the β-lactamases are enhanced by a large number of correctly linked inter- and intra-chain disulfide bonds. It is a highly complex immunoglobulin that depends on

[0007] In U.S. Patent Application Publication No. 2005 / 0123532, antibodies are activated with a reducing agent. or by culturing antibody-producing cells in serum-free medium supplemented with L-cysteine. This provides a method for producing antibodies with free cysteines. When cell culture methods are used, subsequent processing steps, such as filtration, viral inactivation and cloning, may be required. The chromatography leads to the oxidation of free cysteine ​​produced by cell culture methods. In such cases, the free cysteines should ideally be oxidized by oxidizing the free thiol groups. The oxidizing agent itself can then be modified to be protected during subsequent steps, and subsequently used in a variety of techniques, e.g. For example, filtration is used to remove the particles (see U.S. Patent Application Publication No. 2006 / 0257393). For commercial production, such methods require large amounts of reducing agents in the original medium, Large amounts of oxidizing agent are required during subsequent processing, and additional filtration methods are required to remove the oxidizing agent. This requires additional time and increases production costs.

[0008] U.S. Pat. No. 7,928,205 describes IgG obtained from mammalian cell cultures. Preference for using redox pairs in refolding of 2 antibodies, and the effect of 146B7 (Ig G1) Method for decysteinylation of free cysteines in the variable region of an antibody The corresponding research publication, Banks et al. (2008) J. Pharmaceutical Sci. 7:764-779, a recombinant monoclonal IgG1 antibody (MAB007) with a mutated IgG1 domain Decysteinylation of sulfhydryl pairs is taught. Banks et al. Decysteinylation of cysteinyl requires the use of a strong denaturant (GdnHCL) and a reducing agent (cysteine). is required or selective reduction can occur in the presence of cysteine ​​only? The authors investigated whether cysteinylation was regulated by MAB00 in the presence and absence of denaturants. It was determined that it would be effectively removed from 7.

[0009] None of the above references state whether selective reduction of CysL97 in secukinumab is possible. None of the above references teaches whether secukinumab is effective in first-line or second-line treatment. and tertiary structure; the location and location of the oxidized CysL97 in secukinumab (e.g., solvent exposed or not exposed); as well as the relative strength of the conserved disulfide bonds in the antibody. degree (e.g., whether CysL97 reacts first with a given reducing agent or whether the conserved cysteine The choice of CysL97 in secukinumab depends on whether it reacts only after reduction Furthermore, none of these references teach the reagents and conditions required for selective reduction. , selective reduction of the oxidized CysL97 in secukinumab improves antibody structure (e.g., folding), Changes in chemical composition (e.g., deamidation) or properties (binding activity, tendency to aggregate or degrade) However, it has not been reported whether these changes will result in the development of a commercial-scale sex hormone. May make selective reduction of the mab impossible / difficult to achieve. Summary of the Invention

[0010] To maintain maximum secukinumab antigen binding activity, we performed the following treatments for secukinumab: In the present study, CysL97 was masked without significantly reducing the conserved disulfide bond. It has been determined that it is necessary to convert the active form (1) into the free form (2) (see I below). Otherwise, less active and inactive lower molecular weight variants would be produced along the chain. The resulting product is formed by decoupling (H2L2 → H2L, HL2, HL, H and L ). (I) H2L2-Cys-SX+ reagent → H2L2-Cys-SH+X- reagent (1) (2)

[0011] The introduction of reducing conditions in commercial-scale antibody preparations is not considered common sense (e.g., Various reagents and methods have been developed to prevent antibody disulfide bond reduction during cell culture production of antibodies. see Trexler-Schmidt et al. 2010 Biotech and Bioengineering 106:452-61 Nonetheless, the present inventors have demonstrated large-scale commercial production in mammalian cells. Selective reduction of CysL97 of secukinumab during denaturation significantly altered the antibody. It has been determined that this is possible without the need for a hologram. The IL-17 antibodies (and fragments thereof), particularly secukinumab, disclosed herein Disclosed herein are methods for selectively reducing CysL97 in the original binding site. The method of the present invention facilitates the recovery of the binding activity of these antibodies and thus the bioavailability of their preparations. In addition, these methods increase intact antibody levels, and This helps improve the uniformity of the preparations of these antibodies. The antibody-reducing agent ratio in the system during the reaction is dependent on the combined effect of controlled oxygen transfer rate. do.

[0012] Thus, the Cy in preparations of IL-17 antibodies recombinantly produced by mammalian cells A method for selectively reducing sL97, comprising the steps of: a) contacting the preparation with at least one reducing agent in the system to form a reducing mixture; and b) Within the system: ≦ approx. 0.37 h -1 The oxygen mass transfer coefficient (k L a * ) must be maintained. Incubating the reducing mixture wherein k L a * was calculated by fitting the dissolved oxygen curve to the saturation curve; Each of the IL-17 antibodies has the V H The three complementarity determining regions (CDRs) of Containing immunoglobulin heavy chain variable domain (V H ) and V as set forth in SEQ ID NO:10 L The three C's The immunoglobulin light chain variable domain (V L ) and Prior to step a), the initial oxygen saturation percentage of the preparation was measured using a calibrated oxygen probe at 25°C. The method disclosed herein is at least about 60% when measured using a lobe. .

[0013] Selecting CysL97 in a preparation of IL-17 antibody recombinantly produced by mammalian cells A method for selectively reducing a) administering to the preparation an acid selected from cysteine / cystine and cysteine / cystamine; with a set of reducing / reducing reagents to form a reducing mixture; and b) Incubate the reducing mixture under anaerobic conditions at a temperature of about 37° C. for at least 4 hours. or incubate the reducing mixture at a temperature of about 18-24°C for about 16-24 hours. Steps to take Including, Each of the IL-17 antibodies has the V H The three complementarity determining regions (CDRs) of Containing immunoglobulin heavy chain variable domain (V H ) and V as set forth in SEQ ID NO:10 L The three C's The immunoglobulin light chain variable domain (V L Also disclosed herein is a method comprising: do.

[0014] Also provided is a purified preparation of secukinumab, comprising intact secukinumab in the preparation. The levels of β-amycin were measured by sodium dodecyl sulfate capillary electrophoresis (CE-SDS). When determined, the activity level of secukinumab in the preparation is at least about 90%. Purified preparations are also available that are at least about 90% purified as measured by TAMIN-CEX. Disclosed herein. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 shows the percentage of intact antibody (LHHL) over time after exposure to different reducing agents. [Figure 2-1] Figure 2A shows the percentage of intact antibody (LHHL) over time at room temperature after selective reduction with 8 mM cysteine ​​under anaerobic conditions. Figure 2B shows the percentage of intact antibody (LHHL) over time at room temperature after selective reduction with 8 mM cysteine ​​under aerobic conditions. [Figure 2-2] Figure 2C shows the percentage of intact antibody (LHHL) over time at 37°C after selective reduction with 8 mM cysteine ​​under anaerobic conditions. Figure 2D shows the percentage of intact antibody (LHHL) over time at 37°C after selective reduction with 8 mM cysteine ​​under aerobic conditions. [Diagram 3] FIG. 1 shows the percentage of intact antibody over time at 37° C. after selective reduction with 8 mM cysteine ​​at various dissolved oxygen concentrations. [Figure 4]Figure 4A shows a 4D contour plot of activity by CEX and Figure 4B shows a 4D contour plot of purity for CE-SDS. The plots in Figure 4 analyze the effect of cysteine ​​concentration, protein content and cysteine / cystine ratio on the output parameters activity by CEX and purity by CE-SDS, as well as the interaction between cysteine ​​concentration, protein content and cysteine / cystine ratio. [Diagram 5] Figure 5A shows a 4D contour plot of activity by CEX and Figure 5B shows a 4D contour plot of purity for CE-SDS. The plots in Figure 5 analyze the effect of cysteine ​​concentration, protein content and cystine concentration on the output parameters activity by CEX and purity by CE-SDS, as well as the interaction between cysteine ​​concentration, protein content and cystine concentration. [Figure 6] FIG. 4D contour plot of activity by CEX looking at the effects and interactions of pH, time, temperature and cysteine ​​concentration. [Figure 7] FIG. 4D contour plot of purity by CE-SDS looking at the effects and interactions of pH, time, temperature and cysteine ​​concentration. [Figure 8] FIG. 13 shows the dissolved oxygen chart for cysteine ​​treatment in confirmation run 1. [Figure 9] FIG. 13 shows the dissolved oxygen chart for cysteine ​​treatment in confirmation run 2. [Figure 10] FIG. 13 shows the dissolved oxygen chart for cysteine ​​treatment in confirmation run 3. [Figure 11] FIG. 13 shows the dissolved oxygen chart for cysteine ​​treatment in confirmation run 4. [Figure 12] FIG. 11 compares the reaction kinetics of validation run 3 and validation run 4 in terms of activity by CEX and purity by CE-SDS. [Figure 13]Figure 13A shows a scaled and centered coefficient plot of REACT.P CEX activity. Figure 13B shows a 4D contour plot of REACT.P CEX activity. The plots in Figure 13 analyze the effect of cysteine ​​concentration, protein content and stirrer speed on the output parameters CEX activity and CE-SDS purity, as well as the interaction between cysteine ​​concentration, protein content and stirrer speed. [Figure 14A] FIG. 1 shows dissolved oxygen profiles for process characterization runs at 0 rpm (numbers in legend indicate run number, agitator speed and cysteine ​​and antibody concentrations). [Figure 14B] FIG. 1 shows dissolved oxygen profiles for process characterization runs at 50 rpm (numbers in legend indicate run number, agitator speed, and cysteine ​​and antibody concentrations). [Figure 14C] FIG. 1 shows dissolved oxygen profiles for process characterization runs at 100 rpm (numbers in legend indicate run number, agitator speed and cysteine ​​and antibody concentrations). [Figure 14D] FIG. 11 compares the dissolved oxygen profiles of two runs, one without antibody and one with 12.7 g / L antibody, at 50 mL scale, 50 rpm and 6.0 mM. [Figure 15] FIG. 1 shows the kinetics of selective reduction of activity by CEX at different incubation temperatures. [Figure 16] FIG. 1 shows an overlay of dissolved oxygen charts from a scaled-down model qualification run with indications of the timing of the various steps of the process (cysteine ​​addition, heating, incubation, cooling and pH adjustment). [Figure 17] FIG. 1 shows the kinetics of activity by CEX from manufacturing scale trials and scaled down model trials during selective reduction. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The purpose of the present disclosure is to provide certain IL-17 antibodies, such as secukinumab, or their antigens. By providing a method for selectively reducing CysL97 in the antigen-binding site of a binding fragment, "Selectively reducing" refers to the ability of the disclosed IL-17 antibody or antigen-binding fragment thereof to CysL97 in the cysteine ​​residues of these antibodies is converted to the oxidized form without reducing the conserved cysteine ​​residues. The conserved cysteine ​​residues are located in the hinge region of classical IgG1 antibodies. 2 disulfide bridges in the region, 2 interchain disulfide bridges (one in each Fab) , four intrachain disulfide bonds in the Fc region, and eight in the Fab portion of the antibody. During the selective reduction process, one Transient reduction of conserved cysteines in some antibodies may occur. However, upon completion of the reaction, The vast majority of the transiently reduced conserved cysteines are reoxidized to form the residues found in typical antibodies. Conservative disulfide bonds are formed, resulting in selectively reduced preparations of antibodies ( Thus, the purity and activity of the purified preparation is increased. Selectively reduced preparations (i.e., purified preparations) are 100% pure of intact antibody. % but instead, the selectively reduced preparations are not expected to contain CE-SD Ideally, intact antibody should be at least 100% (relative to the theoretical maximum) as measured by S. Also about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 , 93, 94, 95, 96, 97, 98, 99 or about 100%.

[0017] The term "comprising" includes "including" as well as "consisting of." For example, a composition "comprising" X may consist exclusively of X or may contain any additional For example, X+Y may be included.

[0018] The term "about" in reference to a numerical value x means, for example, + / - 10%. When used before a list of numbers, the term "about" applies to each number in the series, e.g. The phrase "about 1 to 5" should be interpreted as "about 1 to about 5" or, for example, "about 1 , 2, 3, 4" should be interpreted as "about 1, about 2, about 3, about 4, etc."

[0019] The relative molecular weight of secukinumab based on the translated amino acid sequence is 147,944 daltons. This molecular weight (i.e., 147,944 daltons) will be referred to as secukinumab throughout this disclosure. Used to calculate molality values ​​and molar ratios. However, during production in CHO cells, Generally, the C-terminal lysine is removed from each heavy chain. The relative molecular weight of secukinumab is 147,688 daltons. Preparations of secukinumab have The present disclosure therefore contains a mixture of molecules with and without a C-terminal lysine residue. The secukinumab molar concentration values ​​used in this study (and the ratios using these molar concentration values) are estimates. and the terms "about," "approximately," and the like, in reference to these numerical values, at least represent relative values. This disclosure encompasses this variability in the quantities and the resulting calculations made using them.

[0020] The term "substantially" does not exclude "completely", for example, "substantially free" of Y means Y may be completely absent. Where necessary, the term "substantially" may be used interchangeably with the definition of the present disclosure. It may also be deleted.

[0021] The term "antibody" as used herein includes whole antibodies and any antigen-binding portion or portions thereof. A naturally occurring "antibody" is made up of a single chain linked together by disulfide bonds. It is a glycoprotein that contains at least two heavy (H) chains and two light (L) chains connected together. Each heavy chain comprises a heavy chain variable region (referred to herein as V H and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2 and CH3. It comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. consists of one domain, CL. V H and V L The region is the framework region (F The hypervariable or complementarity determining regions (CDRs) are interspersed with more conserved regions. Each V can be further subdivided into regions of hypervariability called H and V L There are three and four FRs, which are arranged in the following order from the amino terminus to the carboxy terminus: Arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 The heavy and light chain variable regions contain a binding domain that interacts with an antigen. The domains are involved in the regulation of various cells of the immune system (e.g., effector cells) and the first branch of the classical complement system. Mediating the binding of immunoglobulins to host tissues or factors, including the component (C1q) can be done.

[0022] The term "antigen-binding fragment" of an antibody, as used herein, refers to a fragment that binds an antigen (e.g., an IL-1 receptor). -17) refers to a fragment of an antibody that retains the ability to specifically bind to the antigen. It has been demonstrated that fragments of full-length antibodies can act as "antigen-binding portions" of antibodies. Examples of binding fragments encompassed by the term include V L , V H , from the CL and CH1 domains Fab fragments are monovalent fragments consisting of a Fab fragment linked by disulfide bonds in the hinge region; F(ab)2 fragment, a bivalent fragment containing two Fab fragments; H and CH1 Dome Fd fragment consisting of V of a single arm of an antibody L and V H Fv fragment consisting of domains; V H domain-based dAb fragments (Ward et al., 1989 Nature 341:544-546); and single Exemplary antigen-binding sites include those represented by SEQ ID NOs: 1-6 and 11-13. The Fv comprises the CDRs of secukinumab as set forth in Table 1, preferably the heavy chain CDR3. The two domains of the fragment are V L and V H are encoded by separate genes However, they can be combined into a single protein chain by recombinant methods using synthetic linkers. In this case, V L and V H The domains are paired to form a monovalent molecule. (known as single chain Fv (scFv)) (see, e.g., Bird et al., 1988 Science e 242:423-426, and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883. (See, for example, U.S. Pat. No. 6,393,363.) Such single chain antibodies are also intended to be encompassed by the term "antibody." Single chain antibodies and antigen-binding portions can be obtained using conventional techniques known to those of skill in the art.

[0023] An "isolated antibody," as used herein, is an antibody that is distinct from other antibodies having different antigen specificities. (e.g., an isolated antibody that specifically binds IL-17) (However, it is substantially free of antibodies that specifically bind to antigens other than IL-17.) "Monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a single The term "human antibody" as used herein refers to a preparation of antibody molecules of a molecular composition. Antibodies with variable regions in which both the framework and CDR regions are derived from human sequences A "human antibody" is intended to include any antibody produced by a human, human tissue, or human cells. The human antibodies of the present disclosure do not necessarily have to be produced by amino acids that are not encoded by human sequences. acid residues (e.g., by random or site-directed mutagenesis in vitro, By in vivo addition of N-nucleotides to junctions during recombination of antibody genes or mutations introduced in vivo by somatic mutation. In some embodiments of the processes and compositions for the treatment of IL-17, the IL-17 antibody is a human antibody, a monoclonal antibody, or a The antibodies are isolated antibodies and / or monoclonal antibodies.

[0024] The term "IL-17" refers to IL-17A, formerly known as CTLA8, and various Wild-type IL-17A from various species (e.g., human, mouse, and monkey), IL-17A The present disclosure includes the IL-17A polypeptide and / or the IL-17A polypeptide. The functional equivalent of the IL-17A is preferably at least partially similar to wild-type IL-17A (e.g., human IL-17A). at least about 65%, 75%, 85%, 95%, 96%, 97%, 98%, or even It has 99% complete sequence identity and has the ability to induce IL-6 production by human dermal fibroblasts. Substantially retain.

[0025] The term “K D " is intended to refer to the off-rate of a particular antibody-antigen interaction. The term "K D " as used herein means K d K a (i.e., K d / K a ) expressed as a molar concentration (M) The K of the antibody D Values ​​are determined using methods established in the art. The K D A method for determining the or using a biosensor system such as the Biacore® system. In some embodiments, the IL-17 antibody or antigen-binding fragment, e.g., Kukinumab has a K of approximately 100-250 pM for human L-17. D has.

[0026] The term "affinity" refers to the strength of interaction between an antibody and an antigen at a single antigenic site. Within the body, the variable regions of the antibody "arms" bind to numerous sites via weak non-covalent forces. It interacts with antigens, and the greater the interaction, the stronger the affinity. Standard assays for assessing the binding affinity of an antibody to a target protein include, for example, ELISA, wet These methods are known in the art, including stain blots and RIAs. (e.g., binding affinity) can also be determined by standard assays known in the art, e.g., , can be assessed by Biacore analysis.

[0027] These IL-17 functional characteristics are determined according to methodologies known in the art and described herein. activity (e.g., biochemical activity, immunochemical activity, cellular activity, physiological activity or other biological activity) An antibody that "inhibits" one or more of the activities (e.g., activities) of a given protein that are not inhibited in the absence (or an unrelated protein) of the antibody. A statistically significant increase in a specific activity compared to that seen in the presence of a control antibody of the same specificity. Antibodies that inhibit IL-17 activity are statistically associated with a significant decrease in A significant decrease, e.g., at least about a 10% decrease in the measured parameter, at least 50%; Certain embodiments of the disclosed methods and compositions result in an 80% or 90% reduction In the present study, the IL-17 antibody used was designed to inhibit 95%, 98% or 99% of the functional activity of IL-17. It may also inhibit more than %.

[0028] "Inhibiting IL-6," as used herein, refers to the inhibition of IL-6 production from primary human skin fibroblasts. An IL-17 antibody or antigen-binding fragment thereof that reduces IL-6 production (e.g., Seckinumab The production of IL-6 in primary human (skin) fibroblasts is associated with the induction of IL-17. (Hwang et al., (2004) Arthritis Res Ther; 6:R120-128). In the presence of various concentrations of IL-17 binding molecules or human IL-17 receptor having the c domain, Mouse skin fibroblasts were stimulated with recombinant IL-17. t® (basiliximab) may be used as a negative control. After 16 hours of stimulation Supernatants are harvested and assayed for IL-6 by ELISA. The antigen-binding fragment, e.g., secukinumab, when tested as described above, i.e., The activity of hu-IL-17 on IL-6 production induced by hu-IL-17 in human skin fibroblasts Inhibition of IL-6 production (in the presence of 1 nM human IL-17) as measured by IC of about 50 nM or less (e.g., about 0.01 to about 50 nM) 50 Generally speaking, In some embodiments of the methods and compositions, the IL-17 antibodies or their antigen-binding Fragments, such as secukinumab, and functional derivatives thereof, are IL-1-as defined above. 6 production inhibition is about 20 nM or less, more preferably about 10 nM or less, even more preferably IC50 of about 5 nM or less, more preferably about 2 nM or less, more preferably about 1 nM or less 50 has.

[0029] The term "derivative" refers, unless otherwise indicated, to amino acid sequence variants and to the compounds of the present disclosure. For example, a specific sequence of an IL-17 antibody or antigen-binding fragment thereof, such as secukinumab, may be used. Covalent modifications (e.g., PEGylation, deamidation, hydroxylation) of sequences (e.g., variable domains) The term "functional derivatives" is used to define the functional groups that are involved in the synthesis of a protein (e.g., silyl, phosphoryl, methylation, etc.). As well as the disclosed IL-17 antibodies, these include molecules with equivalent biological activity. Conductors include fragments and peptide analogs of the IL-17 antibodies disclosed herein. It includes regions within the sequence of a polypeptide according to the present disclosure, e.g., of a specified sequence. Functional derivatives of IL-17 antibodies (e.g., functional derivatives of secukinumab) are described herein. The IL-17 antibodies and antigen-binding fragments thereof disclosed in the document H and / or V L array (For example, V in Table 1 H and / or V L sequence) and at least about 65%, 75%, 85% , 95%, 96%, 97%, 98% or 99% total sequence identityH and / or or V L The polypeptide preferably contains a polypeptide domain and has the ability to bind to human IL-17, e.g., IL-1 7-induced human dermal fibroblasts.

[0030] The phrase "substantially identical" refers to a related amino acid or nucleotide sequence (e.g., H or V L domain) will be identical compared to a particular reference sequence or (e.g. This means that the sequences are predicted to have insubstantial differences (by conservative amino acid substitutions). Non-substantial differences include minor amino acid changes, e.g., changes in the amino acid sequence of a specified region (e.g., V H or V L Domestic In the case of antibodies, the secondary antibody contains one or two substitutions within the five amino acid sequence of the same The sequences and embodiments disclosed herein have the same specificity and at least 50% of the affinity. Qualitatively identical sequences (e.g., at least about 85% sequence identity) are also part of the present application. In some embodiments, a derivative IL-17 antibody (e.g., a derivative of secukinumab, e.g. The sequence identity of the secukinumab biosimilar antibody is approximately 90% or more compared to the disclosed sequence. Above, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% %, or 99% or more.

[0031] "Identity" to the native polypeptide and its functional derivatives is determined by aligning the sequences. and, if necessary, introducing gaps to achieve the maximum percent identity. The corresponding native polypeptide, in which any conservative substitutions are not considered part of the sequence identity. is defined herein as the percentage of amino acid residues in a candidate sequence that are identical to the residues in Neither N-terminal extensions, C-terminal extensions nor insertions shall be considered to reduce identity. Methods and computer programs for alignment are well known. The alignment can be performed using standard alignment algorithms, e.g., Altshul et al. ((1990) J. Molecular Biology, 2001). Biol., 215: 403-410) LAST: Basic Local Alignment Search Tool), Needleman et al. ((1970) J.M. ol. Biol., 48: 444 453) or the algorithm of Meyers et al. ((1988) Comput. Appl. The parameter set can be determined by the algorithm of has a gap penalty of 12, a gap extension penalty of 4, and a frame shift The matrix may be a Blosum 62 score matrix with a soft gap penalty of 5. The percent identity between amino acid or nucleotide sequences is based on the PAM120 residue mass table, The ALIGN program was used with a gap length penalty of 2 and a gap penalty of 4. The paper by E. Meyers and W. Miller (198 9) It can also be determined using the algorithm in CABIOS, 4:11-17).

[0032] "Amino acid" refers, for example, to all naturally occurring L-α-amino acids, including D-amino acids. The phrase "amino acid sequence variants" refers to those amino acid sequences that are variants of the present disclosure. The term refers to a molecule that has some difference compared to the sequence of the antibody according to the present disclosure, e.g., of a specified sequence. The amino acid sequence variants are capable of binding to human IL-17, e.g., I The amino acid sequence of the compound is intended to inhibit IL-6 production in I-17-induced human skin fibroblasts. Variants include substitution variants (wherein at least one amino acid residue is removed and the variants are consistent with the present disclosure). (a variant in which a different amino acid has been inserted in place of the same position in a polypeptide) (wherein one or more amino acids are directly related to the amino acid at a particular position in a polypeptide according to the present disclosure) and deletion variants (one of the polypeptides according to the present disclosure). or one or more amino acids removed).

[0033] The phrases "free cysteine," "non-traditional cysteine," and "unpaired cysteine" are used interchangeably. Synonymously, it refers to a cysteine ​​that is not involved in a conserved antibody disulfide bond. Free cysteine may be present in the antibody framework region or in the variable region (e.g., within the CDRs). In the case of secukinumab, the light chain variable region shown as SEQ ID NO: 10 may be present. Amino acid 8 of L-CDR3, set forth as SEQ ID NO:6, corresponds to amino acid 97 of the CysL97) is a free cysteine. There are two such free cysteine ​​residues - each V L Domain contains one -. The process is capable of selectively reducing both free cysteine ​​residues of secukinumab. In some embodiments, the disclosed IL-17 antibody or antigen-binding fragment thereof has a deletion in the light chain. Due to deletions and / or substitutions, a free cysteine ​​is not present at position CysL97. In such cases, the corresponding free cysteine ​​is the target of a selective reduction reaction, hence the term "C It is included in "ysL97".

[0034] IL-17 Antibodies and Their Antigen-Binding Fragments The various processes disclosed herein involve the production of certain IL-17 antibodies or antigen-binding fragments thereof (e.g., For example, the selective reduction of secukinumab. or an antigen-binding fragment thereof, comprising at least one CDR comprising hypervariable regions CDR1, CDR2 and CDR3. At least one immunoglobulin heavy chain variable domain (V H ), wherein the CDR1 comprises the amino acid sequence 1, and said CDR2 has the amino acid sequence SEQ ID NO:2, and said CDR3 has the amino acid sequence SEQ ID NO:3, 3 has the amino acid sequence SEQ ID NO: 3. In one embodiment, the IL-17 antibody or The antigen-binding fragment of the present invention comprises at least one CDR1', CDR2' and CDR3' hypervariable region. Each of them contains one immunoglobulin light chain variable domain (V L’ ), wherein the CDR1' comprises an amine The CDR2' has the amino acid sequence SEQ ID NO: 4, and the CDR2' has the amino acid sequence SEQ ID NO: 5, DR3' has the amino acid sequence SEQ ID NO:6. In one embodiment, the IL-17 antibody or or an antigen-binding fragment thereof, comprising hypervariable regions CDR1-x, CDR2-x and CDR3-x. At least one immunoglobulin heavy chain variable domain (V H ), wherein the CDR1 -x has the amino acid sequence SEQ ID NO: 11, and said CDR2-x has the amino acid sequence SEQ ID NO: 12, and said CDR3-x has the amino acid sequence SEQ ID NO:13.

[0035] In one embodiment, the IL-17 antibody or antigen-binding fragment thereof is Epidemiological globulin V H domain and at least one immunoglobulin V L Includes the domain, a)V HA domain can be (e.g., in order): i) hypervariable regions CDR1, CDR2 and CDR3; DR3 (the CDR1 has the amino acid sequence SEQ ID NO: 1, and the CDR2 has the amino acid sequence 2, and said CDR3 has the amino acid sequence SEQ ID NO: 3) or ii) Hypervariable regions CDR1-x, CDR2-x and CDR3-x (CDR1-x is an amino acid sequence of and said CDR2-x has the amino acid sequence SEQ ID NO:12; the CDR3-x has the amino acid sequence SEQ ID NO: 13), and b) V L Domestic The inclusions are (e.g., in order) hypervariable regions CDR1', CDR2' and CDR3' ( The CDR1' has the amino acid sequence SEQ ID NO: 4, and the CDR2' has the amino acid sequence SEQ ID NO: No. 5, and said CDR3' has the amino acid sequence SEQ ID NO:6).

[0036] In one embodiment, the IL-17 antibody or antigen-binding fragment thereof comprises: a) an IL-17 antibody or antigen-binding fragment thereof as set forth in SEQ ID NO:8 The immunoglobulin heavy chain variable domain (V) contains the amino acid sequence H );b) SEQ ID NO:10 An immunoglobulin light chain variable domain (V) comprising the amino acid sequence described herein. L );c) SEQ ID NO:8 Immunoglobulin V containing the amino acid sequence described H Domains and amino acids listed in SEQ ID NO:10 Immunoglobulin V containing sequences L Domains; d) SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 Immunoglobulin V comprising a hypervariable region according to H Domain; e) SEQ ID NO: 4, SEQ ID NO: 5 and and an immunoglobulin V comprising the hypervariable region set forth in SEQ ID NO:6. L domain; f) SEQ ID NO:1 1. Immunoglobulin V comprising the hypervariable regions set forth as SEQ ID NO:12 and SEQ ID NO:13 H domain; g) comprising the hypervariable regions set forth in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 Immunoglobulin V H Domains and hypervariable sequences as set forth in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 Immunoglobulin V containing domain L domain; or h) SEQ ID NO:11, SEQ ID NO:12, and Immunoglobulin V comprising the hypervariable region set forth as SEQ ID NO: 13 H Domain and SEQ ID NO:4 , an immunoglobulin V comprising the hypervariable regions set forth in SEQ ID NO:5 and SEQ ID NO:6 L Domain include.

[0037] For ease of reference, the following are based on the Kabat definition and on X-ray analysis and Secukinumab monoclonal antibody was determined using the approach of Chia and coworkers. The amino acid sequences of the hypervariable regions of the polypeptides are provided in Table 1 below.

[0038] [Table 1]

[0039] In a preferred embodiment, the constant region domain is a suitable human constant region, such as "Seq. Uences of Proteins of Immunological Interest", Kabat EA et al, US Department o f Health and Human Services, Public Health Service, National Institute of Health The DNA encoding the VL of secukinumab is preferably set forth in SEQ ID NO: 9. The V of secukinumab H The DNA encoding this is set forth in SEQ ID NO:7.

[0040] In some embodiments, an IL-17 antibody or antigen-binding fragment thereof (e.g., secukinumab) is In another embodiment, the IL-17 antibody comprises the three CDRs of SEQ ID NO: 10. or an antigen-binding fragment thereof comprising the three CDRs of SEQ ID NO:8. The IL-17 antibody or antigen-binding fragment thereof comprises the three CDRs of SEQ ID NO: 10, and The CDRs of SEQ ID NO:8 and SEQ ID NO:10 are shown in Table 1. The free cysteine ​​in the light chain (CysL97) can be found in SEQ ID NO:6. This can be done.

[0041] In some embodiments, the IL-17 antibody or antigen-binding fragment thereof has the sequence of SEQ ID NO: 14. In another embodiment, the IL-17 antibody or antigen-binding fragment thereof comprises a C-terminus In another embodiment, the heavy chain comprises a heavy chain of SEQ ID NO: 15, with or without a terminal lysine. The IL-17 antibody or antigen-binding fragment thereof may comprise a light chain of SEQ ID NO: 14 and a C-terminal lysine In some embodiments, the IL-1 7 antibody or antigen-binding fragment thereof comprises the three CDRs of SEQ ID NO: 14. In the present invention, the IL-17 antibody or antigen-binding fragment thereof comprises the three CDRs of SEQ ID NO: 15. In another embodiment, the IL-17 antibody or antigen-binding fragment thereof is selected from the group consisting of 3 of SEQ ID NO: 14. SEQ ID NO: 14 and SEQ ID NO: 15. The 5 CDRs can be found in Table 1.

[0042] The hypervariable regions are preferably of human origin, but may be any type of framework region. Suitable framework regions are described in Kabat EA et al, ibid. A preferred heavy chain framework is a human heavy chain framework, e.g., It is the Numab antibody, which is, in order, for example, FR1 (amino acids 1 to 3 of SEQ ID NO: 8) 0), FR2 (amino acids 36 to 49 of SEQ ID NO:8), FR3 (amino acids 67 to 69 of SEQ ID NO:8) FR3 (amino acids 117-127 of SEQ ID NO: 8) region. Taking into account the hypervariable regions determined by X-ray analysis of the mab, another preferred heavy chain frame was The framework is, in order, FR1-x (amino acids 1 to 25 of SEQ ID NO: 8), FR2-x (sequence 8), FR3-x (amino acids 61-95 of SEQ ID NO: 8), and The light chain FR4 region (amino acids 119 to 127 of SEQ ID NO: 8) was synthesized in a similar manner. The framework is composed of, in order, FR1' (amino acids 1 to 23 of SEQ ID NO: 10), FR2' (sequence 10), FR3' (amino acids 58-89 of SEQ ID NO: 10), and and FR4' (amino acids 99 to 109 of SEQ ID NO: 10) region.

[0043] In one embodiment, an IL-17 antibody or antigen-binding fragment thereof (e.g., secukinumab a) a variable domain comprising hypervariable regions CDR1, CDR2 and CDR3 of a human heavy chain in that order; An immunoglobulin heavy chain or a fragment thereof (such as the above C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C31, C42, C53, C64, C7, C8, C9, C12, C13, C14, DR1 has the amino acid sequence SEQ ID NO:1, and said CDR2 has the amino acid sequence SEQ ID NO:2. and b) a CDR3 having the amino acid sequence SEQ ID NO:3 of a human light chain. A variable domain, which in turn comprises the variable regions CDR1', CDR2' and CDR3', and a constant domain or a fragment thereof, wherein the CDR1' is an amino acid sequence selected from the group consisting of: the CDR2' has the amino acid sequence SEQ ID NO:5, the CDR3' has the amino acid sequence SEQ ID NO:6, the CDR4' has the amino acid sequence SEQ ID NO:7, the CDR5' has the amino acid sequence SEQ ID NO:8, the CDR6' has the amino acid sequence SEQ ID NO:9, the CDR7' has the amino acid sequence SEQ ID NO:10, the CDR8' has the amino acid sequence SEQ ID NO:11, the CDR9' has R3' is a human IL-17 antibody having at least the amino acid sequence SEQ ID NO:6. are selected.

[0044] In one embodiment, the IL-17 antibody or antigen-binding fragment thereof comprises an antigen-binding site a) a single chain antibody or an antigen-binding fragment thereof, the antigen-binding site being selected from a) a hypervariable A first domain comprising the regions CDR1, CDR2 and CDR3, said CDR1 being 1, and said CDR2 has the amino acid sequence SEQ ID NO:2, and said CDR3 has the amino acid sequence SEQ ID NO:3, 3 has the amino acid sequence SEQ ID NO: 3); b) the hypervariable regions CDR1', CDR2, a second domain comprising, in order, CDR1' and CDR3' (said CDR1' having the amino acid sequence No. 4, said CDR2' having the amino acid sequence SEQ ID NO:5, and said CDR3' having having the amino acid sequence SEQ ID NO:6); c) the N-terminal end of the first domain and the N-terminal end of the second domain or is linked to the C-terminal end of the first domain and the C-terminal end of the second domain. It contains a peptide linker attached to the N-terminal extremity of the main.

[0045] Alternatively, the IL-17 antibody or antigen-binding fragment thereof when used in the disclosed methods is , derivatives of the IL-17 antibody sequences described herein (e.g., PEGylation of secukinumab) Alternatively, the IL-17 antibody used in the methods of the present disclosure may include a cytosine-binding domain (a cytosine-binding domain), ... or an antigen-binding fragment thereof V H or V L The domains are the V H or V L V domains substantially identical to those set forth in SEQ ID NOs: 8 and 10. H Ma or V L The human IL-17 antibodies disclosed herein may have a (C Substantially identical to that set forth in SEQ ID NO: 15 (with or without a terminal lysine) and / or a light chain substantially identical to that set forth in SEQ ID NO:14. The human IL-17 antibodies disclosed herein include those having a C-terminal lysine or no C-terminal lysine. The present invention may include a heavy chain comprising SEQ ID NO: 15 (not including SEQ ID NO: 15), and a light chain comprising SEQ ID NO: 14. The human IL-17 antibody disclosed herein comprises a) an amino acid sequence substantially identical to that shown in SEQ ID NO:8. and b) one heavy chain comprising a variable domain having the sequence of The definition of a human light chain and a variable domain having substantially the same amino acid sequence as that shown in FIG. It may contain one light chain containing the constant region.

[0046] Alternatively, the IL-17 antibody or antigen-binding fragment thereof used in the disclosed methods may be The present invention also provides an IL-17 antibody that is an IL-17 antibody that is an IL-17 antibody. The present disclosure provides that the VH or VL domain of secukinumab is One or more of the main amino acid residues (but not CysL97), usually just A small number (e.g., 1-10) of the sequences may be, for example, mutations, e.g., site-specific mutations in the corresponding DNA sequence. IL-17 antibodies or antigen-binding fragments thereof that have been altered by allergy (e.g., In all such cases, derivatives and variants of IL-17 antibodies or an antigen-binding fragment thereof, the antigen-binding fragment may have an affinity for said molecule of about 50 nM or less, about 20 nM or less, about 10 nM or less At a concentration of about 1 nM or less, about 5 nM or less, about 2 nM or less, or more preferably about 1 nM or less, M (=30gn / ml) can inhibit the activity of human IL-17 by 50%, and the inhibitory activity The properties are as described in Example 1 of WO 2006 / 013107. Based on the IL-6 production induced by hu-IL-17 in human skin fibroblasts, It is measured based on.

[0047] In some embodiments, an IL-17 antibody or antigen-binding fragment thereof, such as a sec- Numab is a marker for Leu74, Tyr85, His86, Met87, Asn88, and Val12. 4, including Thr125, Pro126, Ile127, Val128, and His129; In some embodiments, the IL-17 antibody binds to an epitope on mature human IL-17. For example, secukinumab has Tyr43, Tyr44, Arg46, Ala79, and Asp8 In some embodiments, the IL-17 antibody binds to an epitope of mature human IL-17, including IL-17 antibodies, such as secukinumab, bind to an IL-17 homing agent that contains two mature human IL-17 chains. The monodimer binds to an epitope that is located at Leu74 and Tyr85 on one chain. , His86, Met87, Asn88, Val124, Thr125, Pro126, Ile127, Val128, and His129 on the other chain, and Tyr43, Tyr44, and A These epitopes include rg46, Ala79, and Asp80. The residue numbering scheme used indicates the residue that is the first amino acid of the mature protein (i.e. , lacking the 23 amino acid N-terminal signal peptide and beginning with glycine, IL-17A) The sequence of immature IL-17 is based on Swiss-Prot entry Q16552. In some embodiments, the IL-17 antibody is about 100-200 pM K D In some embodiments, the IL-17 antibody has a human I IC of approximately 0.4 nM for in vitro neutralization of the biological activity of L-17A 50 Have In some embodiments, the absolute bilirubin of the subcutaneously (sc) administered IL-17 antibody is The availability ranges from about 60% to about 80%, for example about 76%. In an embodiment, the IL-17 antibody, secukinumab, is administered for about 4 weeks (e.g., about 23 to about 35 days). In some embodiments, the elimination half-life is about 23 to about 30 days, for example, about 30 days. IL-17 antibodies (e.g., secukinumab) have a T of approximately 7–8 days. max has.

[0048] Particularly preferred IL-17 antibodies or antigen-binding fragments thereof for use in the disclosed methods include , human antibodies, in particular those described in Example 1 of WO 2006 / 013107 and Secukinumab is currently used to treat immune-mediated inflammatory conditions. A recombinant high affinity, fully human monoclonal antibody of the IgG1 / kappa isotype in clinical trials It is a monoclonal anti-human interleukin-17A (IL-17A, IL-17) antibody. Kinumab (see, e.g., WO 2006 / 013107 and WO 2007 / 11774 (see brochure no. 9) has a very high affinity for IL-17, i.e. , K of about 100-200 pM D and about 0.67 nM human IL-17A biological activity IC of approximately 0.4 nM for in vitro neutralization of 50 Therefore, the section Kinumab inhibits the antigen at approximately a 1:1 molar ratio. Due to its high binding affinity, secukinumab Mabs are particularly well suited for therapeutic applications. Furthermore, the secukinumab antibody has very long half-chain amino acids. It has a half-life of approximately 4 weeks, which allows for extended periods between doses. This is an exceptional property when treating chronic lifelong disorders such as rheumatoid arthritis. .

[0049] Preparations of the above-mentioned IL-17 antibodies and antigen-binding fragments thereof (e.g., secukinumab) Disclosed herein is a process for selectively reducing CysL97 in The method is conveniently performed on a preparation of an antibody (e.g., an IL-17 antibody, e.g., secukinumab). A "preparation" of an antibody is a composition having multiple antibody molecules. A "preparation" refers to a solution (e.g., a solution) that contains an IL-17 antibody or antigen-binding fragment thereof. This includes any liquid composition. Thus, the preparation may be, for example, in water or buffer, in a column The IL-17 antibody or antigen-binding fragment thereof, e.g., in the eluate, dialysis buffer, etc. In some embodiments, the initial preparation of the antibody may include a buffer (e.g., For example, Tris (e.g., 1 mM to 1 M Tris, pH 6.0 to 8.0) or WFI and a pool of IL-17 antibodies and antigen-binding fragments thereof, such as secukinumab, in Before adding a reducing agent to the antibody, it is recommended to change the dissolved oxygen level, solution pH, antibody concentration, etc. In some embodiments, the preparation may be adjusted prior to the addition of the reducing agent. The concentration of the antibody (e.g., secukinumab) in the solution is about 4 mg / ml to about 19.4 mg / ml, For example, about 10 mg / ml to about 19.4 mg / ml, about 10 mg / ml to about 15.4 mg / ml, about 12mg / ml to about 15mg / ml, or about 13.5mg / ml. In some embodiments, prior to the addition of the reducing agent, the percent oxygen saturation in the preparation is reduced by at least At least about 60% (measured using a calibrated oxygen probe at 25°C), e.g. In some embodiments, prior to the addition of the reducing agent, the pH of the preparation is adjusted to about 7.3 to about 8.5, for example, about 7.8 to about 8.2, for example, about 7.9 to about 8.1, for example The antibody concentration, pH and oxygen level are adjusted immediately after the addition of the reducing agent (or Furthermore, it can be adjusted during addition) and therefore should be interpreted as equivalent.

[0050] Preparation of IL-17 antibodies or antigen-binding fragments thereof for use in the disclosed processes The subject invention may be used in any mammalian cell line, such as Chinese hamster ovary (CHO) cells. , mouse myeloma NS0 cells, baby hamster kidney (BHK) cells, human fetal kidney cells HEK-293 human retinal cell line, Per.C6 (Crucell, NL), and HKB1 1 cell clone (hybrid cell fusion of HEK293S and Burkitt's lymphoma line 2B8) ) or the like, and produced recombinantly by any mammalian cell. "Recombinantly produced in mammalian cells" means that the This means that the production of antibodies against the IgG antibody has been achieved using recombinant DNA technology. The IL-17 antibody preparation was purified by centrifugation (with or without subsequent clarification). It may be a pool of antibodies harvested from mammalian cells. Alternatively, it may be subjected to selective reduction. The purified IL-17 antibody preparation was purified by further downstream chromatography steps. Pooling, e.g., affinity columns (e.g., Protein A columns), cation exchange It may be an eluate from a column, an anion exchange column, etc. Alternatively, it may be a product of selective reduction. The exposed IL-17 antibody preparation may be subjected to a downstream filtration step, e.g., depth filtration, nanofiltration, The antibody may be a pool of antibodies obtained by selective reduction, ultrafiltration, etc. The IL-17 antibody preparation exposed to Even if the antibody is a pool from a downstream step that has been treated to inactivate the virus, In one embodiment, the IL-17 antibody preparation subjected to selective reduction is This is the tein A eluate pool.

[0051] Depending on the process conditions selected (e.g., temperature, length of reduction time, pH, etc.), In some embodiments, the antibody concentration in the original preparation will be altered. The concentration of the IL-17 antibody to be administered is about 2 mg / mL of the IL-17 antibody or antigen-binding fragment thereof. Between about 3.8mg / ml and about 19.5mg / ml, between about 4ml and about 20mg / ml between about 10 mg / ml and about 19.4 mg / ml, For example, between about 10 mg / ml and about 15.4 mg / ml, for example, between about 12 mg / ml and about Between 15 mg / ml, for example about 13.5 mg / ml. Adjust the antibody concentration in the initial antibody preparation as desired using water (WFI) or a buffer of choice. you will be able to adjust it.

[0052] The selective reduction process described herein may be carried out in vessels of any size. In this embodiment, the container is laboratory-scale (e.g., 1 L to 2 L). In a further embodiment, the vessel is pilot-scale (e.g., 12 L to 20 L). In this case, the container is commercial-scale (e.g., more than 10,000 L, e.g., 14,000 L, 15 ,000L, 16,000L, etc.).

[0053] Reducing Agent A reducing agent is a substance that can donate electrons in a redox (reduction-oxidation) reaction. In one embodiment, such an agent is an IL-17 antibody or an antigen-binding fragment thereof (e.g., Seckinumab). It is useful for providing hydrogen to masked cysteines present in antibodies. The process disclosed herein uses a reducing agent for the selective reduction of IL-17 antibodies. Each of the reducing agents referred to herein may be used in combination with their derivatives (e.g., salts, esters and amides). Thus, for example, reference to "cysteine" includes cysteine ​​and cysteine. and references to "TCEP" include TCEP and TCEP-HCL. and reference to sodium thioglycolate includes thioglycolate esters, etc. Reducing agents for use in the disclosed methods include sodium bisulfite, ammonia , triethylsilane, glycylcysteine, sodium cyanoborohydride, thioglycol Ammonium thioglycolate, calcium thioglycolate, sodium thioglycolate, aspartate Corbic acid, hydroquinone, aminomethanesulfonic acid, cysteic acid, cysteine ​​sulf Tauric acid, ethanedisulfonic acid, ethanesulfonic acid, homotaurine, hypotaurine, isetate thiourea, mercaptoethanesulfonic acid, N-methyltaurine (MTAU), TCEP bis(2-carboxyethyl)phosphine hydrochloride), NN-dimethyl-NN-bis(methyl 2-Mercaptoacetyl)hydrazine (DMH), dithiothreitol (DTT), beta-mercaptoethanol, 2-mercaptoacetic acid (thioglycol Acid, TGA), Cysteine ​​(L-cysteine), Cysteamine (beta-mercaptoethanol), Some of these include methylaminoethylamine, or MEA, glutathione, and combinations thereof. In an embodiment, the reducing agent for use in the disclosed process is a thiol-containing reducing agent (i.e., compounds having an R-SH group), for example, organosulfur compounds. In some embodiments, the reducing agent for use in the disclosed process is, for example, dithiothreitol. diethyl ether (DTT), 2-mercaptoethanol, 2-mercaptoacetic acid, cysteine, cysteamine These are amine, glutathione and combinations.

[0054] The strength of a reducing agent is determined by its oxidation-reduction potential (redox potential), E 0 and oxidation-reduction The potential is given in volts (V) and is traditionally determined at pH 7 and 25°C. For example, CS Standard redox potential E for H / CSSC 0 is about -0.20V to about -0.23V (p H7, 25℃) (PC Jocelyn (1967) Eu.J. Biochem 2:327-31, Liu “T he role of Sulfur in Proteins,” in The Proteins, 3rd Ed.(ed. Neurath) p. 250-25 2 Academic Press 1977). The standard redox potential of DTT, E 0 is about -0.33V (pH7 , 25℃) (MJO'Neil, ed. by (2001). Merck Index: an encyclopedia o f chemicals, drugs, & biologicals: 13th ed. (13. ed. ed.) United States: MERCK & CO INC., Liu, supra). The standard redox potential of glutathione, E 0 is about -0.24V or is approximately -0.26 V (pH 7, 25°C) (Rost and Rapoport (1964) Nature 201:185; Gilbert (1990) Adv.Enzymol.Relat.Areas Mol.Biol.63:69-172, Giles (2002) Gen. Physiol Biophys 21:65-72, Liu, supra). Standard oxidation of 2-mercaptoethanol Reduction potential E 0 is approximately -0.26 V (Lee and Whitesides (1990) J. Org. Chem. 2003). m 58:642-647). In some embodiments, the reducing agent is a standard redox reaction analogous to cysteine. Electric potential E 0 (For example, about -0.20V to about -0.23V, about -0.20V to about -0.22V , about -0.20V to about -0.21V, about -0.21V to about -0.23V, about -0.21V ~-0.22V, approx.-0.22V~approx.-0.23V, approx.-0.20V, approx.-0.21V, Approximately -0.22V, approximately -0.23V).

[0055] Standard redox potential E of thiol-containing compounds 0 Thermal analysis, reduction of NAD+, polarogram raffin, reaction with Fe++, or thiol-disulfide exchange studies. (Jocelyn, supra, Borsook et al. (1937) J. Biol. Chem 117:281, Ghosh et al. (19 32) J. Indian Chem.Soc.9:43, Kolthoff et al. (1955) J. Am. Chem.Soc.77:4739, Tana ka et al.(1955) J. Am. Chem.Soc.77:2004, Kolthoff et al.(1955) J. Am. Chem.Soc.7 7:4733, Eldjarn (1957) J. Am. Chem. Soc. 79:4589). Original potential E 0 The reaction was carried out by thermal analysis, polarography, reaction with Fe++, or thiol-disulfide. For example, preferably by thiol-disulfide exchange studies. In some embodiments, the standard redox potential E 0 Determine at pH 7 and 25°C. .

[0056] The reducing agent, when combined with the antibody preparation, forms a "reducing mixture." The reducing mixture includes the reducing agent In addition to the IL-17 antibody, excipients may also be included. For example, in certain embodiments In this case, a small molar ratio of the oxidized form of the reducing agent (e.g., cysteine, cystamine) is added to the reducing mixture. The reducing agent may be added to the mixture simultaneously with the reducing agent or sequentially, e.g., at the start of the incubation period. For example, when cysteine ​​is the reducing agent, Alternatively, a small amount of cysteine ​​may be added to the reducing mixture, for example, simultaneously with the cysteine, or For example, cysteine ​​may be combined with an IL-17 antibody or antigen-binding fragment thereof. , may be added 30 minutes later. Thus, in some embodiments, the reducing mixture may include This includes oxidizer / reductor pairs. "Oxidizer / reductor pair" refers to a redox pair or A redox couple is an oxidizing agent and a reducing agent that appear on opposite sides of a half-reaction (e.g., The parent species and its corresponding oxidized form, e.g. Fe2+ / Fe3+, cysteine / cystine, Cysteamine / Cystamine)

[0057] Reaction conditions (temperature, length of reaction time, amount of IL-17 antibody or its antigen-binding fragment, pH The concentration of reducing agent used in the specific reducing mixture and selective reduction reaction depends on In some embodiments, the amount of reducing agent used in the reducing mixture is In some embodiments, the reducing mixture may contain a 5 mM to 10 mM glycerol. The concentrations of reducing agents used are approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13. , 14, 15 or 16 mM. In one embodiment, a reducing agent (e.g., cysteine The amount of ) is between about 4 mM and about 8 mM, for example, 5.9 mM, 6 mM, 7.7 mM, 7.9 mM, 8 mM.

[0058] In one embodiment, the reducing agent is beta-mercaptoethanol. In an embodiment, beta-mercaptoethanol is used at a concentration of about 2.0 mM to about 8.0 mM. do.

[0059] In one embodiment, the reducing agent is glutathione. Tathione is used at a concentration of about 2.0 mM to about 5.0 mM.

[0060] In one embodiment, the reducing agent is cysteamine. Theamine is about 1.0 mM to 20 mM, about 4.0 mM to about 19 mM, about 2.0 mM to about 8. 0 mM, about 4.0 mM to about 8.0 mM, about 4.8 mM to about 8.0 mM, about 5.5 mM to about Used at a concentration of 6.7 mM, or about 6.0 mM.

[0061] In one embodiment, the reducing agent is cysteine. About 1.0mM to 20mM, about 4.0mM to 19mM, about 2.0mM to 8.0mM M, about 4.0 mM to about 8.0 mM, about 4.8 mM to about 8.0 mM, about 5.5 mM to about 6. 7 mM, or about 6.0 mM. For example, 120 mM cysteine-HCL A stock solution can be used to adjust the cysteine ​​concentration.

[0062] Each IL-17 antibody has two CysL97 residues that require selective reduction. Therefore, the amount of reducing agent used must be such that it does not irreversibly reduce traditional disulfide bonds. Thus, a significant portion of the IL-17 in the antibody preparation can be removed without simultaneously over-reducing the antibody. The amount should be sufficient to selectively reduce both CysL97 residues of the antibody. Depending on the reaction conditions (presence of oxidizing agent, temperature, length of reaction time, pH, etc.), certain reducing mixtures may be The molar ratio of the compound and reducing agent:IL-17 antibody used in the selective reduction reaction may vary. The present inventors have demonstrated that the reductant (e.g., cysteine):antibody (e.g., secukinumab) The molar ratios ranged from about 11:1 (Example 5.2) to about 546:1 (Example 6.2). In some embodiments of the disclosed methods, a reducing agent (e.g., silyl) may be used. The molar ratio of stain:antibody (e.g., secukinumab) is between about 11:1 and about 462:1 (e.g., about 21:1), about 31:1 to about 545:1 (e.g., about 31:1 to about 156: 1), between about 21:1 and about 296:1, or between about 46:1 and about 91:1. In other embodiments, the reducing agent (e.g., cysteine): antibody (e.g., secukinumab) The molar ratio of is between about 23:1 and about 91:1 (for example, between about 23:1 and about 57:1), 44:1 to about 66:1 (e.g., about 44:1 to about 66:1), preferably about 46:1 to about 118:1 (e.g., about 56 The ratio is preferably between about 54:1 and about 82:1, more preferably between about 54:1 and about 82:1. In an embodiment, the reducing agent (e.g., cysteine):antibody (e.g., secukinumab) molar ratio is The ratio is approximately 66:1.

[0063] Using a higher molar ratio of reducing agent (e.g., cysteine):antibody (e.g., secukinumab) When using a reducing agent (meaning an excess of reducing agent compared to the antibody), a small amount of the corresponding oxidizing agent (e.g. Addition of a reducing agent (e.g., cystine or cystamine) reduces the This can be particularly beneficial in anaerobic environments. Thus, in some embodiments, an oxidizing agent / reducing agent set is used (e.g., In some embodiments, the selective reduction is carried out in an aerobic or anaerobic environment, preferably in an anaerobic environment. In the original mixture, about 2:1 to about 80:1, about 4:1 to about 80:1, about 26:1 to about 80:1 , about 2:1 to about 10:1 (for example, about 6:1 to about 10:1), about 4:1 to about 28:1 (for example, 4:1 to about 18:1), about 27:1 to about 53:1 (e.g., about 27:1) of reducing agent (e.g., cysteine):oxidizing agent (e.g., cystine) molar ratio to effect selective reduction. In certain embodiments, the reducing mixture contains the oxidizing agent cystine or cystamine (preferably In some embodiments, about 4 mM to 14 mM cystine is used in combination with cysteine. cysteine ​​(e.g., about 7.7 mM to about 8.0 mM cysteine) and about 0.1 to about 1 mM cysteine. cystine (e.g., about 0.1 to about 0.3 mM cystine) is used in the reducing mixture. In one particular embodiment, the reducing mixture contains about 8.0 mM systemic erythrocytes. about 7.9 mM cysteine ​​and about 0.1 mM cystine, about 7.9 mM cysteine ​​and about 0.1 mM cystine or about 7.7 mM cysteine ​​and about 0.3 mM cystine. When an oxidizing agent, e.g. cystine, is used in combination with a reducing agent, e.g. cysteine, For example, after combining cysteine ​​with the IL-17 antibody or antigen-binding fragment thereof, an oxidizing agent, e.g. It will be understood that, for example, cystine may be added. For example, an IL-17 antibody or or an antigen-binding fragment thereof is combined with cysteine ​​to form a reducing mixture, and then, for example, 15-3 The reaction is incubated for 0 min, after which cystine may be added to the reaction.

[0064] Dissolved oxygen As used herein, "dissolved oxygen," "dO2," and "DO" refer to the amount of dissolved oxygen (DO) in a given medium. This refers to the amount of oxygen dissolved or carried in a liquid medium. DO can be measured using an oxygen probe such as a Tode. DO is expressed as a concentration (milligrams per liter). It is reported either as milligrams per liter (mg / L) or as "percent saturation." Per liter is the amount of oxygen in 1 liter of solvent, also equal to parts per million = ppm. Oxygen Percent saturation is the amount of oxygen in a solution relative to the total amount of oxygen the solution can hold at a particular temperature. The quantity.

[0065] As used herein, "initial oxygen saturation percent" refers to the percentage of oxygen saturation in a subject receiving an IL-17 antibody (e.g., The amount of dissolved oxygen in a preparation of secukinumab (secukinumab) when the preparation is contacted with a reducing agent in a container. The initial oxygen saturation percentage refers to the amount of oxygen before it is mixed with the oxygen to form a reducing mixture. This can be done directly (e.g., by sparging) or indirectly (e.g., by stirring) before the process begins. The oxygen level can be adjusted (by adjusting the oxygen supply) to achieve a desired oxygen level. For example, in some embodiments, The initial oxygen saturation percentage in the IL-17 antibody preparation is at least 10% prior to contact with the reducing agent. to 40%, 50%, 60%, 70%, 80%, 90% or even 100% Once the reducing agent is added to the IL-17 antibody preparation to form a reducing mixture, This adjustment can be made to weaken the reducing agent, thereby reducing the Partial or partial disulfide splitting of the traditional disulfides of antibodies, which would result in reduced activity and purity. In a preferred embodiment, the initial oxygen saturation of the IL-17 antibody preparation is A percent oxygen content of at least 6 (measured using a calibrated oxygen probe at 25°C) In a preferred embodiment, the initial oxygen saturation percentage of the IL-17 antibody preparation is adjusted to 0%. Adjust the oxygen content to at least 80% (measured with a calibrated oxygen probe at 25°C). Adjust.

[0066] The inventors have demonstrated that increasing the oxygen level during the cysteine ​​treatment step increases the reducing power of cysteine. This is likely due to the fact that oxygen inhibits secukinumab, resulting in insufficient reduction of C97. They concluded that this problem of oxygen uptake from the atmosphere could have a significant detrimental effect on antibody activity. Varying the amount of reducing agent and the reducing agent:antibody molar ratio may be useful, especially when working at a production scale. by using a defined mixing rate, or even by using stirring pauses. The more reducing agent (e.g. cysteine) is available, the better it can handle. It will be appreciated that the oxygen level in the reducing mixture is high. During the incubation step of the selective reduction process, the oxygen saturation in the reducing mixture is generally A very low percentage (e.g., less than about 40%, less than about 30%, less than about 20%, less than about 15% less than, less than about 10%, less than about 5%.

[0067] The reduction in reducing power of the reducing agent (e.g., cysteine) is due to the fact that the This is likely due to incomplete deblocking of CysL97-SH at early time points during incubation. In the later part of the incubation step (or during the cooling step), residual reducing agents (e.g. In the absence of a cysteine, reoxidation of the deblocked Cys97L-SH can occur. Therefore, ideally, the low oxygen saturation percentage should be maintained for at least about 60 minutes to about 33 minutes. 0 minutes, for example, at least about 60 minutes, at least about 90 minutes, at least about 120 minutes, at least At least about 150 minutes, at least about 180 minutes, at least about 210 minutes, at least about 240 minutes at least about 270 minutes, at least about 300 minutes, or at least about 330 minutes. In some embodiments, this low oxygen saturation percentage is used throughout the entire incubation period. This will be maintained not only during the annealing step but also as part of the cooling step.

[0068] During incubation of the reducing mixture, the percent oxygen saturation was measured directly (e.g., by The desired oxygen level can be adjusted either directly (e.g., by stirring) or indirectly (e.g., by agitation). In the case of an aerobic environment, intermittent (rather than continuous) mixing of the reducing solution, e.g. For example, by using <15 min / h, e.g. 2 min / h, or continuous at low rotation speeds. With continuous agitation, a low percentage of oxygen saturation can be achieved. There is no (or very little) oxygen present which would lead to consumption of the reducing agent.

[0069] Oxygen mass transfer capacity (k L a * ) When selective reduction is performed under aerobic conditions, the oxygen level in the reaction is not directly controlled, but rather controlled by other processes. The physical settings of each reaction are also controlled by the reaction mixer. It affects the level of oxygen present in the solution. Identify variables that can be compared during specific antibody processing steps across clinical settings. It is important to determine the L a * " (e.g., Garcia-Ochoa and G omez (2009) Biotechnology Advances 27:153-176, Bandino et al. (2001) Biochem.Eng ineering J. 8:111-119, Juarez and Orejas (2001) Latin Am. Appl.Res.31:433-439, Y See Ange and Wang (1992) Biotechnol. Prog. 8:244-61. L a * The spa This represents the amount of oxygen that is transferred into the solution over time through the headspace without pulsation. The values ​​are specific to each setup and scale and include agitator type, agitator speed, fill volume, and The surface area of ​​the solution in contact with the headspace (this is influenced by the individual geometry of each container) The k for each physical setting depends on L a * Although the differences are different, the solubility of the solution during the selective reduction process Since the oxygen level in the solution affects the activity and integrity of secukinumab, the inventors , the selective reduction process has a similar k L a * If done with a system that shows the range, the same quality can be achieved. The present invention is expected to result in a preparation of secukinumab that is

[0070] As used herein, the term "system" refers to a system that is capable of dissolving in the headspace without sparging. Therefore, the physical settings (container, stirring type, etc.) that affect the oxygen transferred to the solution over time and process conditions (filling volume, rotation speed, etc.), i.e., scale, agitator speed, filling volume, headspace and surface area of ​​solution in contact (this varies depending on the individual geometry of each container) This includes factors such as the influence of

[0071] As used herein, the term "vessel" refers to any vessel in which a selective reduction reaction is carried out. The vessels include, but are not limited to, bioreactors used in pilot and commercial scale antibody production. reactors (e.g., steel, stirred tank, disposable or non-disposable, etc.); Common laboratory vessels include, for example, flasks, test tubes, etc. In some embodiments, The container may be at least about 2 liters, at least about 100 liters, at least about 500 liters at least about 1000 liters, at least about 2000 liters, at least about 5000 liters, at least about 10,000 liters, at least about 15,000 liters The bioreactor is capable of holding a volume of 1000 liters or more.

[0072] k L a * cannot be determined directly in oxygen transfer experiments. Instead, the d O2 was replaced with nitrogen and the increase in dO2 over time was monitored using a calibrated dO2 probe. This experimental dO2 curve can then be generated. Saturate the curve according to the equation shown below (e.g., using Mathcad®). By curve fitting, the k L a * The value for a particular system Calculate:

[0073] [ka] (Where, DO = measured dissolved oxygen, C = oxygen saturation value (1 when saturation is reached with infinite stirring) 00%), which corresponds to Euler number e = 2.718281... and t = DO value. and t0 = the start time). This equation describes the transfer of oxygen into the solution (k L a * value) This represents the empirical formula in integral form established for the determination of (Doran, PM 1995. Bioprocess Engineering Principles, Academic Press, 2003) ess, San Diego, California).

[0074] The inventors have found that the heating and cooling steps of the selective reduction process generally favor the in Higher k than incubation step L a * It was concluded that there is a high possibility of acceptability for However, elevated oxygen levels during the incubation step may have a detrimental effect on antibody activity. This is because the reducing power of reducing agents (e.g., cysteine) can be suppressed to reduce the It is likely that increased oxygen transfer results in insufficient reduction of C97 of kinumab. In the selective reduction process, the increase in stirring speed (or stirring time) for the reducible mixture is k L a * Increase the higher k L a * Continuous stirring of the reducing mixture resulting in a value of During the heating and cooling steps, it is acceptable. k in the top L a * (separately), about 0.12h -1 ~about 1.69h -1 , about 0.08h - 1 ~about 0.69h -1 , about 0.24h -1 ~ approx. 0.44 hours -1 , about 0.39h -1 ~about 0 .69h -1 In some embodiments, the temperature in the system during the heating and cooling steps is k L a * ≦ 0.69h -1 and said k L a * The saturation curve is converted to a dissolved oxygen curve. Calculate by fitting.

[0075] During the incubation step, the mixture is stirred, for example, at a low rotation speed or preferably at 100° C. is using intermittent mixing, k L a * In some embodiments, it is preferable to keep ≦about 0.37h -1 , ≦about 0.27h -1 , or ≦ 0.18h -1 Oxygen mass transfer volume Quantity coefficient (k L a * ) is maintained in the system while incubating the reducing mixture, k L a *is calculated by fitting the saturation curve to the dissolved oxygen curve. k in the system during the incubation step of the reaction L a * 0.37h -1 Less than (<0 .37h -1 ), the molar ratio of reducing agent (e.g., cysteine):antibody is (more Both shorter and longer incubation times, e.g., about 210 to 330 minutes) 46.11:1 (approx. 46:1) to 118.36:1 (approx. 1 18:1). The system during the incubation step of the selective reduction reaction k in L a * The molar ratio of reducing agent (e.g., cysteine):protein is Incubation time, e.g., for incubations up to about 240 minutes)6 Between 9.89:1 (approx. 70:1) and 118.36:1 (approx. 118:1) or longer For short incubation times, e.g. up to about 300 minutes )76.85 (approximately 77:1) to 118.36:1 (approximately 118:1), 0. 37h -1 It is not too high (≦0.37h -1 ) may occur.

[0076] The entire selective reduction process (i.e., heating step, incubation step and The cooling step is carried out at a molar ratio of reducing agent (e.g., cysteine):antibody of about 46:1 to about 11 If the ratio is between about 8:1, an incubation time of about 210 to about 330 minutes (e.g., about 2 Incubation times of 40 to 300 minutes) are included, <0.37 h -1 k L a * Use A totally selective reduction process (i.e., no heating step, no inert step) can generally be carried out using the The incubation and cooling steps are carried out by adding a reducing agent (e.g., cysteine):antibody. The molar ratio of the β-amyloides is between about 70:1 and about 118:1, and the incubation time is up to about 240 or when the molar ratio of reducing agent (e.g., cysteine):antibody is about 77:1 between about 118:1 and incubation is up to about 300 minutes, k L a * ≦0.37h -1 This can generally be done using

[0077] Further process components The present disclosure relates to the selection of CysL97 in certain IL-17 antibodies, such as secukinumab. A method for selective reduction of an antibody comprising contacting the antibody with a reducing agent to form a reducing mixture. The present invention provides a method for producing a reduced mixture comprising an IL-17 antibody or antigen-binding fragment thereof and a reduced It will be understood that the reconstituting mixture may contain components other than the agent. (e.g., a buffer, such as a Tris buffer), reducing mixture, salts, May also contain reagents used to increase or decrease pH, such as EDTA Thus, the reducing mixture comprises an IL-17 antibody (e.g., secukinumab) and a reducing agent. Although the reducible mixture will necessarily contain In some embodiments, the reducing mixture may or may not contain a metal chelator ( For example, EDTA, DMSA, DMPS. The mixture may be at a concentration of 1.3 mM to about 0.8 mM, about 1.1 mM to about 0.9 mM, or about 1.0 mM Contains EDTA (e.g., disodium EDTA).

[0078] Reaction conditions (temperature, length of reaction time, amount of IL-17 antibody, concentration of reducing agent, reducing agent:antibody) The pH of a particular antibody preparation may vary depending on the pH of the antibody (e.g., pH ratio). Based on the reaction conditions present, such conditions can be varied to achieve the desired selective reduction. In some embodiments, the pH of the antibody preparation prior to contact with the reducing agent is about 6.5 to about 7. The range may vary from about 7 to about 9.5, for example from about 7 to about 9. The pH of the antibody preparation is about 7.4 to about 8.5, about 7.8 to about 8.2, about 7.9 to about 8. 1, or about 8.0. In some embodiments, the pH of the antibody preparation is After gassing to adjust the initial oxygen saturation percentage, a buffer solution, e.g., 1M T Adjust the pH using Tris buffer (e.g., 1M Tris buffer pH 10.8). There is.

[0079] A preparation containing an IL-17 antibody or antigen-binding fragment thereof (e.g., secukinumab) After contact with a reducing agent, an intact IL-17 antibody or antigen-binding fragment thereof (HLLH) The term "intact" refers to the absence of all conserved disulfides. Antibodies with conserved disulfide bridges (e.g., 16 conserved disulfide bridges in the case of classical IgG1 antibodies) The various IL-17 antibody fragments, namely H2L, HL2, HL, H and L antibodies, are The formation of the intact H2L2 (HHLL) band, as well as the intact H2L2 (HHLL) band, is known in the art. Using various analytical tools (e.g., SDS-PAGE, cation exchange HPLC) Preferably, the intact IL-17 antibody or its anti-IL-17 antibody in the mixture is The levels of intact bound fragments were analyzed by sodium dodecyl sulfate capillary electrophoresis (CE-SDS). CE-SDS separates proteins according to their molecular weight in an electric field. Non-reduced CE-SDS can be used to assess size variants in antibody preparations. In some embodiments, the intact IL-17 antibody or its antigen in the mixture The level of bound fragments is determined approximately 1-30 minutes (e.g., approximately 15 minutes) after addition of the reducing agent to the antibody preparation. ) is reduced by at least about 80% as measured by CE-SDS. In an embodiment, the level of intact IL-17 antibody or antigen-binding fragment thereof in the mixture is The amount of chlorine is reduced by at least about 83% as measured by CE-SDS. In one embodiment, the level of intact IL-17 antibody or antigen-binding fragment thereof in the mixture is In some embodiments, the intact protein in the mixture is reduced by between about 75% and about 87%. Levels of IL-17 antibodies or antigen-binding fragments thereof as measured by CE-SDS , at least about 38, 39, 40, 41, 45, 50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 8 5, 86 or 87%.

[0080] CE-SDS analysis was performed using a Beckman Coulter PA-800 capillary electrode. This can be done using a gas electrophoresis system. The inner diameter is 50 μm and the length is 30 cm (reduced C The separation ranges for E-SDS and non-reduced CE-SDS analysis were 20 cm and 25 cm, respectively. An uncoated fused silica capillary with a 10 cm diameter and 1 cm width was used for the analysis. Separation was monitored by UV detection. For non-reduced CE-SDS analysis, dilute the antibody with water for 6 h. 0 mg / mL and diluted with non-reducing CE-SDS sample buffer (0.1 M Tris / 1.0 % SDS, pH 7.0) and 250 mM iodoacetamide at 20 / 75 / 5 (v / v The mixture was thoroughly mixed at a ratio of 1:1 / v and heated at 70°C for 10 minutes to perform disulfide bridge shuffling. The capillary temperature is set to 25°C for separation. Electrophoresis is performed at 15 kV. Run at constant voltage in normal positive polarity mode for 20 minutes.

[0081] In some embodiments, the reducing mixture is heated. Heating is performed prior to the application step. In some embodiments, the reducing mixture is heated to about 32° C. to about 42° C. In some embodiments, the mixture is heated to a temperature between about 35° C. and about 39° C., such as to about 37° C. Heat for about 30 to 120 minutes, about 45 to 90 minutes, about 45 to 75 minutes, or about 6 During heating, any means for stirring may be used, for example, continuously or intermittently. The reducible mixture may be agitated. The agitation may be axial (e.g., by means of a pitched blade impeller). It may be of the radial type (using a rotor, for example, a Rushton turbine). In some embodiments, during heating, the reducing mixture is rotated at a speed of 65 to 200 rpm (e.g., 50 rpm, Stir constantly at 65 rpm, 75 rpm, 85 rpm, 100 rpm or 200 rpm do.

[0082] During the incubation step, the reducing mixture generally contains free cysteines (e.g., Incubate for a period of time to allow selective reduction of the free cysteine ​​of secukinumab. In some embodiments, the incubation is performed after heating the reducing mixture. The reaction conditions (reducing agent, temperature, amount of IL-17 antibody or its antigen-binding fragment, pH, etc.) Depending on the concentration of the reducing mixture, the incubation time of the reducing mixture may vary. In certain embodiments, this time period may vary from about 1 to about 24 hours (e.g., , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 16, 18, 20 In some embodiments, the incubation is for about 200 minutes to about 500 minutes. , about 210 minutes to about 420 minutes, about 210 minutes to about 330 minutes, about 240 minutes to about 300 minutes, about It will last for 250 minutes.

[0083] During the incubation step, the target free cysteines (e.g., antigen binding sites of secukinumab) Incubation was performed at a predetermined temperature that allows selective reduction of the binding site, CysL97. The reaction conditions (reducing agent, time, amount of IL-17 antibody or antigen-binding fragment thereof, In some embodiments, the incubation temperature will vary depending on the temperature, the concentration, and the amount of the solution. In some embodiments, the predetermined temperature may vary between about 20 and about 42° C. In the present embodiment, the predetermined temperature is between about 32°C and about 42°C, between about 35°C and about 39°C, or about 37°C. It will be.

[0084] During the incubation step, a reducing agent is added to the IL-17 antibody preparation, e.g., Seckinumab. Stir the reducing mixture while incubating with the flask to ensure homogeneity of the product. However, during this part of the selective reduction reaction, the reduction of IL-17 by the reducing agent is not observed. The oxygen level in the vessel should be kept low to allow selective reduction of CysL97 in the antibody. Under aerobic conditions, continuous stirring should be avoided, for example by using intermittent stirring, e.g. For example, hypoxia can be achieved by using a rate of ≦15 min / hr, e.g., ≦2 min / hr. Under anaerobic conditions, oxygen transfer is limited and stirring can be continued for longer periods. In addition, under anaerobic conditions, oxygen transfer is reduced. Tight control of the mixing speed (e.g., by controlled sparging) also allows for longer mixing periods (continuous This makes it possible to apply the following techniques:

[0085] In some embodiments, the mixture is cooled after the incubation step. In some embodiments, the mixture is cooled to room temperature (e.g., a temperature between about 16° C. and about 28° C.). In an embodiment, the cooling is performed for about 30 minutes to about 120 minutes, about 45 minutes to about 90 minutes, or about 45 minutes to about 7 minutes. 5 minutes, approximately 60 minutes. During cooling, use any means for stirring, e.g., constant The reducible mixture may be stirred continuously or intermittently. The stirring may be axial (e.g. For example, using pitched blade impellers) or may be radial (for example, Rushton Turbo In some embodiments, the reducible mixture is rotated at 65-200 rpm (e.g., 35-400 rpm) during cooling. For example, 50 rpm, 65 rpm, 75 rpm, 85 rpm, 100 rpm or 200 rpm. Stir constantly at 300°C (250°F).

[0086] Selective reduction reactions can be carried out, for example, using iodoacetamide or o-phosphoric acid (e.g. The reaction can be stopped using a stock solution of 0.3M o-phosphoric acid. In some embodiments, quenching occurs after a cooling step. By adjusting H to about 5.0 to about 5.5, about 5.1 to about 5.3, or about 5.2, Selective reduction is stopped. pH adjustment can be achieved using o-phosphoric acid.

[0087] The phrase "purified preparation" refers to a selectively reduced IL-17 antibody or preparations thereof. Refers to an antigen-binding fragment. After selective reduction is complete, intact IL-17 in the purified preparation The level of antibody will be increased. The levels of antibody can be determined by a variety of well-known techniques (e.g., non-reducing SDS PAGE, CE-SDS PAGE, size exclusion chromatography (SEC), and HPLC In some embodiments, the level of intact antibody is measured by CE-SDS. In some embodiments, after the selective reduction is complete (e.g., after a cooling step), a purified The levels of intact IL-17 antibodies or antigen-binding fragments in the preparations were determined by CE-SD At least about 80% as measured by S. After reduction, the levels of intact IL-17 antibody or antigen-binding fragment thereof in the purified preparation were The elution is at least about 80, 81, 82, 83, 84 as measured by CE-SDS. , 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, In some embodiments, the purified preparation is The levels of intact IL-17 antibodies or antigen-binding fragments were determined by selective reduction followed by CE-S. At least about 90% as measured by DS.

[0088] In the preparation before selective reduction, in the preparation during selective reduction, or in the preparation after selective reduction ( The activity (e.g., biological activity) of the antibody (i.e., in a purified preparation) can be assayed using various peripheral Techniques of Knowledge (e.g., International Publication No. 2006 / 013107, International Publication No. Pamphlet No. 07 / 117749, Shen and Gaffen (2008) Cytokine.41(2): 92-104 In certain embodiments, the antibody can be measured by ELISA. Based assays or cell-based assays (e.g., IL-6 or GROalpha a) of IL-17-dependent release from, for example, C-20 / A4 chondrocytes or the BJ cell line. In some embodiments, cystamine-CEX (cation inhibition) is used to measure activity. The activity is measured by the cystamine-CEX method. Derivatization of antibodies with 2,2'-dithiobis(ethylamine) followed by cationic The present invention also includes analytical separations using cation exchange chromatography (CEX). The activity of antibodies (e.g., secukinumab) is reduced when CysL97 is in an oxidized form. Therefore, derivatization of CysL97 with cystamine was used as a proxy to measure antibody activity. In other words, if selective reduction is successful, the reduced CysL97 is transferred to cystamine. However, if selective reduction fails, the oxidized CysL97 can be derivatized with cysteine. Cystamine derivatization is possible with one free Cys97 residue per free Cys97 residue. The resulting derivatized forms of secukinumab (e.g. The derivatives (e.g., +2, +1 charges) are then separated from the underivatized form and purified by CEX. Two sigma-binding domains bound to unpaired Cys97 on both light chains can be quantified using the The cystamine-containing, cystamine-derivatized secukinumab molecule is theoretically 100% biologically active. One cytochrome P450 bound to the unpaired Cys97 on one light chain can be considered as an active site. Cystamine-derivatized secukinumab molecules with cystamine added were 50% more biologically active. It is possible to consider a cystamine-derivatized cystamine derivative that does not have any cystamine bound to the molecule. Cukinumab can be considered biologically inactive. Therefore, antibody preparations (e.g. The level of cystamine derivatives in a preparation of secukinumab antibody was measured by measuring the cystamine content of the preparation. The theoretical maximum level of amine derivatization (e.g., expressed as a percentage of the theoretical maximum) (obtained) can be used as a measure of the activity of the preparation.

[0089] Briefly, cystamine-CEX can be performed as follows: Antibody samples (50 μl) g) was first treated with carboxypeptidase B (1:40 w:w) to obtain the C-terminal linker in the heavy chain. The synthase was then removed and the cells were incubated for 4 h in 5 mM sodium acetate, 0.5 mM EDTA, pH 4.7. Derivatize with 2 mM cystamine for 2 h at room temperature by adding 2 μL of 1 M phosphoric acid. Derivatization is stopped. A ProPac™ WCX-10 analytical column (4 mm x 250 CEX was performed on cystamine-derivatized antibody samples using a 1.0 mm ELISA kit (Dionex). 12.5 mM to 92.5 mM in 25 mM sodium phosphate, pH 6.0, at a flow rate of 1 / min A 20 mM sodium chloride gradient is used for the separation. Absorbance at 220 nm is monitored by a UV detector (Ag The results are recorded by a spectrophotometer (HPLC 1200).

[0090] Some initial preparations of IL-17 antibodies had oxidized free cysteines, up to 45% In some embodiments, prior to initiation of the selective reduction process, The activity level of an IL-17 antibody or antigen-binding fragment thereof in a preparation may be determined by the following steps (e.g., cystadenoma, When measured by the Min-CEX method, less than about 80%, less than about 75%, less than about 70%, The percentage is less than 65%, less than about 60%, less than about 55%, less than about 50%, or less than about 45%. During selective reduction, the activity level of the IL-17 antibody in the purified preparation will increase. In some embodiments, the IL-17 antibody or antigen-binding fragment thereof in the antibody preparation. The activity level of the antibody preparation is about 6 after contacting the antibody preparation with a reducing agent to form a reducing mixture. Within 0 minutes, at least about 15 pacifi one-cent point (e.g., from about 60% to at least about 75%), at least about 20 percent 100% (e.g., from about 60% to at least about 80%), at least about 25 percent -cent point (e.g., from about 60% to at least about 85%) or at least about 3 0 percentage points (eg, from about 60% to at least about 90%).

[0091] After selective reduction, the purified preparation contained IL-17 antibodies with a reduced form of CysL97. The resulting product is enriched for α-aminobutyric acid and exhibits increased activity levels compared to the initial preparation. In some embodiments, after the selective reduction is complete, the IL-17 antibody or The activity levels of the antigen-binding fragments of the antibodies were determined by cystamine-CEX, ELISA, or cell culture. When measured by cell-based binding assays (e.g., cystamine-CEX assay) , at least about 80% (compared to the theoretical maximum). After completion of the selective reduction, the activity of the IL-17 antibodies or antigen-binding fragments thereof in the purified preparations was The levels of affinity were determined using CEX, ELISA, or cell-based binding assays (e.g., cystamivir activator assays). At least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108 4, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 , 98, 99, or about 100%. In some embodiments, after the selective reduction is complete, purification The activity levels of IL-17 antibodies or antigen-binding fragments thereof in the prepared preparations were determined by the Cystamine-CEX, ELISA, or cell-based binding assays (e.g., Cystamine-CEX) X assay).

[0092] Thus, the Cy in preparations of IL-17 antibodies recombinantly produced by mammalian cells A method for selectively reducing sL97, comprising the steps of: a) contacting the preparation with at least one reducing agent in the system to form a reducing mixture; and b) ≦ 0.37 h calculated by fitting the dissolved oxygen curve to the saturation curve -1 The oxygen mass transfer coefficient (k L a * ) in the system while injecting the reducing mixture into the ink. Steps to Introduce Including, Each of the IL-17 antibodies has the V H The three complementarity determining regions (CDRs) of Containing immunoglobulin heavy chain variable domain (V H ) and V as set forth in SEQ ID NO:10 L The three C's The immunoglobulin light chain variable domain (V L ) and Prior to step a), the initial oxygen saturation percentage of the preparation was measured using a calibrated oxygen proton pump at 25°C. Disclosed herein is a method, wherein the luminal density is at least about 60% as measured using a probe.

[0093] Thus, the Cy in preparations of IL-17 antibodies recombinantly produced by mammalian cells A method for selectively reducing sL97, comprising the steps of: a) administering to the preparation an acid selected from cysteine / cystine and cysteine / cystamine; with a set of reducing / reducing reagents to form a reducing mixture; and b) Incubate the reducing mixture under anaerobic conditions at a temperature of about 37° C. for at least 4 hours. or incubate the reducing mixture at a temperature of about 18-24°C for about 16-24 hours. Steps to take Including, Each of the IL-17 antibodies has the V H The three complementarity determining regions (CDRs) of Containing immunoglobulin heavy chain variable domain (V H ) and V as set forth in SEQ ID NO:10 L The three C's The immunoglobulin light chain variable domain (V L Also disclosed herein is a method comprising: do.

[0094] Therefore, the C in preparations of secukinumab antibody recombinantly produced by mammalian cells A method for selectively reducing ysL97, comprising the steps of: a) The concentration of secukinumab in the preparation is between about 4 mg / ml and about 19.4 mg / ml, e.g. For example, about 10 mg / ml to about 19.4 mg / ml, for example, about 10 to about 15.4, for example The concentration is adjusted to about 12 mg / ml to about 15 mg / ml, for example, about 13.5 mg / ml. Top, b) increasing the oxygen saturation percentage of the preparation to at least about 60%, e.g., at least about 80%. %, c) adjusting the pH of the preparation to about 7.4 to about 8.5, for example, about 7.8 to about 8.2, for example, about 7.9 to about 8.1, for example, about 8.0; d) contacting the preparation with cysteine ​​in a container to form a reducing mixture; The concentration of cysteine ​​in the reducing mixture is about 4.0 mM to about 8.0 mM, for example, about 4.8 5.5 mM to about 6.7 mM, for example, about 6.0 mM. Step, e) The reducing mixture is heated to a temperature between about 32° C. and about 42° C., for example, about 35° C. to about 39° C. For example, the heating is continued for about 45 to about 90 minutes, for example, for about 45 to about 7 5 minutes, for example about 60 minutes, f) The reducing mixture from step e) is heated at a temperature between about 20° C. and about 42° C., for example between about 32° C. and about Incubate at a temperature of 42° C., for example, at about 35° C. to about 39° C., for example, at about 37° C. The incubation is performed for about 210 to about 420 minutes, for example, about 21 The reaction is carried out for 0 to about 330 minutes, for example, for about 240 to about 300 minutes, for example, for about 250 minutes. , in container ≦0.37h -1 Oxygen mass transfer capacity coefficient (K L a * ) is maintained, and the K L a * is calculated by fitting the saturation curve to the dissolved oxygen curve, g) Cool the resulting mixture from step f) to a temperature of about 16° C. to about 28° C. The cooling is then carried out for about 45 to about 90 minutes, for example, about 45 to about 75 minutes, for example, about 60 minutes. Steps, and h) adjusting the pH of the mixture resulting from step g) to between about 5.1 and about 5.3; For example, adjust the step to about 5.2 Also disclosed herein is a method comprising:

[0095] A purified preparation of secukinumab, comprising: The elution was measured by sodium dodecyl sulfate capillary electrophoresis (CE-SDS). and the activity level of secukinumab in the preparation is at least about 90%. Purified preparations, which are at least about 90% as measured by -CEX, are also disclosed herein. Disclose in writing.

[0096] General rules In some embodiments of the above methods, the IL-17 antibody or antigen-binding fragment thereof comprises: i) an immunoglobulin heavy chain variable domain (VH) comprising the amino acid sequence set forth in SEQ ID NO:8; ) an immunoglobulin light chain variable domain (VL) comprising the amino acid sequence set forth in SEQ ID NO: 10; iii) an immunoglobulin VH domain comprising the amino acid sequence set forth in SEQ ID NO: 8 and SEQ ID NO: 10) an immunoglobulin VL domain comprising the amino acid sequence according to SEQ ID NO:1, the sequence an immunoglobulin VH domain comprising, in order, the hypervariable regions set forth in SEQ ID NO:2 and SEQ ID NO:3; v) an immunoglobulin comprising the hypervariable regions set forth in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, in that order; vi) the Brin VL domain as set forth in SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13 vii) an immunoglobulin VH domain comprising, in order, a hypervariable region having the following structure: an immunoglobulin VH domain comprising, in order, the hypervariable regions set forth in SEQ ID NO:2 and SEQ ID NO:3; An immunoglobulin comprising the hypervariable regions set forth in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, in that order. and viii) the VL domain as set forth in SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13. and an immunoglobulin VH domain comprising, in order, the hypervariable regions set forth in SEQ ID NO: 4, SEQ ID NO: 5 and the hypervariable region set forth in SEQ ID NO:6, in that order. In some embodiments of the disclosed methods, the IL-17 antibody or antigen-binding fragment thereof is In some embodiments of the disclosed methods, the antibody is a human antibody of the IgG1 isotype. is secukinumab.

[0097] The details of one or more embodiments of the disclosure are set forth above in the accompanying description. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. In the present disclosure, the preferred methods and materials are described below. The features, objects, and advantages of the present invention will become apparent from the description and from the claims. In the specification and the appended claims, the singular forms "a," "an," and "the" are used unless the context clearly dictates otherwise. Unless otherwise defined, all references herein include plural referents. All technical and scientific terms are commonly understood by those of ordinary skill in the art to which this disclosure belongs. All patents and publications cited herein are incorporated by reference. The following examples more fully illustrate preferred embodiments of the present disclosure. These examples are provided to illustrate, but not to limit the scope of the appended claims. The present disclosure should not be construed as limiting the scope of the patented subject matter, which is defined by the claims. No. [Example] EXAMPLES

[0098] Reduction of Secukinumab Protein A Intermediate with Different Sulfhydryl Reagents Example 1.1 Various sulfhydryl-containing reducing agents (e.g., dithiothreitol (DTT), 2-methyl- Mercaptoethanol, 2-mercaptoacetic acid, cysteine, cysteamine, glutathione) We present a study on their use in the selective reduction of the oxidized Cys97 of the light chain of secukinumab. In the first set of experiments, the initial fermentation process after the Protein A capture step was A 1 mL aliquot of the inactive starting material from the process was diluted with various concentrations of β-mercaptoethanol, The stain and glutathione were incubated at 37°C at various pH levels. After specific time points, samples were desalted by gel filtration into 20 mM acetate pH 6 buffer and the activity was analyzed. Recovery was assessed by ELISA, and the content of free sulfhydryl groups was determined by Ellman's assay. The deblocking of sulfhydryl groups was determined by experimental (The results are expected to give H values.) The results are listed in Table 2 below.

[0099] [Table 2]

[0100] As a result of these studies, the pH range of 7-9, concentrations of 1-20 mM, and 20-40°C Mercaptoethanol and cysteine ​​at a temperature of 100° C. have been found to be most suitable.

[0101] During further investigation, it was discovered that the antibody was over-reduced during exposure to reducing agents, resulting in the formation of interchain disulfides. They noticed that antibodies with partially reduced cross-links were formed. This over-reduction For example, the antibodies can be purified by diafiltration or by chromatography as described above. is isolated from the reaction mixture and is reversible under atmospheric conditions because Dissolved oxygen causes spontaneous reoxidation, resulting in reformation of intact antibodies. L-SS-HH-SS-L→L-SH+HS-HH-SS-L→L-SS-HH-SS- L (intact antibody) (reduced interchain disulfide bridges) (intact antibody)

[0102] Example 1.2 (Anaerobic conditions) In the second set of experiments, we investigated the processing of secukinumab antibodies obtained after the Protein A capture step. The reduction results were performed under anaerobic conditions by using a degassed solution and an argon or nitrogen atmosphere. In short, the effect of dissolved oxygen on the sera from the Protein A capture step was eliminated. The cukinumab solution was adjusted to pH 8.0 by the addition of Tris base stock solution. The solution was diluted with cysteine / EDTA stock solution (e.g., 200 mM / 12.5 mM EDTA) to a cysteine ​​concentration of 8 mM The EDTA concentration in the reducing solution was adjusted to 4 mg / mL (approximately The mixture was incubated at room temperature for an additional 24 hours. At different time points, samples were withdrawn and spatulated with excess iodoacetamide. This iodoamide inactivates the reducing agent and the sulfhydryl groups of the antibody. The reaction is stopped by.

[0103] The same setup was used for 2-mercaptoethanol, 2-mercaptoacetic acid, cysteine, and cystamine. DTT was used at a concentration of 1 mM. Ta.

[0104] The samples were subjected to capillary electrophoresis with SDS in non-reducing mode (CE- The analysis was performed using the antibody's different reaction products (HHL, HH, H The antibodies (L, H and L species) were separated from the intact antibodies (LHHL) and analyzed by online U V is detected and quantified by area integration of the resulting signal.

[0105] In Figure 1, the reduction treatment was performed for 0, 3, 15 minutes, 1 hour, 2 hours, 4 hours, and 20 to 24 hours. The data from the intact antibody (LHHL) from the group showed that DTT and β-mercaptoacetic acid inhibited the intact antibody. It shows that the antibody is completely reduced without re-equilibration with the tact antibody. β-Mercaptoethanol also showed significant reduction, but with intact antibody (LHHL). Cysteine ​​was able to induce re-equilibration of the antibody by approximately 50%, which then The data for cysteamine show that this reagent causes little reduction. This indicates that re-equilibration does not occur or occurs very rapidly (within a time period of less than 3 minutes).

[0106] The order of reduction determined (i.e., DTT>β-mercaptoacetic acid>β-mercaptoethanol) (Glutathione) is a published data on redox potential or disulfide interchange. T. Liu in “The Proteins, 3rd Edition, Volume 3 (1977) p. 239 Please refer to this reference, which provides the following comments and data: Redox potential E = E0 + 0.059 × log [R-SS-R] / [R-SH] The standard redox potential (E0 in volts) cannot be measured directly because the electrode is poisoned by sulfur. ). The redox potential was therefore deduced from an indirect measurement: Dithiothreitol DTT / DTTox: E0 approx. -0.33V (pH 7, 25℃) Glutathione GSH / GSSG: E0 approx. -0.24V (pH7, 25℃) Cysteine ​​CSH / CSSC: E0 approx. -0.22V (pH7, 25℃) EXAMPLES

[0107] Cysteine ​​selectively reduces secukinumab under aerobic and anaerobic conditions. 8 mM cysteine, 4 mg / ml secukinumab, pH 8 (cysteine:antibody molar ratio approximately 295.88:1) for further experiments at room temperature (RT) (approximately 25 °C) or 37 °C. The reaction was carried out under aerobic conditions (i.e., solutions were prepared and handled under normal air). (i.e., solutions were prepared and processed without degassing and then (The experiments were carried out under oxygen exclusion or reduction by working under an argon or nitrogen atmosphere at 100°C.)

[0108] Under anaerobic conditions (e.g., nitrogen or argon atmosphere with 0% dissolved oxygen), At this stage, there is substantial degradation of the antibody with the formation of antibody fragments - a sign of over-reduction. Approximately 40% The greatest decrease in intact antibody (LHHL) to 100 μg / mL was observed at room temperature (Figure 2A) or 37°C (Figure 2C) ) for experiments performed in either location, the time period is approximately 15 minutes. Re-equilibration with the antibody resulted in approximately 17.1% over-reduced variant remaining in the room temperature sample, and 3 The 7° C. sample shows 12.1% over-reduced variant remaining.

[0109] Under aerobic conditions, i.e. in the presence of dissolved oxygen, the reaction is milder. For the experiment (Figure 2B), the maximum residual level of intact antibody (LHHL) was approximately 80%. For the experiments performed at 37°C and 40°C (Fig. 2D), the maximum reduction to about 73% was approximately 15%. Re-equilibration with intact antibody occurs in minutes. Only 5% of the antibody remains in the room temperature sample after 20 hours. .8% over-reduced variant, and only 9.3% over-reduced variant remaining in the 37°C sample after 8 hours. The reduced variant is shown.

[0110] Thus, under anaerobic conditions, the initial reduction is greater and the re-equilibration with intact antibody is more rapid. Not as complete as under aerobic conditions. EXAMPLES

[0111] Effect of dissolved oxygen and cystine on selective reduction Cysteine ​​(Cys-SH) is oxidized to cystine (Cys-SS-Cys) in the presence of air. Therefore, the difference observed between aerobic and anaerobic conditions is due to the A small amount of cysteine ​​formed during the preparation and also during the actual processing of secukinumab with cysteine. The selective reduction of secukinumab by dO2 and cystine (also To assess the effect of cystamine levels, we performed several studies involving the isolation of antibodies. A few further preparative experiments were carried out.

[0112] For some samples, selective reduction of 4mg / ml secukinumab was performed at pH 8.0. The antibody was incubated at 37°C in a solution containing 8 mM cysteine ​​(295.88:1 molar ratio of cysteine:antibody). Under aerobic conditions, 100% dO2 (without nitrogen fumigation), 50%, and 20% dO2 (Ambient atmosphere was fumigated with nitrogen to ensure that dissolved oxygen remained at the target level throughout the experiment. agitation), as well as anaerobic (0% dO2) conditions without cystine (degassed solution, full nitrogen atmosphere) ) and in the presence of approximately 0.1 mM cystine [cysteine:cysteine In one embodiment, 0.3 mM cysteine ​​was added, and the This was followed by an initial 30 min incubation under anaerobic conditions with 8 mM cysteine. In another experiment, selective reduction was performed under anaerobic conditions with 7.7 mM cysteine ​​and 0.3 mM cysteine. The study was carried out using cysteine ​​[cysteine:cystine ratio = 25.66:1]. 0.1 mM cystamine was added in place of cystine.

[0113] For all these examples, samples were drawn at different time points. At the end of the first 30 min (30 min), the pH of the bulk solution was adjusted to 5.0 and the cation exchanger binding to the antibody was added. The antibody was isolated by loading it onto a column. After washing to remove the reducing agent, the antibody was desalted. and / or pH gradient elution and CE-SDS, SE-HPLC and CEX Therefore, we analyzed it.

[0114] Figure 3 shows the C of iodoacetamide quenched samples taken at different processing times. The E-SDS analysis shows that the lower the dO2, the more reduction there is in the initial stage of selective reduction, and the better the reduction. The equilibrium in the later stages of the incubation was slow. ) were significantly lower (70%) at the end of treatment. The reaction rate increased to more than 90% when the dissolved oxygen concentration was 50% or higher. The addition of sulin (or cysteamine) reduced the initial reduction of the antibody.

[0115] Table 3 shows the results of the reactivation of samples from the different reactivation treatments (anaerobic and aerobic conditions) using SP-Sepharose The activity and activity of the antibody obtained after purification using subsequent chromatography on FF were Indicates purity.

[0116] [Table 3]

[0117] Regarding biological activity, in all cases, the enzymes underwent non-selective reduction with only 45% activity. Fully active material (86-108% of theoretical maximum) was obtained for the secukinumab starting material. Regarding CE-SDS purity, the antibody obtained under complete anaerobic treatment lacking cystine was A decrease in purity (82.3%) was observed under these conditions, which was most likely due to (Note: Addition of cystine to the anaerobic reaction was measured by CE-SDS. This increased the purity of the sample because of the smaller reduction potential. (This is unlikely due to superreduction). However, all other selective reduction processes have been shown to produce smaller and The inventors have demonstrated that SP-Sepharose chromosomes can be used to obtain high antibody quality and activity. Some CE-SDS purity values ​​before the chromatography step were The values ​​were slightly lower than those obtained after treatment (e.g., 82% after treatment vs. 65% before treatment). It was also noted that (data not shown) further reoxidation These results suggest that CEX activity may occur during the fermentation step. The reduction was lower under these conditions because there was more oxygen, which weakened the reducing power of cysteine. Addition of cystine and cystamine, which are essential stimulants, under aerobic conditions further reduced CEX activity. Ta.

[0118] Summary and conclusions drawn from Examples 1-3 The inventors have demonstrated that CysL97 of secukinumab can be cleaved using, for example, guanidine HCl. Available for reduction in solution without the need for partial unfolding of the complete antibody structure Furthermore, secukinumab was found to be generally susceptible to selective reduction and to be in a controlled manner. The conditions are expected to allow activation of the antibody without substantial degradation.

[0119] Cysteine ​​exhibits rapid equilibration and only slight overactivation, so It was found to be particularly ideal for the selective reduction of kinumab. Within an hour of commencing catalytic reduction, the antibody is partially reduced, e.g., under anaerobic conditions. It reduces the nitrification rate by up to 60% and by about 30% under fully aerobic conditions. Numab slowly reoxidizes to the intact antibody. Reoxidation of the sample proceeded much slower at room temperature than in reactions carried out at 37°C. (Compare 8 hours for equilibration at 37 °C vs. 21 hours for re-equilibration at room temperature) The room temperature samples generally retained intact DNA compared to the selective reduction reactions performed at 37°C. A smaller maximal reduction in antibody was also observed.

[0120] Under anaerobic conditions, we found that the initial reduction of the antibody was greater and that intact secukinumab It was noted that the re-equilibration of cystine with ethanol was not as complete as under aerobic conditions. Addition of cystine to the aerobic reaction resulted in improved purity and solubility compared to anaerobic reactions performed in the absence of cystine. Thus, the aerobic reaction process was controlled by the oxidation of a small molar ratio of the reducing agent (e.g. In the case of cysteine, cystine is used as a reducing agent to simulate anaerobic conditions. Furthermore, the present inventors have found that the addition of cystine increases the accelerated equilibration of intact antibodies even when they were not present during the first 30 min of the reaction. Therefore, the selective reduction can be carried out in the presence of air and in an inert gas atmosphere. (e.g., nitrogen or argon) in the absence of dissolved oxygen When operating under completely anaerobic conditions, the addition of a small molar ratio of the oxidized form of a reducing agent is useful. EXAMPLES

[0121] Cysteine ​​selective reduction step development study: Proof of concept (DoE1) Example 4.1 - Study Design and Methods The primary purpose of the cysteine ​​treatment step is to facilitate cell culture, harvesting and / or Protein A cleavage. The -SH group of the cysteine ​​at position 97 of the light chain can be determined during chromatography. Restoration of full biological activity of secukinumab antibody by masking. , and antibody purity was assessed by a non-reducing CE-SDS method to monitor antibody integrity ("CE-SDS"). The antibody was analyzed for purity by cystamine-CEX chromatography. The biological activity of cystamine-CEX chromosomes (referred to as "CEX activity") was analyzed. In the cystamine-based cystamine chromatography, cystamine was added to the antibody sample, and all of the secukinumab was detected. The free cysteines are then derivatized. The cystamine-derivatized antibody species then carries an additional positive charge. Analysis by cation exchange chromatography, which has a cation exchange ratio of 0.1 to 0.2 and elutes after the underivatized antibody species The sample was then subjected to a quantitative separation and the chromatogram was then compared to that of the untreated sample. The species in the chromatogram of the treated sample, which had a shifted elution position, were compared with those in the original sample. It is a direct measure of the abundance of free CysL97-SH in the cytoplasm and serves as a surrogate marker of activity. can be used.

[0122] The primary objective of the proof-of-concept study (DoE1) was to improve the cysteine ​​processing step. A first set of parameter ranges is tested to identify key influencing factors and support the definition of the operating ranges. The objective of DoE1 was to confirm the applicability of the experimental research plan to the Whether the determination is applicable to detect the effect of input parameters on output parameters. The purpose of this study was to evaluate whether the MD 9.0 (Umetrics) software could be used. was used to analyze this concept study.

[0123] The input parameters are shown in Table 4. The redox potential is the ratio of reduced to oxidized forms, in this case the system The ratio of cystine to cystine is affected by the addition of cystine (oxidized form) as an additional input. The parameters tested were cysteine / cystine ratio and cysteine ​​concentration at three levels. The antibody concentration (ALC content) was examined for each of the two levels. The product quality output parameters, activity by CEC and purity by CE-SDS, were determined. It was determined.

[0124] [Table 4]

[0125] This design is a 3-step design without a center point trial. 2 ×2 multilevel factorial design. Cysteine Select this plan to obtain data and process understanding for improving processing steps. This type of design uses the input parameters cysteine / cystine ratio and cysteine ​​concentration It supports the calculation of mathematical models with linear terms, interaction terms and quadratic terms for The experimental design plan is given in Table 5.

[0126] [Table 5]

[0127] Selective reduction reactions were carried out in 15 mL sealed polyethylene test tubes placed in a water bath and heated to 37°C. The protein solution was diluted to the target concentration with WFI and diluted to pH 8.0 with 1 M Tris. After the addition of the cysteine ​​and cystine solutions, the test tubes were adjusted and incubated in a water bath for 4 hours. Then, samples were taken for the determination of ALC content. Directly after incubation, a 10-fold excess (with respect to the cysteine ​​concentration) of iodine was added to the CE-SDS was performed using samples whose reaction was stopped by adding acetamide. The solution is cooled to ambient temperature, its pH is adjusted to pH 5.0-5.5, and CEX and SE A sample was withdrawn for C analysis.

[0128] This design was carried out according to the actual experimental design in Table 6.

[0129] [Table 6]

[0130] Example 4.2 - Results of a Proof-of-Concept Study The values ​​of the output parameters are listed in Table 7 along with the input parameters. All trials at 0.5 mM cysteine ​​showed higher The reason for this is most likely the low cysteine ​​concentration. This is because the complete unmasking of Cys97 cannot be achieved due to the high number of nucleotides. When the antibody: antibody ratio was increased from about 11:1 to about 21:1, only a small amount of activity was observed with CEX. Improvement was observed (REACT00_17, _14 and _12). and a slightly higher cysteine:cystine ratio (REACT007_14 and _12) (high The ones with a low degree of certainty (representing a slight decrease in the reducing power of cysteine) were It also had good purity by parameters. Thus, the series of trials was The difference between the two is small (92.4–93.1%) and is within the analytical precision range with a high degree of certainty. This makes it difficult to interpret the differences between groups. Finally, one set of four trials showed the highest activity (9 3.7–95.1%). Notably, these trials showed that the highest cysteine / ta The protein ratio (462.33) was not significant.

[0131] [Table 7]

[0132] For good model fit, contours are used to understand the main influencing parameters of the system. It was possible to use plots. The quality of the model was evaluated using four tools: 2 , Q 2 , expressed as the model validity and standard deviation (SD) of the replicates. The model has an R close to 1.0. 2 and Q 2 , and model validity above 0.25 Models with less statistical significance will have a lower R 2 and Q 2 Value The model diagnosis for the quality output parameters is based on the activity of CEX and CE-SD The models for purity by S showed high statistical significance (R 2 =0.8 8 and Q 2 = 0.73, and for purity by CE-SDS, R 2 =0.84 Yobi Q 2shows having (=0.63). The contour plot for the activity by CEX (Figure 4A) teaches that when the input parameter cysteine concentration is between 4.0 mM and 0.9 mM, activity values by CEX of 93.0% or more are achieved. Furthermore, the content by ALC, which is the input factor, can vary from a low level (3.25 mg / mL) to a high level (6.5 mg / mL) when the cysteine / cystine ratio, which is the input factor, is set at the center point of 6. To improve the activity by CEX, the model indicates that the content by the input parameter ALC should be higher than the investigated upper level of 6.5 mg / mL.

[0133] Trials are listed in Table 8 according to the increase in CE - SDS purity. Trials with low purity (<=70%) to which trials with a high molar ratio of 462 mol / mol belong; trials with medium purity (70 - 83%); and trials with high purity (>83%) can be seen as three groups. For good model fitness, it is also possible to use a contour plot to understand how the input parameters affect the purity values by CE - SDS shown in Table 8. According to the contour plot of CE - SDS in Figure 4B, the region closest to the theoretical optimum is located in the lower right corner. This implies that the cysteine / cystine ratio should be set at a higher level and the cysteine concentration should be set at a lower level. In addition, a higher content by ALC can be used to increase the purity by CE - SDS.

[0134]

Table 8

[0135] Example 4.3 - Summary of Proof-of-Concept Studies Proof-of-concept studies to evaluate and refine input parameters for the cysteine ​​processing step We confirmed the applicability of the experimental setup to the high affinity of cysteine ​​to proteins. It was noted that the molar ratio could lead to low purity values ​​by CE-SDS. Increasing protein and cysteine ​​concentrations improved the purity by CE-SDS and CEX. They also suggested that the activity of cystathion may be improved by The product quality characteristics of the parameter range increase for the concentration of amines and the content by ALC. The effect of the CE-SDS on the activity of the CE-SDS-based ELISA kits was investigated in the following study (Responding Authors). A more detailed evaluation was performed using a surface area design1). EXAMPLES

[0136] Cysteine ​​selective reduction step development study: Response phase design 1 Example 5.1 - Study Design and Methods Based on the data from Example 4, the input parameters of the selective reduction step were adjusted. This development study was analyzed using Modde 9.0 (Umetrics) software. The input parameters are listed in Table 9. Each factor was investigated at three levels. Proof of concept with adjustment of the level of content and cysteine ​​concentration by input parameters ALC The input parameter "cysteine / cystine ratio" was The data was replaced by "concentration of cystine" to simplify the experimental setup and further evaluation of the data. did.

[0137] [Table 9]

[0138] This design consisted of two trials with four center trials in the center condition. 3 Center Composite This type of design involves linear terms, interaction terms, and quadratic This supports the calculation of a mathematical model with terms. The experimental design plan is given in Table 10.

[0139] [Table 10]

[0140] Selective reduction reactions were carried out in 15 mL sealed polyethylene test tubes placed in a water bath and heated to 37°C. The experiment was carried out according to the actual experimental design in Table 11.

[0141] [Table 11]

[0142] Example 5.2 - Results of Response Surface Design 1 The input and output parameter values ​​are listed in Table 12. They are listed according to increasing activity. Trials with higher CEX activity were treated with higher cysteine ​​concentrations. It can be seen that a single trial with lower activity (React016_13) was used. had a high protein concentration, a low cysteine ​​concentration, and a high cystine concentration.

[0143] Model diagnostics of product quality output parameters are performed using R for activity by CEX. 2 =0.95 , Q 2 = 0.76, SD = 0.20, and model validity = 0.78, CE-SD R for purity by S 2 =0.79, Q 2= 0.53, SD = 2.91, and model The model validity was 0.83, which indicates that the model was significant for both output parameters. To get insight into the influence of input parameter settings and their effect on the activity of CEX, Contour plots were calculated (Figure 5).

[0144] [Table 12]

[0145] As shown in the contour plot of Figure 5, the most important factors for achieving high activity with CEX are The most impactful input parameter is the cysteine ​​concentration, which should be set to its high level. However, even at low levels of cysteine, the content and If the cystine concentration is not at the upper limit, high CEX activity (>93%) can be achieved. The content and cystine concentration measured by the meter ALC are also related to the activity measured by the output parameter CEX. , but to a lesser extent.

[0146] Table 13 lists the trials according to increasing CE-SDS purity. These trials were divided into three groups: A series of six trials with low purity (<80%) (mainly the 14 mM trial) A series of trials with intermediate purity (85-91%) and a series with higher purity (>91%) were There was a series of trials in which the cysteine ​​was added to the 4 mM cysteine ​​solution, and the latter ones were all 4 mM cysteine ​​trials.

[0147] [Table 13]

[0148] Based on the results, to achieve higher purity values ​​by CE-SDS, cysteine The concentration should be low, and the ALC content and cystine concentration should be high. be.

[0149] Experiments React016_6, React016_7, React016_8 and React016_9 Act016_11 has high values ​​for both quality output parameters. Estimating the failure risk of the cysteine ​​processing step based on the model established by oE To do this, we ran a design space estimator using Monte Carlo simulation. The spatial estimator determines the range within which the factors can vary and the output parameters are consistent with the product quality. and the target range of the process performance output parameter is obtained. This is This suggests that the C content should be set to 12 mg / mL as is ideal. The cystine concentration is ideally 4.0 mM for this particular experiment. The idea is 0.32mM.

[0150] The results of this study show that the cysteine ​​treatment step is primarily dependent on the cysteine ​​concentration. High cysteine ​​concentrations promote high activity by CEX, but at the same time, CE-SDS results in low purity. According to this design space estimator, the cysteine ​​concentration should be kept at 4 mM, and the ALC content should be kept at 12 mg / mL (approximately 49 cysteines). The ratio should be set to 1:1 (to protein ratio).

[0151] For validation of the results of this statistical plan, air overlay and open headspace were used. The target run was carried out in a stirred 2 L bioreactor with stirring during incubation. was not applied. The process parameters are listed in Table 14 and the analytical results are shown in Table 15.

[0152] [Table 14]

[0153] [Table 15]

[0154] All three trials yield similar results in terms of activity by CEX. Previous DoE studies have shown that M-cysteine ​​is sufficient to restore antibody activity. The third targeted trial (REACT021) did not include cystine; As a result, the purity by CE-SDS was comparable to that of Trial 1 (REACT019). This is because under the conditions tested, cystine contributes only slightly to the cysteine ​​processing step. The results suggest that 8.0 mM (cysteine ​​to protein) Increasing the cysteine ​​concentration to approximately 91% of the total cysteine ​​concentration leads to higher activity values ​​by CEX. Not only that, it also leads to lower purity by CE-SDS in the pool, resulting in As a result, the development goal of 90% was not achieved. Cystine showed significance in this statistical plan. However, no positive effect was detected in the target trials. In addition, in this statistical design The effect of CEX on activity was small (see Figure 5), whereas the effect of the cystine-containing buffer was small. Preparation is difficult due to the low solubility of cystine in non-basic buffers. can be omitted from the cysteine ​​treatment buffer. EXAMPLES

[0155] Cysteine ​​selective reduction step development study: Response surface design 2 Example 6.1 - Study Design and Methods The primary objective of response surface design 2 (DoE3) was to evaluate the effect of cysteine ​​treatment in addition to cysteine ​​concentration. The process parameters temperature, time and pH were investigated in the steps. Data was also used to support the definition of the operational scope and to identify key influencing factors. Modde 9.0 (Umetrics) software was used to analyze this development study. The input parameters are found in Table 16.

[0156] [Table 16]

[0157] The design is a central composite facet (CCF) design with three center point trials. The plan is given in Table 17. The cysteine ​​treatment solution was improved in the previous experiment, so further plans were This design was chosen to evaluate the effect of process parameters. It is the most influential input parameter of the input processing step and has interactions with the input parameters. Cysteine ​​concentration was analyzed again because it was expected that cysteine ​​would be useful. This type of design allows for linear terms, crosstalk, and Supports the calculation of mathematical models that include interaction terms and quadratic terms.

[0158] [Table 17]

[0159] Selective reduction was performed in a 15 mL sealed polypropylene test tube placed in a water bath and heated to 37°C. The reaction was carried out.

[0160] Example 6.2 - Response Surface Design 2 Results The design was carried out according to the actual experimental design in Table 18.

[0161] [Table 18]

[0162] The values ​​for the product quality output parameters are collected in Table 19 in ascending order for the CEX activity results. The model diagnosis of product quality output parameters is shown in R for activity by CEX. 2 =0 .93, Q 2 = 0.80, model validity = 0.79, and SD = 0.42, and CE - Purity by SDS is R 2 =0.96, Q 2 = 0.89, model validity = 0.6 7 and SD=0.00.

[0163] [Table 19]

[0164] Table 19 shows the three main groups of trials: low activity (<93%), medium activity (93-95%) and high activity. Due to the good model fit, the system It is possible to use contour plots to identify the main influencing parameters of Figure 6. Figure 6 shows the effect of medium to high incubation times (e.g., 4 to 7 hours) and high cysteine. The results show that the incubation with 100 mM ethanol at 25°C is beneficial for increasing the activity of CEX. Temperature and process pH have little effect on the activity according to the output parameter CEX. Nevertheless, the areas for 95% or greater purity were significantly higher at pH 8.0 and 8. 5 plot, suggesting that this is the optimum operating range. do.

[0165] Table 20 lists the trials according to increasing CE-SDS purity. Trials were divided into 3 groups: low purity (< 82%), trials with intermediate purity, and trials with high purity (>90%). All trials with high purity by CD-SDS were performed using 2 mM sys. Tein was used.

[0166] [Table 20]

[0167] To understand the process and key influencing input parameters for good model fit Contour plots can be used (Figure 7). Figure 7 shows the results for medium to high incubation Showing that time and low cysteine ​​concentration increase purity by CE-SDS. The parameters incubation temperature and process pH were adjusted for purity by CE-SDS. had only a minor impact.

[0168] Summary and conclusions drawn from Example 6 The temperature may vary from moderate to high, the pH may vary from moderate to high, and the time The interval may vary from medium to high, but when the cysteine ​​concentration is kept at a low level, the opening The development goal is achieved. To facilitate the estimation of input parameter ranges that lead to high product quality. To do this, we implemented a design space estimator using Monte Carlo simulation. For the purpose of purification, we have achieved >93% for CEX activity and >93% for CE-SDS purity. A target of 90% was set. Monte Carlo simulation showed that the temperature was 38.6°C to 3 These qualities may vary between 39.1°C and 9.7°C, but ideally should be 39.1°C. The goal was predicted to be achieved. The incubation time was 4.0 to 6.0 hours. The ideal pH is 8.2, although the ideal pH range may vary between 1.0 and 5.0 hours. The final input parameter is cysteine ​​concentration, which can vary from 8.15 to 8.25. For , the predicted ideal is 2.4mM, varying from 2.2mM to 2.5mM. Within these ranges, the design space estimator determines the product quality output parameter CE The predicted DPMO (defects per million operations) value for activity with X is 110. The DPMO value for purity by CE-SDS was found to be 20. ,The target limit for activity by CEX was not reached 110 times out of 1 million, The E-SDS purity target limit of 20 out of 1 million was not met. This means:

[0169] According to the results of the contour plots and the previous development studies (DoE1 and DoE2), Taking the results into account, the incubation temperature target was set at 37°C. The synthesis time target was set at 4 hours (240 minutes), and similar to the setup of this experiment, the synthesis time target was set at 4 hours (240 minutes), and the synthesis time target was set at 4 hours (240 minutes), and similar to the setup of this experiment, ... The heating and cooling times were not included in the calculation. Together with the pH adjustment interval, a total process time of approximately 6 hours applies. DS had no detectable effect on purity, and CEX had only a small effect on activity. Therefore, the target was set at 8.0, which is well within the range of the study and is not expected to be related to the activity of CEX. The development goal of the CEX system can be achieved safely. This reduces the risk of forming acidic variants (which are often mutated as genomic DNA). Cysteine ​​concentration results show a low optimal level of 2.4 mM, but 2 mM cysteine ​​was used. Most experiments using CEX have shown that the activity is lower than desired. In conjunction with the results of the validation trial, the goal of cysteine ​​concentration was set higher during the validation trial, i.e., 4m This suggests that it should be M. EXAMPLES

[0170] Process Comparison and Analysis of Continuous Mixing Example 7.1 - Comparison of Process B2 and Process C The considerations outlined in Example 6 are important in determining whether selective reduction has been performed on a manufacturing scale until now (this Process B2 in the specification) Higher protein concentration and lower cysteine ​​concentration (4- 6 mM) can be preferably performed. Table 21 shows the previous process B2 and The input parameters for the proposed process C are compared. Comparative trials were carried out in duplicate at the 50 mL scale in warmed polypropylene test tubes. The results are shown in Table 22.

[0171] [Table 21]

[0172] [Table 22]

[0173] Table 22 shows that the improved cysteine ​​treatment of Process C resulted in a significant increase in CE-SDS purity. This shows that the selective reduction step results in a very slight decrease in activity due to CEX. Increased protein concentration and decreased cysteine ​​concentration (i.e., cysteine ​​in proteins) A smaller molar ratio of 1:1 to 1:1 results in higher purity values ​​by CE-SDS. Although this leads to a lower reducing potential, this reducing potential is not sufficient for the proper activity of secukinumab. It is still sufficient to ensure the reliability.

[0174] Example 7.2 - Testing for continuous stirring during incubation During clinical manufacturing with process B2, heterogeneity during the incubation step at 37°C was Intermittent mixing (2 min per hour) was applied to avoid The feasibility of using continuous stirring to ensure homogeneity was examined in a 2 L bioreactor ( The experimental set-up and process conditions are shown in Table 23.

[0175] [Table 23]

[0176] The purity of REACT029 was 96% by CE-SDS and the activity was confirmed by CEX. The activity was 92.7%. This activity was confirmed by a small-scale trial, REACT030, without stirring. and with lower CEX activity than that shown by REACT031 (proposed process C). Therefore, the increased oxygen transfer to the cysteine-treated solution due to continuous mixing is Nevertheless, the reduction potential of CEX in the cysteine-treated pool was suppressed. The activity of the catalyst was close to the quality target. The oxygen transfer and surface / volume ratios improved with expansion to the production scale. This experiment was considered as the worst case for high oxygen transfer because the process The pilot-scale trial ("Process C") included continuous mixing to ensure homogeneity of the solution. was used.

[0177] Summary and conclusions drawn from Examples 5-7 We were able to increase the protein concentration three-fold during cysteine ​​treatment, which improved the process. We determined that lower cysteine ​​concentrations significantly reduced tumor volume. Similar product activity and increased product purity after cysteine ​​treatment by CE-SDS, depending on the concentration Therefore, a lower cysteine:protein ratio can be achieved. This ensures high quality products.

[0178] The data show that temperature and pH have only a small effect on the cysteine ​​treatment step. In order to obtain a high-quality product, it is necessary to add oxidized cysteine, cystine. The incubation time was shown to be ideal at 5 hours, but the present inventors The goal was set at 4 hours, and similar to the experimental setup, this must be taken into consideration during production-scale applications. The experiment did not include heating and cooling times that were not required. Although the use of stirring in selective reduction appeared to be feasible, the inventors later As shown in the examples, oxygen transfer to the solution should be limited (e.g., by using low / gentle stirring). It is. EXAMPLES

[0179] Pilot-scale process validation The proposed refinement conditions after process improvement were tested by process validation on a 7L pilot scale. A first scale-up was performed to obtain a first impression of the reproducibility and robustness of the process. The selective reduction step with sucrose C (Table 21) was carried out in a pilot-scale experiment (REACT035). Continuous stirring was applied. Dissolved oxygen concentration was measured by a pO2 probe.

[0180] Activation of REACT035 by CEX after the selective reduction step in validation trial 1 The analysis of the dissolved oxygen curve (see Figure 8) showed that the It was found that there was a steady decrease in pO2 during the first 3 hours, which was due to agitation. This shows that oxygen consumption occurs in solution faster than the oxygen transfer into the solution that is induced. The oxygen consumption is either converted to cystine in the cysteine ​​reagent (Cys-SH) or converted to cysteic acid (Cy Therefore, the oxygen in the final reaction time is The increase in the level is due to the consumption of the cysteine ​​reagent and the increase in the solution due to agitation. This shows that the reducing power of cysteine ​​is the key to the success of this treatment. This implies that the final period is not valid, and therefore the activity reduction obtained from this trial I can explain a little.

[0181] Example 8.1 - Investigation of the Cysteine ​​Step in Validation Run 1 Low activity determined by CEX in the first pilot-scale trial, REACT035 To investigate the root causes of the mortality (90.4%), possible influencing factors were evaluated. As mentioned above, the low cysteine ​​concentration, the introduction of oxygen during the reaction due to prolonged stirring, and the regulation of the A pseudo-change (reaction volume increase) may result in insufficient reducing power. The agitator speed and cysteine ​​concentration during the incubation were evaluated.

[0182] The experimental plan and activity by output parameter CEX are shown in Table 24. In this experiment, Dissolved oxygen concentration was measured by a pO2 probe.

[0183] [Table 24]

[0184] Three smaller reactions (REACT038, REACT039 and REACT04 0) All showed improved activity with CEX compared to validation trial 1 (REACT035). However, the corresponding activity by CEX was not observed in experiments with elevated cysteine ​​concentrations. In the previous experiment (REACT040), no continuous mixing was performed during the incubation period. In the REACT038 study, the incidence of schizophrenia was not increased compared to REACT039. Therefore, the cysteine ​​concentration and the stirring during incubation were the main influencing factors at this point. Nonetheless, pilot scale verification trial 1 (REA The dissolved oxygen (dO2) profile of CT035 (Figure 8) was observed after approximately 3 hours of incubation. After incubation, the dO2 increased, indicating that the reducing power of cysteine ​​was depleted. Two smaller scale experiments, REACT039 and REACT039, did not involve continuous stirring. The dO2 chart for T040 showed no increase in dO2 towards the end of treatment (data not shown). This suggests that the reducing power of cysteine ​​was not depleted in these smaller scale experiments. This indicates that.

[0185] Example 8.2 - Investigation of the Cysteine ​​Step in Validation Run 2 The filling volume was 14 L instead of 7 L as in validation trial 1, and two agitators (bottom A radial flow stirrer at the bottom and an axial flow stirrer at the top were used for stirring. The temperature (°C) (mmol) and stirrer speed (100 rpm) were the same as in validation run 1, REACT035. After selective reduction, the activity of the antibody solution was only 85.5% as measured by CEX. According to the dO2 chart shown in Figure 9, the reducing power of cysteine ​​was higher than that of the control in the first test. The oxygen was consumed even early (Figure 8). This is because oxygen transfer to the solution by continuous stirring was the main cause of this trial. This suggests that the

[0186] The trend of the dO2 curves in (Figures 8-9) is across the entire headspace with continuous mixing. Oxygen transfer from 1 to 2 was the primary underlying cause of the lower activity after selective reduction in validation runs 1 and 2. For the 7 L filling volume in validation trial 1, only one agitator (bottom The reactor (100 mm diameter radial flow agitator) was submerged in the process solution for the cysteine ​​treatment step. For 14L filling volume, two agitators (radial flow agitator at the bottom and axial flow agitator at the top) ) was submerged in the process solution, resulting in even greater oxygen transfer to the solution. Therefore, the much poorer results of Confirmatory Trial 2 are most likely due to increased oxygen transfer to the process solution. This can be seen from the dO2 curve (Figure 9). Due to the higher oxygen transfer to the solution, the reducing power of cysteine ​​is depleted earlier, As a result, the activity by CEX is lower. Therefore, oxygen transfer to the process intermediates is Limitations can be achieved, for example, by adjusting the mixing (speed, duration and frequency) during incubation. It should be.

[0187] Example 8.3 - Investigation of the Cysteine ​​Step in Validation Run 3 The fill volume was 16 L instead of 14 L as in validation run 2, and the mixing profile was Instead of continuous stirring during cysteine ​​treatment, the previous process B2 was adapted. Only 2 min mixing was applied per hour. Measured by CEX after selective reduction step. The activity of the antibody solution was 92.5%.

[0188] The lack of continuous mixing in the cysteine ​​treatment step limits oxygen transfer, resulting in The dO2 chart shown in Figure 10 ensures sufficient reducing power and enhances the activity of CEX. The experiment shows a rapid decrease in oxygen levels - to almost zero percent - at the beginning of the treatment. The amount of oxygen introduced during the cysteine ​​treatment ensures proper reaction conditions, and As a result, it will be demonstrated that high activity is ensured by CEX.

[0189] Example 8.4 - Investigation of the Cysteine ​​Step in Validation Run 4 Based on the results of validation run 3, a fourth pilot-scale validation run (REACT040) was conducted. The setup and process conditions were identical to those in validation run 3. However, the process for validation run 4 was The amount of oxygen in this pool is increased so that the process can handle the higher oxygen transfer to the solution. Cysteine ​​treatment was increased from 4mM to 6mM. This increased reducing power and possible antibody A balance must be struck between over-reduction and over-reduction. Based on the results of the study (see Table 24), this indication represents a change with a low risk of failure. After the selective reduction step, the activation of REACT040 by CEX was 91.9%. I did.

[0190] Figure 11 shows the dO2 chart for validation trial 4. As with validation trial 3, dO2 was After addition of cysteine, the levels decreased and remained at low levels. The raw material is not consumed and meets the product purity requirements as determined by CE-SDS. Shows.

[0191] Figure 12 shows that the activity by CEX and the purity by CE-SDS were confirmed in the confirmation run 3. The kinetics of CEX-induced activity was comparable in both experiments. A plateau was reached after approximately 2 hours, after which the increase in activity for the following 2 hours of treatment was The CE-SDS purity curves showed higher cysteine ​​levels at the early stages of the reaction. The difference was slight, with the drop in purity being more pronounced in the run at 6 mM. However, since both procedures yielded equivalent product quality at the end of the cysteine ​​treatment, both However, oxygen levels in process solutions are often not sufficient to ensure adequate product quality. Considering the effect of increasing the concentration of cysteine, the process applied in confirmation run 4 is more robust to variations in oxygen transfer to the process solution during cysteine ​​treatment. Finally, the cysteine ​​concentration of 6 mM (13.5 mg antibody = 65.75:1 cysteine:antibody molar ratio [i.e., about 66:1]) under the conditions of Confirmation Run 4 We chose a cysteine ​​treatment step at the same manufacturing scale as that of the cysteine ​​treatment step ... CEX activity in trials with a molar ratio of cysteine:protein of 1:1 (1 h) Faster kinetics (reaching a plateau during the first 2 h) were also noted (data not shown).

[0192] Summary and conclusions drawn from Example 8 The inventors have demonstrated that increasing the oxygen level during the cysteine ​​treatment step increases the reducing power of cysteine. This is likely due to the fact that oxygen inhibits secukinumab, resulting in insufficient reduction of C97. They concluded that this oxygen uptake from the atmosphere could have a significant detrimental effect on the activity of CEX. When working at production scale, it is possible to vary the cysteine / protein ratio and set a defined stirring speed. This can be countered by using agitation interruptions or even Cut.

[0193] Based on the results of the validation run, the cysteine ​​concentration was increased to 4 mM (the ratio of cysteine ​​to protein). From the molar ratio of 43.84, 6 mM ( The molar ratio of cysteine ​​to protein was increased to 65.75. To reduce the level of dissolved oxygen present during the incubation stage of the original reaction, Continuous mixing was replaced with 2 min of mixing per hour during incubation. was changed during process verification, but no obvious root cause (oxygen transferred to solution during selective reduction) was identified. (level of cysteine) in the drug substance by the adjusted cysteine ​​processing step. Ensured proper activity by X. EXAMPLES

[0194] Characterization of the selective reduction step The input parameters for the selective reduction step were protein concentration, cysteine ​​concentration and stirring. The effect of the agitator speed was evaluated by a statistical design. The output parameters CEX activity and CE - Purity by SDS was used to assess product quality after the selective reduction step and to determine the input parameters The output parameter ranges were determined according to a previous planning process. Using these constraints, we defined the input parameter ranges.

[0195] In addition, the results of three scaled-down model qualification trials and seven selections made at manufacturing scale are presented. The results of the quantitative reduction cycles were used to selectively determine the best / worst case for reduction power studies. The product quality after reduction was evaluated and the incubation time and temperature were examined. We evaluated a dedicated trial for

[0196] Example 9.1 - Process Characterization Response Surface Design Example 9.1.1 - Experimental Design and Methods Protein content, cysteine ​​concentration, and agitation mode during incubation (agitation (no stirring, continuous stirring at 50 or 100 rpm) were selected as input parameters for the response surface design. See Table 25.

[0197] [Table 25]

[0198] Various cysteine ​​concentrations, expressed as "dilution factors by TITR3 addition", were used. reflects the process conditions and potential variations, e.g., inaccuracies in weighing, This is caused by inaccuracies in the addition of solutions. The antibody concentration is determined by the ratio of cysteine ​​to protein. Because antibody concentrations contribute to the ratios of 100–1500 antibody responses, they were also examined in this study to detect potential interactions. In addition, the stirrer speed during incubation was tested as an input parameter. As shown in Example 8, oxygen transfer from the headspace to the solution is a selective reduction step. Because it has an impact.

[0199] Use a Central Composite Face Design with three center point trials This type of design is used to design mathematical models that have linear, interaction and quadratic terms. Secukinumab, INAKT.F( The buffer used for the process characterization was Ti tration Buffer AIN457-TITR1 (1M Tris base, pH 10.8 [pH range ≥ 10.0; conductivity 0.10-0.30 mS / cm]), Titra tion buffer AIN457-TITR2 (0.3M o-phosphate, pH 1. 4 [pH range ≦2.0; conductivity range 19.5-22.7 mS / cm]), Titrati On buffer AIN457-TITR3 (120 mM cysteine-HCL + 20 mM disodium EDTA, pH 8.0 [pH range 7.8-8.2; conductivity range 14. 7~18.3mS / cm].

[0200] Using a qualified scale model (described in detail in Example 10), a stirred bioreactor (Maximum capacity 2L), at ambient atmosphere, with free air exchange, pH, dissolved oxygen, agitator speed and The experiment was carried out while monitoring the temperature and temperature. The experimental plan is shown in Table 26. All input parameters not part of the study were held constant at their respective target values ​​according to Table 27. I did.

[0201] [Table 26]

[0202] [Table 27]

[0203] Thaw INAKT.F in a hand warm water bath and dilute to the target concentration with WFI. After pH adjustment, the solution was diluted to 0.05%. 1 L of solution was transferred to the flask. The concentration was calculated to achieve the target cysteine ​​concentrations listed in Table 28. Selected cysteine ​​samples were then added to the wells of the selected wells in TITR3 buffer. The solution was incubated at 37°C for 60 min under agitation at 50 rpm. The mixture was then heated to the desired temperature at 50 or 100 rpm continuously according to the experimental plan. Incubate the solution at 37°C for 300 min with or without stirring (0 rpm). After 300 minutes of reaction time (60 minutes of heating and 240 minutes of incubation), the test The sample was then removed (REACT.PT300). A further 60 min incubation at 37°C was performed. After the reaction (60 min heating and 300 min incubation), the solution was stirred at 50 rpm. The material was cooled to ambient temperature within 60 minutes at the machine speed and another sample was taken (REACT.P, whole roll). Response time 420 minutes. This procedure is planned at two points ("REACT.PT300" and "RE This allows for the evaluation of the ACT.P (referred to as “ACT.P”) and the potential impact of incubation time. do.

[0204] Example 9.1.2 - Response surface design output parameters Product quality output parameters for samples REACT.PT300 and REACT.P The values ​​of Table 28 (ascending order of CEX activity in REACT.PT300 sample) and Table 29 ( The data are listed in ascending order of CEX activity in the REACT.P samples. Purity by SDS was high in all trials (>90%), but some trials were CEX The results show that the REACT.PT300 sample had lower activity (<90%) than the Regarding CEX activity in the 100-mg / kg bw (Table 28), there was one trial with low activity (89.4%). In this trial (REACT085), the cysteine ​​concentration was at the lower limit and the antibody concentration was high (hence the lowest molar ratio of cysteine ​​to protein) and the highest stirring speed ( High oxygen transfer rate). Also, high activity ( It can be seen that there is one group of 8 trials with a median survival rate of 1:1 (>96%).

[0205] [Table 28]

[0206] As shown in Table 29, the CEX activity in the REACT.P sample was lower. There was one group of 6 trials that yielded a high (<94%) yield. It is worth noting that all trials without stirring had the highest CE X activity (>96%). CEX activity was 100 rp in the REACT.P sample. The CEX activity in the REACT.PT300 sample was decreased in the experiment using Table 29 also shows that the run speed was lower (although less pronounced) at 100 rpm and 50 rpm. The highest reductions in these trials were seen at the lower end of the cysteine ​​molar ratio range. (For example, the molar ratio in REACT085 was about 46:1, from 89.4 to 84. 0%, and REACT075, REACT084 and REACT072 (similar decline in

[0207] [Table 29]

[0208] The statistical diagnostics (Table 30) show that the purity by CE-SDS at both time points is A statistically significant model for the activity of REACT.P by CEX at the end of selective reduction The model for the activity of REACT.PT300 with CEX shows lower statistical significance. Significance (Q of 0.15) 2 ) is shown.

[0209] [Table 30]

[0210] Example 9.1.2.1 - Product quality output parameters at time point 1 (REACT.PT300) Modeling evaluation of Coefficient plot and contour plot for CEX activity at time point REACT.PT300 The lot was input parameter stirrer speed during incubation (stir) by CEX. The results clearly show that the protein concentration affects the activity of the β-glucosamine derivatives (data not shown). (contA) and cysteine ​​concentration (dil-c) were significantly higher than those of CEX in the range investigated. To achieve high activity with CEX, The agitator speed during incubation should be low.

[0211] As shown in Table 29, all experiments at time point REACT.PT300 were performed according to the CE-SDS The coefficient plots (data not shown) show that the three It is demonstrated that all input parameters affect the purity by CE-SDS. ALC There are also quadratic terms for the content by (contA) and the stirrer speed (stir). Alternating dilution factor (dil-c, which represents cysteine ​​concentration) and stirrer speed with TITR3 addition There is an action term, and there is also an interaction term between the dilution factor due to the addition of TITR3 and the content due to ALC. The model coefficients, as visualized in this contour plot (data not shown), are High agitator speed and high dilution factor with TITR3 addition (respectively, low cysteine ​​concentration) , and moderate to high content of ALC positively impacts purity by CE-SDS. All of these settings reduce the reducing power of cysteine, which has a positive effect on antibody integrity. Nevertheless, all trials had high purity by CE-SDS. The entire input parameter range investigated yielded adequate purity by CE-SDS after selective reduction. Suitable for securing.

[0212] Example 9.1.2.2 – Modeling product quality output parameters at time point 2 (REACT.P) ng The coefficient plots in FIG. 13A and FIG. 13B for CEX activity at time point REACT.P The contour plot of Fig. 1 shows that the input parameter stirrer speed (stir) affects the activity of CEX. Therefore, the high activity of CEX was confirmed. To achieve this, minimal agitation is required during incubation to allow oxygen transfer to the solution. In addition, the low dilution factor (dil-c) by adding TITR3 and high A low cysteine ​​concentration is beneficial for activity by CEX after selective reduction. The interaction between the rate and dilution with TITR3 addition was small but significant for the activity of CEX. The interaction between the ALC content and the dilution factor due to the addition of TITR3 had a significant effect. The effect of ALC on the content is not significant. These results are Conditions that increase the force, e.g., limited oxygen transfer due to minimum stirring speed and lower dilution It was clear that higher cysteine ​​concentrations by dilution resulted in higher activity by CEX. show.

[0213] As shown in Table 29, all experiments at time point REACT.P showed slight cytotoxicity by CE-SDS. The purity of the CE-SDS samples was at least 90%. The plot (data not shown) shows the relationship between the content (protein concentration) and the input parameter ALC. dilution factor by adding TITR3 (representing cysteine ​​concentration, dil-c), and and the stirrer speed (stir, representing oxygen transfer) were small relative to the purity by CE-SDS. The agitator speed shows a second-order effect, and the TITR3 dilution The interaction between the coefficient of interpretation and the agitator speed is statistically significant. -Improved by high agitator speed and high dilution factor with TITR3 addition. This reduces the reducing power of cysteine ​​and has a positive effect on antibody integrity. All inputs investigated met the purity range specified by the CE-SDS. The parameter range was suitable to ensure adequate purity by CE-SDS after the selective reduction step. is.

[0214] Example 9.1.2.1 - Relationship between Dissolved Oxygen Level and Output Parameters The oxygen profile of the reaction shown in Table 26 was analyzed using the input parameters agitation speed, antibody content The effect of the amount of cysteine ​​and the concentration of cysteine ​​on oxygen levels was determined. A graph of the results is provided in Figure 14 (Note: the x-axis, 0 to 1.00, indicates that stirring was performed in all trials). The time from 1.00 to 6.00 corresponds to the 1-hour heating stage, where the stirring was 0 rpm. m [i.e., no stirring], and the 5-hour incubation was either 50 or 100 rpm. The range from 6.00 to 7.00 corresponds to the mixing stage, and the stirring was 50 rpm in all trials. All trials correspond to a 1-h cooling phase where the initial low levels of dissolved oxygen were It is worth noting that this decrease was observed in trials with low antibody content. The difference was not so significant (0 rpm series trials REACT090 and REACT080 (Figure 14A), REACT086 of the 50 rpm series (Figure 14B) and 100 rpm m series REACT084 and REACT091 (see FIG. 14C ); This was most evident in trials with high antibody content (trial at 15.4 mg / mL in Figure 14). (See the graph profile.)

[0215] In all trials without stirring during incubation (Figure 14A), the dO2 level The RE remained below about 20% during both the incubation and cooling stages. Trials using 50 rpm agitation during incubation, except for ACT086 (Figure 14 B) Oxygen transfer during the incubation phase enabled by 50 rpm agitation. Nevertheless, dO2 levels remained below approximately 20% during both the incubation and cooling phases. However, the cysteine ​​concentration was low (4.8 mM), in trial REACT072. A slight increase in oxygen levels can be seen during the final cooling phase. Trials using 100 rpm agitation during mixing (Figure 14C) showed a very different profile. First, as mentioned above, trials with low antibody content (10 mg / mL) (RE ACT084 and REACT091) showed a slower dO2 decrease and The decrease continued during the conversion phase. However, at a lower cysteine ​​concentration (4.8 mM), In trial REACT084, the final time of incubation was at time 5.00 and During 6:00, the dO2 level increased to nearly saturation levels. Such an increase was observed in the trial REA CT085 was also observed at an earlier time point (time 4.00). A high antibody content (15.4 mg / mL) but low levels of cysteine ​​were used. An increase in the bell was also observed during the cooling phase (times 6.00–7.00) in run REACT075. The cysteine ​​concentration in trial REACT075 was at a moderate level (6.0 mM). .

[0216] These profiles indicate the amount of oxygen that is absorbed into the headspace from the reaction mixture. The antibody concentration in the trial was 1.1% and 1.2%, respectively. This also indicates that the antibodies themselves can induce dO2 consumption. This suggests that in later stages of selective reduction in trials with low cysteine ​​levels The increase in oxygen is most likely due to the reaction of oxygen with cysteine ​​(Cys-SH) as follows: This shows that the 4Cys-SH+O2→2Cys-SS-Cys+2 H2O and / or 2Cy-SH + 3O2 → 2Cys-SO3H. In fact, the rate Approximately 4 hours later in row REACT085 and 5 hours later in attempt REACT084 The rate was so fast that all cysteine ​​was consumed (Figure 14C). After cysteine ​​was consumed, O2 levels were restored to saturation levels (100% O2).

[0217] To study the effect of antibodies on oxygen consumption, we used 6 mM cysteine ​​and Two further trials were carried out at a 50 mL scale using a stirrer speed of 50 rpm and 50 mL. In this trial, the antibody concentration was 12.7 mg / ml (cysteine:antibody molar ratio = 69.89: In one trial, the antibody concentration was 0.1 (i.e., approximately 70:1), and in the other trial, the antibody concentration was zero. As shown in Figure 1, the no-antibody trial showed a decrease in dO2 to approximately 75% within the first hour. During this time the reaction temperature was increased from 20°C to 37°C. The % decrease was from a reported 8.9 mg / L at 20°C to 6.6 mg / L at 37°C. This is in good agreement with the decrease in dO2 saturation concentration (US Geological Survey TWRI Book 9, April, 98). The dO2 level remains constant for the remainder of the incubation phase. When the cooling phase began (after 6 hours), the dO2 level increased due to the decrease in temperature from 37°C to 20°C. Thus, in the absence of antibodies, there is no oxygen consumption in the solution and the system The consumption of oxygen by the inflow occurs slowly, and therefore the headspace caused by the agitation is lost. In contrast, in trials with antibodies, the maximum oxygen transfer was approximately There is a steady decrease in dO2 levels until 5.5 hours. At 0.5 hours, there was a slight increase in dO2 levels, followed by a decrease in dO2 levels during the cooling layer (i.e., at time 6 There is therefore a stronger increase in dO2 levels in the 10-7% group. Almost certainly, but not all, cysteine ​​was consumed after 5.5 hours. Amino acid analysis (data not shown) revealed a significant increase in cystine, corresponding to almost complete consumption of cysteine. The results showed increased levels of nicotinamide.

[0218] This information can be used to explain the low activity of CEX observed in some trials by dO2 protease inhibitors. It is possible to correlate the files with the trial REACT085 (Figure 14C) (System The molar ratio of cysteine ​​to antibody (approximately 46:1) was such that cysteine ​​was consumed after the 4.00 time point, and the antibody Since complete reduction was achieved, CE was performed with REACT.PT300 (sample from 5.00 hours). X had 89.4% activity (see Table 28). Later time point REACT. REACT085 (Figure 14C) in P (sample from the end point 7.00 hours) High oxygen levels in the final stage of the reaction in the presence of oxidative degradation of the antibody. It had even lower activity than CEX (84.0%, see FIG. 29). Rows REACT084 (Figure 14C) (cysteine:antibody molar ratio of approximately 71:1) and REA CT075 (FIG. 14C) (cysteine:antibody molar ratio of approximately 71:1) showed a higher cysteine ​​concentration at the later RE time point. Two separate trials had relatively low activity in ACT.P, but activity was faster in the R In these trials, antibody reduction was higher at time point 5.0. It seemed to be completed at 0 hours (REACT.PT300), but the increased dO2 levels Due to the absence of acetylcholine and cysteine, degradation occurred by a later time point, REACT.P. Similarly, for trial REACT072, the relatively later time point REACT.P was observed. The low activity (90.2%) (cysteine:antibody molar ratio approximately 56:1) was observed during the cooling phase of the reaction. This is explained by the increased oxygen levels in the 0 rpm trials (high antibody content and system The cysteine ​​concentration is low [cysteine:antibody molar ratio approximately 46:1], even in trial REACT082 The high activity obtained is due to the low oxygen transfer rate and therefore the very little cysteine ​​consumption. and thus, complete reduction (and also This happened because the protection provided by the company was guaranteed.

[0219] In essence, oxygen consumes a reducing agent (cysteine), and that consumption is mediated by the antibody itself. Since oxygen transfer in the reduction system is reduced (or accelerated), oxygen transfer in the reduction system should be kept low. This cysteine ​​consumption therefore has a twofold effect: 1) a decrease in the reducing power of cysteine; We note that the incomplete deblocking of CysL97-SH at the earlier time point, REACT.PT300, 2) The time from REACT.P300 to REACT.P If no remaining cysteines are available to protect during Reoxidation of L-SH may occur.

[0220] Example 9.2 - Process Characterization: Worst / Best Case Scenarios The primary objective of the worst / best case study was to determine whether ,Selective reduction steps were assessed as potentially important in the process risk analysis. The data were used to define the acceptable ranges and to characterize the input factors for the and to assist in the classification of input parameters based on their impact on product characteristics. Used.

[0221] Example 9.2.1 - Experimental Design and Methods The increase in heating time was evaluated as potentially significant. The increase in heating time was due to the long duration of stirring. The selective reduction of cysteine ​​increases oxygen transfer to the solution over time, resulting in the reduction of cysteine and therefore less selective reduction of the antibodies. For the same reason, increasing initial dO2 levels may be considered significant. was evaluated as a possible input parameter.

[0222] The oxidation of sulfhydryl groups of cysteine ​​is pH dependent. were evaluated as potentially important input parameters.

[0223] High reducing power can result in over-reduction of the antibody, and long cooling times can lead to poor antibody reconstitution. Therefore, longer cooling times may also be beneficial. was evaluated as an input parameter.

[0224] Temperature was already tested during previous process development (32-42°C) and may be significant. Typically, the rate of a chemical reaction decreases at lower temperatures. Upper and lower dynamics were determined in the trial.

[0225] Incubation times were also investigated. Shorter incubation times were associated with selective reduction processes. Conversely, over time, The incubation time was lower with CEX as shown by the response phase design results. In some cases, this led to activation (see Table 29, REACT.PT300 and R (compare CEX activity of EACT.P sample).

[0226] Define three worst / best case sequences to selectively reduce the parameters described above. The effect of the steps was evaluated. To evaluate the reductive power of the worst / best case sequences, The input parameters for this are shown in Table 30. A second worst / best case sequence is created to The effect of temperature on the selective reduction step was evaluated (Table 31). The impact is evaluated in a third worst / best case study according to the input parameters listed in Table 32. The product quality outputs used to evaluate the results of these worst-case and best-case studies were The parameters are activity by CEX and purity by CE-SDS.

[0227] [Table 31]

[0228] [Table 32]

[0229] [Table 33]

[0230] Dedicated trials were performed to evaluate the influence of further parameters that may be of importance. Secukinumab from large-scale trial B008530, INAKT.F (stored below -60°C) The buffers used were AIN457-TITR1 and AIN457-TITR2. and AIN457-TITR3 as described in Example 10. A suitable scaled-down model of an open stirred bioreactor (maximum 2 L volume) (see Example 10) The pH, dissolved oxygen, agitator speed and temperature were monitored using a All trials applied the process parameters from Table 27. All input parameters not part of the standard sequence are set to the target values ​​in Table 27 within the operating range. was kept constant.

[0231] Example 9.2.2 – Worst / Best Scenario Results Experimental setup to investigate the effects of heating time, cooling time, starting dO2 and process pH The screening plan and output parameters (purity by CE-SDS and activity by CEX) are shown. 33. Experimental design plan and output for investigating the effect of incubation temperature The parameters (purity by CE-SDS and activity by CEX) are given in Table 34. Experimental design plan and output parameters to investigate incubation time (CE-SDS The purity by CEX and activity by CEX are shown in Table 35.

[0232] As shown in Table 33, duplicate trials with high reducing power as well as those with low reducing power Duplicate runs of the same enzyme also yielded high activity with CEX and high purity with CE-SDS. Therefore, this process was carried out using the input parameters heating time and cooling time within the test range. This can cover variations in time (including mixing), starting dO2 levels, and process pH. can.

[0233] [Table 34]

[0234] The results of the experiment to evaluate the effect of incubation temperature are shown in Table 34. Purity by SDS was always high, but high activity by CEX was only observed at 32°C or higher. It becomes clear that...

[0235] [Table 35] In addition, the kinetics of CEX-induced activity was determined at different incubation temperatures. The results are shown in FIG. 15. The kinetics at 42°C was faster than that at 32°C, especially after 60 minutes. However, at both temperatures, the same reaction time was observed at 240 min and REACT.P. At 18°C, the reaction rate is slower and, as a result, the reaction continues for the steps tested. Over time, the increase in activity due to CEX becomes smaller.

[0236] The results of the experiment to evaluate the effect of incubation time are shown in Table 35. In this case, high activity and purity were obtained.

[0237] [Table 36]

[0238] Example 9.3 - Classification and Justification of Process Parameters Example 9.3.1 - Response Surface Design Study: Tolerance Range Purity by CE-SDS was measured at time point 1 (REACT.PT300 ) and at the end of selective reduction (REACT.P) was at least 90%. To achieve a high level of purity with S, the input parameters TITR3 and dilution factor with addition The number of particles should be set high and the agitator speed should be set high from the center. The reason for this is due to the lower cysteine ​​concentration and increased oxygen transfer throughout the headspace. Therefore, it corresponds to a low reducing power. The content by the input parameter ALC, i.e., the antibody concentration had a significant effect at time point 1 (REACT.PT300) during the selective reduction step. The ideal is to measure the antibody concentration relative to the absolute purity value by CE-SDS. The effect of ALC on the composition of the sieve is small within the range investigated. The specified purity values ​​were met in all experiments, and the content (antibody concentration) by the parameters ALC, TIT Investigate dilution factor (cysteine ​​concentration) and agitator speed (oxygen transfer) with R3 addition The entire range shown is suitable for ensuring adequate purity by CE-SDS after selective reduction.

[0239] CEX activity was measured for all agonists except REACT085 at time point 1 (REACT.PT300). The results were at least 90% for all samples (see Table 28). Contrary to expectations, two additional experiments were performed at high agitator speeds, indicating good oxygen transfer. Experiments (REACT075 and REACT084, see Table 29) < 93% Since the activity of CEX was shown to be higher than that of the control, a longer incubation time (i.e., further The 60 min, point REACT.P) is particularly useful for activity according to the output parameter CEX. These experiments were carried out at low cysteine ​​concentrations and throughout the headspace. Higher agitator speeds or higher levels of dilution due to TITR3 addition indicate increased oxygen transfer. These results suggest that in order to achieve the highest activity with CEX, The input parameter agitator speed should be set to a low level (implying low oxygen transfer), The dilution factor with TITR3 addition should be set at the midpoint or low level (higher The input parameter ALC, which represents the antibody concentration, is also suggested. The amount of CEX in the culture did not significantly affect the activity of CEX in these experiments.

[0240] The input parameter that mediates oxygen transfer from the headspace to the solution is the agitator speed. It is important for the reduction step that the stirring speed be adjusted accordingly to ensure adequate activity by CEX. Further manufacturing-scale trials (B018838, data presented) should be conducted. A maximum of 15 minutes of stirring per hour was tested according to the selective reduction step. However, the results of Example 8 indicate that oxygen transfer due to agitation is regulated. It is model and configuration specific and indirectly controlled. For example, in the case of process changes or scale-up, agitation conditions during incubation should be evaluated. The stirring during incubation, which indirectly represents the level of dO2 transfer to the reaction solution, was From the process point of view, these are classified as significant input parameters.

[0241] The input parameters ALC content, i.e., antibody concentration, and TITR3 addition The dilution factor, i.e., cysteine ​​concentration, shows a statistically significant effect, but is well controlled and The selective reduction step has little impact on the process. As long as the mixing time during incubation is within acceptable limits, Variations within the range of cysteine ​​and antibody concentrations investigated demonstrated that the antibody was adequately purified after the selective reduction step. The proven input parameters will result in optimal process performance and product quality. A list with acceptable tolerances is given in Table 36.

[0242] [Table 37]

[0243] Example 9.3.2 - Worst / Best Case Study: Tolerance Input parameters Heating and cooling times (including stirring), starting dO2 level and preheat The process pH had a significant effect on the product quality characteristics CEX activity and CE activity within the range of investigation shown in Table 37. -There is no significant effect on purity by SDS. However, due to experience with response phase design However, excessively high oxygen transfer to the selective reduction solution should be avoided since it reduces the reducing power. Oxygen transfer is scale and setting specific, but in aerobic processes, oxygen transfer is much faster than in other processes. It is indirectly controlled by the process conditions, e.g., agitation, airflow into the headspace. Thus, agitation during heating and cooling that affects oxygen transfer is particularly important, e.g. process changes, Any process transfer or expansion must be carefully evaluated. Heating and cooling times (including stirring) are classified as significant parameters from a process point of view. The cooling time (including stirring) was tested in the range of 45 to 90 minutes, but the allowable range was ≦ The heating and cooling time of 90 minutes (including stirring) was set to 90 minutes. The worst case for oxygen transfer that results in adequate activity and purity by CE-SDS. This is because it reflects the

[0244] [Table 38]

[0245] The results of the experiment investigating the input parameter incubation temperature showed that the incubation temperature of 18°C It was shown that the reaction temperature leads to slow reaction kinetics, resulting in poor reactivation. This low temperature inhibited CEX activity by 93% within a 240 min incubation period. It was not enough to increase the product quality attribute CE-S to a level higher than 0.0%. Purity by DS can be achieved within the incubation temperature range of 18-42°C. However, to ensure adequate activity by CEX after the selective reduction step, the samples were run at 18–42 °C. The experimental range was narrowed to 32-42°C. Therefore, the incubation temperature tolerance was The limit is 32°C and the upper limit is 42°C.

[0246] Finally, the incubation time range of 210 to 330 minutes was used to determine the selective reduction step. After the step, proper activity by CEX and proper purity by CE-SDS are ensured.

[0247] Summary and conclusions drawn from Example 9 Based on the results of the process characterization study, the tolerance ranges of the investigated process input parameters The selective reduction step was performed according to two product quality output parameters: purity by CE-SDS, The parameters are classified and the acceptable ranges are shown in Table 3. 8. The cysteine:antibody ratio of PAR is about 46:1 to about 118:1. The cysteine:antibody ratio of R is from about 54:1 to about 83:1.

[0248] [Table 39]

[0249] At the beginning of the selective reduction step, oxygen weakens the reducing power of cysteine, but the introduction of oxygen into the solution As a result of the continuous transfer of oxygen, the oxygen level increases and the antibody activity by CEX decreases. At the end of the selective reduction step, the introduction of oxygen reduces the dimerization of the antibody that dissociated at the beginning of the reaction, which has a high reducing power. Therefore, oxygen can promote the formation of sulfide bonds. It is important to ensure proper purity with E-SDS. The significance is the significant effect of agitator speed on oxygen transfer and therefore product activity via CEX. This is evidenced by the results of a response surface design study in which a significant effect was observed (see Tables 28 and 29). However, the agitator speed may vary depending on the vessel size, agitator size, agitator type, etc. Therefore, the oxygen transferred to the solution is It is important to identify variables that can be used to compare across scientific settings. EXAMPLES

[0250] The oxygen transfer rate (k L a * ) In the previous examples, the inventors have noted, inter alia, that during selective reduction, in particular during incubation The amount of dO2 present during the application step had a strong effect on the amount and activity of secukinumab. When selective reduction is performed under aerobic conditions, the oxygen level in the reaction is directly controlled. It is not controlled by the flow rate but by other operating conditions, e.g., agitation speed and airflow to the headspace. The physical setup of each reaction also influences the level of oxygen present in the reaction mixture (apparatus, and the operating conditions together form a "system." L a * " moves into the solution Oxygen can be used to compare between physical settings during specific antibody processing steps. (For example, Garcia-Ochoa and Gomez (2009) Biotechnology Advances 27:153-176, B andino et al. (2001) Biochem.Engineering J. 8:111-119, Juarez and Orejas (2001) L See atin Am. Appl.Res.31:433-439, Yang and Wang (1992) Biotechnol.Prog.8:244-61. I want to be lit up.L a * in solution over time via headspace without sparging This value is specific to each setup and scale and depends on the agitator type. , agitator speed, fill volume, and the surface area of ​​the solution in contact with the headspace (which is The k for each physical configuration depends on the L a * is different However, the oxygen level in the solution during the selective reduction step can have an adverse effect on the activity and integrity of secukinumab. Since this has a significant effect, we conclude that the selective reduction step can be performed with similar k L a * Range The systems shown result in preparations of secukinumab of similar quality. I predict that.

[0251] k L a * cannot be determined directly in oxygen transfer experiments. Instead, the d O2 was replaced with nitrogen and the increase in dO2 over time was monitored using a calibrated dO2 probe. This experimental dO2 curve can then be generated. Saturate the curve according to the equation shown below (e.g., using Mathcad®). By fitting the curve, k L a * Values ​​are calculated for a particular system:

[0252] [ka] (Where, DO = measured dissolved oxygen, C = oxygen saturation value (1 when saturation is reached with infinite stirring) 00%), e = 2.718281..., t = time point corresponding to the DO value, and t0 = starting time point). This equation describes the transfer of oxygen into the solution (k L a * Experimental study of integral forms established for the determination of the value of This formula has been confirmed by different authors in various experiments (Doran, PM 1995. Bioprocess Engineering Principles, Academic Press, San Diego, California, chapte r 9.10.2, p. 210-213).

[0253] Example 10.1 - Experimental and Statistical Design Methods Dissolved oxygen level is an important factor influencing redox reactions, so the application of reduced models is Oxygen transfer was evaluated for qualification and for comparison to production scale.

[0254] A statistical design was used to determine the volume, airflow into the headspace, agitator speed and agitator pressure. Type of input parameter and k L a * The input parameters are listed in Table 39. Factors were investigated at three levels, each following a statistical design plan. The output parameters were: k L a * value.

[0255] [Table 40]

[0256] k for each sample L a * To determine this value, fill a 2 L bioreactor with water and place at 37 °C. The air flow was controlled by a mass flow meter and applied to the head space. The liquid was sparged with nitrogen gas to remove dO2 from the water. Constant agitation was applied using an oxygen probe (calibrated at room temperature, approximately 18-25°C). The dO2 level in the water was recorded over time using a dO2 detector ( The experimental dO2 curves were then fitted to the saturation curve as described above. TEK L a * values ​​were calculated.

[0257] A face-centered central composite design (CCF) with three center point trials was used. Correlation between parameters and k L a * To determine their impact on We selected a design with linear, interaction, and quadratic terms. Supports the calculation of physical models.

[0258] Example 10.3 - Output parameters (k L a * )value Experimental conditions and process performance output parameters k L a * The values ​​are listed in Table 40.

[0259] [Table 41]

[0260] Example 10.4 - Output parameter k L a * Statistical diagnosis of We evaluate the quality of the model using four tools: 2 , Q 2 , model validity and replication standards The mean mean is expressed as the standard deviation (SD). A model that describes the data well has an R close to 1.0. 2 and Q 2 , and model validity above 0.25. L a * An evaluation of the results shows that without transformation, we can lead to models where the residuals do not meet the requirement of normal distribution. This is a general understanding of bioreactors. L a * What the measurements show There is L a * Indicates that a transformation of values ​​is necessary to express the result in an appropriate way. Model diagnostics for process performance output parameters are 2 =0.98, Q 2 =0.91( Following a transformation by the power of -0.5), model validity = 0.56, and standard deviation of the iterations = 0.06, which indicates a highly statistically significant model.

[0261] Example 10.5 - Explanation of the mathematical model The Residual N Plot plots standardized residuals (residuals of the responses divided by their standard deviations) on the horizontal axis. The normal probability is plotted on the vertical axis. Anomalies are those that fall outside the ±4 standardized residuals. Also, nonlinear plots may require output parameter transformations. A model for designing experiments can be presented (see “Design of Experiments - Principles and Applications s,” (1999-2008) MKS Umetrics AB, ed. Eriksson et al.). Process output parameters k L a * The N plot of residuals (data not shown) for The values ​​fit the regression line well, which indicates that the model transformation is appropriate. This means that the standardized residuals satisfy the normal distribution requirement. In addition, all experimental values ​​are , within ±3 standardized residuals, which indicates that the measurements do not contain any outliers. vinegar.

[0262] The coefficient plots of the process output parameters (data not shown) are The air flow rate, volume and agitator speed are the process parameters k L a * It has a significant impact on Contour plots (data not shown) show the relationship between airflow, volume, and agitator speed. k L a * This illustrates the effect of higher airflow and lower volume ( i.e., the larger the head space, the higher the agitation speed, the L a * Expressed as There is a lot of oxygen transfer throughout the headspace that is created.

[0263] Use the contour plots and coefficient plots to identify the corresponding k L a * Value The predictions were made using MODDE 8.02 using the computational model. The results are shown in Table 41. The k values ​​were examined during process characterization (see Example 9) using a reduced model. L a * This demonstrates the range.

[0264] [Table 42] EXAMPLES

[0265] Production-scale oxygen transfer rate (k L a * ) Constant agitation at production scale (1800 L vessel) (i.e., heating and k in the type of stirring used during the cooling stage L a * It was decided.

[0266] Example 11.1 - k at manufacturing scale at SITE A L a * Determining Values Example 11.1.1 - Experimental and Statistical Design Methods A statistical design is used to determine the k within the range of test input parameters. L a * The dependence of was determined. The input parameters are listed in Table 42 below. Each has three levels of factors following a statistical design plan. The output parameters are k L a * value.

[0267] [Table 43]

[0268] These experiments at SITE A were carried out in a 1.5m-diameter 1.5-liter kettle with a maximum working volume of 1800 L and a height of 2.7 m. The measurements were carried out in a stainless steel vessel with a 0.0 m L a * To determine the value, Water was filled and heated to the indicated temperature. Nitrogen gas was then applied to the headspace to remove the water. dO2 was removed. Constant agitation was then applied (using a propeller stirrer from the bottom). Use an oxygen probe (calibrated at room temperature, e.g., 18-25°C) to ensure that dO2 is below 90°C. The dO2 level in the water was recorded over time until it reached 0.0001%. By fitting the experimental dO2 curve to the saturation curve, k L a * The value was calculated. The meters and corresponding results are shown in Table 43.

[0269] A composite face-centered (CCF) design with four center points was used. The most important input parameters were This design was used to determine correlations between the data and their effects on oxygen transfer. This type of design is a mathematical model with linear, interaction and quadratic terms. Assists in the calculation of.

[0270] Example 11.1.2 - Output parameters (k L a * )value Experimental conditions and process performance output parameters k L a * The values ​​are listed in Table 43.

[0271] [Table 44]

[0272] Example 11.1.3 - Output parameter k L a * Statistical diagnosis of We evaluate the quality of the model using four tools: 2 , Q 2 ,Model Validity and Replication Standards The mean mean is expressed as the standard deviation (SD). A model that describes the data well has an R close to 1.0. 2 and Q 2 , and model validity above 0.25. Dell has a low R 2 and Q 2 It has a value k L a * The evaluation of the results is the residuals without transformation. It was confirmed that the results of the normal distribution do not satisfy the requirements of the normal distribution. To describe the results L a * We show that a transformation of is necessary to obtain a robust model. , the output parameters were transformed by the power of -0.5. Model diagnostics of process performance were performed using R 2 =0.95, Q 2= 0.88, model validity = 0.82, SD = 0.15. This indicates a model with high statistical significance.

[0273] Example 11.1.4 - Description of the mathematical model Process output parameter k L a * N plot of residuals versus values ​​(data not shown) indicates a good fit between the experimental results and the model. The values ​​fit well to the regression line, which is , which means that the model transformation is appropriate (the standardized residuals meet the requirements for normal distribution). In addition, all experimental values ​​were within ±3 standardized residuals, which indicates that the measurements were outliers. Indicates that the value does not include

[0274] The coefficient plot (data not shown) shows that the error bars do not intercept the x-axis, so all inputs The power parameters, i.e., stirring speed, volume and temperature, are the process parameters k L a * value In addition, the interaction between agitator speed and volume is significant. k L a * Contour plots of the values ​​(data not shown) show the relationship between temperature, volume and agitator speed. k L a * 3 illustrates the effect of temperature on capacitance, i.e., the higher the temperature, the lower the capacitance. The higher the agitator speed, the higher the k L a * The total headspace is expressed as There is a lot of oxygen transfer.

[0275] Example 11.1.5 - Results of a dedicated trial at manufacturing scale k L a * The values ​​are determined by dedicated experiments and the model predictions are calculated using MODDE 8.02. The results of the predicted and dedicated trials are shown in Table 44. According to these results, this number The physical model is L a * values ​​could be predicted.

[0276] [Table 45]

[0277] Example 11.1.6 - Prediction of process conditions at SITE A L a * Process conditions used during campaign AT493021 at SITE A k L a * The results are shown in Table 45. The prediction k when applying steady mixing L a * The value is 0.05h -1 ~0.69h -1 is These values ​​indicate that this process step covers a wide range of k due to different process volumes. L a * It is shown to be robust over a range of values. L a * (and therefore oxygen transfer) The parameters that give the total mixing time should be considered in the final evaluation of the process robustness. The spacing has a significant effect on oxygen transfer.

[0278] [Table 46] EXAMPLES

[0279] Qualification trials of scaled-down models and comparison with production scale at Site A Example 12.1 - Methods Reduced model qualification trials conducted under standard conditions (REACT065, REACT066 and REACT067) were compared to seven representative trials at manufacturing scale. For the confirmation trial, manufacturing-scale trial B008530 (campaign AT493021, SI Seckin, INAKT.F (stored at less than -60°C) derived from TE A The loading dose (INAKT.F) was heated in a hand warm water bath (15-35°C) before use. The steps of the complete selective reduction procedure are shown in Table 46.

[0280] [Table 47]

[0281] The process parameters listed in Table 47 were applied in the reduced model qualification runs. The parameters correspond to the process conditions at the manufacturing scale (SITE A) and are It is a seriously reduced version.

[0282] [Table 48]

[0283] The sample was withdrawn and the pH was adjusted to 1.4 (AIN457-TI) with 0.3 M phosphoric acid within 5 min. The TR was adjusted from 2 to 5.2 (acceptable range 5.0 to 5.4), and the samples were then transferred to the analytical laboratory. All retained samples were thawed and stored at ≤-60°C. Quality characteristics: Comparison of activity by CEX and purity by CE-SDS confirmed the effectiveness of the reduced model. Qualification was evaluated. The following time intervals were tested: 0, 10, 20, 30, 45, 60, 120, 240 minutes.

[0284] Example 12.2 - Results Dissolved oxygen charts from three representative production-scale trials (data not shown) and scaled-down models. The dissolved oxygen chart from the pilot qualification trial (Figure 16) shows a characteristic shape of the selective reduction step. These curves show the results when dissolved oxygen is removed from the system by the oxidation of cysteine. Development experience shows that longer stirring times result in higher oxygen transfer and This leads to insufficient activity by EX. Therefore, the selective reduction solution should be added during the incubation. Limit oxygen transfer to the solution by stirring for only 2-15 minutes per hour. The dissolved oxygen level is kept low until selective reduction is stopped. During the cooling phase, the oxygen level is continuously There is a slight increase due to oxygen transfer into the solution by mixing, and at the same time, a certain amount of The cysteine ​​is oxidized, and as a result, the cysteine ​​is no longer available as a reducing agent for dissolved oxygen. become.

[0285] For manufacturing scale trials 3-6 of campaign AT493021 at SITE A, The activity of EX was 96.3% on average with a standard deviation of 0.8%, whereas the activity of CE-SDS was 96.3% on average with a standard deviation of 0.8%. The purity averaged 92% with a standard deviation of 1%. The results of each run were compared to the predictions obtained from Table 45. k L a * The results are shown in Table 48 along with the values.

[0286] [Table 49]

[0287] For the reduced model qualification trials, activity by CEX averaged 1.6% with a standard deviation of 1.6%. The purity by CE-SDS was 94% on average with a standard deviation of 1%. There was (Table 49).

[0288] [Table 50]

[0289] In addition to evaluating the product quality at the end of the selective reduction step, we also performed dynamics of activity by CEX. The state of the model was also monitored, and the results of the reduced model trials (average of REACT065, 66 and 67) The results from the manufacturing scale at SITE A were compared. The results are shown in Figure 17. The data are within the range of production scale variability, which is consistent with the dynamics at large scale (Site A). Demonstrate that the dynamics at small scale are comparable. EXAMPLES

[0290] k from Examples 9 to 12 L a * Composite analysis of values Due to the importance of the dissolved oxygen level in the solution during the selective reduction reaction, it is necessary to remove oxygen from the headspace into the vessel. Controlled oxygen transfer to the cation exchange is important for the selective reduction step. Then, these variables are L a * It can be captured as a value. L a * is the agitator speed, The agitator type, fill volume, temperature, and surface area of ​​the solution in contact with the headspace ( This depends on the individual geometry of each container. L a * depends on each setting From the above experiment, k L a * The range of values ​​is the selective reduction process It is found that this is acceptable to ensure the quality and activity of secukinumab during

[0291] Predictions about the experimental conditions of the reduction study L a *Range 0.18h -1 ~0.37h -1 teeth, It was determined by using a statistical model (Table 41). L a * The values ​​are based on a fixed volume and It decreased as the stirring speed decreased. Response surface design experimental conditions and settings in Example 9 is the same as that used for the reduced model in Example 10, so the predictions in Table 41 k L a * The values ​​can be correlated with the conditions and results in Table 28. This analysis is shown in Table 50. Present.

[0292] [Table 51]

[0293] For experiments with a 240 min incubation period (REACT.PT300) All reactions, except REACT085, were determined by CEX and CE-SDS. It resulted in very high selective reduction performance with adequate product quality (Table 50). 85, 100 rpm stirring speed, 0.37 h of incubation -1 k L a * , and had a very low molar ratio of cysteine ​​to antibody (approximately 46:1). Fast oxygen transfer (high k L a * In combination with the low antibody titer of cysteine, Lower molar ratios were more likely to result in poor reactivation.

[0294] For experiments with a 300 min incubation period (REACT.P), All reactions except CT085, REACT075 and REACT084 were and very high selective reduction performance with adequate product quality as measured by CE-SDS. REACT085, REACT075 and TRACT084 were Agitation speed of 100 rpm, incubation for 0.37 h -1 k L a * , and had low to moderate molar ratios of stain to antibody (approximately 46:1 and approximately 71:1) High oxygen transfer to the container (high k L a * In combination with the anti-cysteine Low to moderate molar ratios of β-lactam to β-lactam were likely to result in insufficient reactivation. The increase in incubation time (relative to the REACT.PT300 sample) was CT.P appeared to contribute to poorer CEX values.

[0295] As can be seen from Table 50, reactions with CEX activity greater than 90% were selective reduction reactions. During the incubation part of the reaction, the k L a * It has the following properties: 1) k in the system during the incubation step of the selective reduction reaction L a * But <0 .37h -1 If so, the molar ratio of cysteine:antibody should be both short incubation times and longer incubation times, e.g., from about 210 to about 330 minutes; For example, for about 240 to about 300 minutes, the ratio may vary from about 46:1 to about 118:1; Call 2) k in the system during the incubation step of the selective reduction reaction L a * Sys The tein:protein molar ratio can be increased (shorter incubation times, e.g., up to about 2 Between about 56:1 and about 118:1 for incubations up to 40 minutes, or For longer incubation times, e.g. up to about 300 minutes, If the ratio is between about 77:1 and about 118:1, it is 0.37h -1 It can be as high as . Of course, k during the heating and cooling phases L a * is the k during the incubation phase L a * Yo For example, Table 54 shows that the -1 As high as k L a * Added It is applied during the heat / cool stage, demonstrating that a high quality product can still be produced.

[0296] In Example 9 (process characterization study), constant stirring was applied during the incubation stage. The output parameters, activity by CEX and purity by CE-SDS, were calculated using a reduced model. In Example 10, the degree of k in a reduced model that also applies steady mixing was examined. L a * Check the value In Example 11, the k value was calculated using continuous mixing at a manufacturing scale. L a * The value was checked. During actual production, continuous stirring is only applied during the heating and cooling stages of the selective reduction reaction; Intermittent mixing (e.g., 2-15 min per hour) is required during the incubation phase of the selective reduction reaction. Therefore, during the production, the selective reduction step is applied k of the system in the phase L a * is the k observed in Examples 10 and 11. L a *More than the value Nevertheless, the results of Examples 9 to 11 combined show that When the stain:antibody molar ratio is between about 46:1 and about 118:1, the stain:antibody molar ratio is between about 240 and about 300. The entire selective reduction step (i.e., heating step, incubation step, etc.) including 0.00 min incubation step was performed. The incubation and cooling steps are generally performed for <0.37 h. -1 k L a * Use It is clear that this can be done by using

[0297] The worst / best experiment in Example 9 used the same qualified scaled model to measure a system Use an in:antibody molar ratio to determine the incubation phase (<0.18 h -1 k L a * ) with very little agitation (i.e., 2 minutes per hour). Worst / Best Example 9 In the case study, all reactions incubated for 210 to 330 minutes produced high-quality products. Since we have generated k L a * The antibody has a low cysteine ​​content and a moderate cysteine:antibody molar ratio (approximately 66:1). In this case, it is expected that the incubation time can be extended to about 210 to about 330 minutes. will be done. EXAMPLES

[0298] Preferred selective reduction process parameters and procedures The preferred process parameters for the selective reduction step are listed in Table 51. The total cysteine:antibody molar ratio is approximately 66:1, with a recommended operating range of approximately 54:1 to approximately 83:1. :1, with a demonstrated acceptable range being from about 46:1 to about 118:1.

[0299] [Table 52]

[0300] The individual steps of the selective reduction procedure are shown in Table 52. First, the protein concentration of the starting material is Adjust to a target concentration of 13.5 mg / mL by adding WFI. Then add 1M Tris Adjust the pH to 8.0 by adding 0.5% ethanol. Check the dO2 and if it does not correspond to the operating range, The concentration is adjusted (e.g., to ≧60%), e.g., by further mixing, aeration in water, etc. The reaction was started by adding tetracycline to 6 mM, and the solution was then incubated for approximately 1 h. The solution is heated to the incubation temperature (approximately 37°C) for approximately 1 hour. Incubate for approximately 250 minutes with 2 min agitation and observe the headspace during the incubation period. Finally, cool the solution to room temperature (for about 1 hour). The reaction is stopped by cooling and adjusting the pH to about 5.2, for example with 0.3 M ortho-phosphoric acid. .

[0301] [Table 53] The present application also relates to the following aspects: (1) CysL in preparations of IL-17 antibodies recombinantly produced in mammalian cells A method for selectively reducing 97, comprising the steps of: c) contacting said preparation with at least one reducing agent in a system to form a reducing mixture; and d) ≦about 0.37 h-1 calculated by fitting the dissolved oxygen curve to the saturation curve The reducing mixture is then infused with the oxygen mass transfer coefficient (kLa*) while maintaining the oxygen mass transfer coefficient (kLa*) in the system. Incubation step Including, Each of the IL-17 antibodies has three complementary determining regions (CDRs) of VH set forth in SEQ ID NO:8. ) and VL as set forth in SEQ ID NO: 10. and an immunoglobulin light chain variable domain (VL) comprising the CDRs of Prior to step a), the initial oxygen saturation percentage of the preparation is measured using a calibrated oxygen saturation test at 25° C. at least about 60% as measured using a probe. (2) The at least one reducing agent is selected from the group consisting of cysteamine, cysteine, and combinations thereof. The method according to (1) above, wherein the compound is selected from the group consisting of: (3) The at least one reducing agent is a thiol-disulfide exchange inhibitor. In this case, the standard redox potential E0 of about -0.20 V to about -0.23 V at pH 7.0 is The method according to (1) above, wherein the reducing agent is an ol-containing reducing agent. (4) The method according to (2) above, wherein the at least one reducing agent is cysteine. (5) kLa* in the system during step b) is ≦about 0.37 h-1; The molar ratio of cysteine:IL-17 antibody in the reducing mixture is about 56:1 to about 118:1. and the reducing mixture is incubated according to step b) for up to about 240 minutes. The method according to (4) above, (6) kLa* in the system during step b) is ≦about 0.37 h-1; The molar ratio of cysteine:IL-17 antibody in the reducing mixture is about 77:1 to about 118:1. and the reducing mixture is incubated according to step b) for up to about 300 minutes. The method according to (4) above, (7) kLa* in the system in step b) is <約0.37h-1であり、前The molar ratio of cysteine:IL-17 antibody in the reducing mixture is about 46:1 to about 118:1. The method according to (4) above, wherein (8) kLa* in the system in step b) is <約0.37h-1であり、前The molar ratio of cysteine:IL-17 antibody in the reducing mixture is about 54:1 to about 82:1. The method according to (4) above, wherein (9) The molar ratio of cysteine:IL-17 antibody or in the reducing mixture is about 66: The method according to (4) above, wherein the compound is 1. (10) kLa* in the system during step b) is ≦about 0.27 h-1, preferably The method according to any one of the above (1) to (9), wherein the h-1 is ≦about 0.18 h-1. (11) Prior to step a), the preparation has a pH of from about 4 mg / ml to about 19.4 mg / ml , for example, about 10 mg / ml to about 19.4 mg / ml, for example, about 10 mg / ml to about 1 5.4 mg / ml, for example, about 12 mg / ml to about 15 mg / ml, for example, about 13.5 The method according to any one of (1) to (10) above, comprising administering to the patient an IL-17 antibody at a concentration of 1 mg / ml. (12) The concentration of cysteine ​​in the reducing mixture is about 4.0 mM to about 8.0 mM, e.g. For example, about 4.8 mM to about 8.0 mM, for example, about 5.5 mM to about 6.7 mM, for example, about The method according to (4) above, wherein the concentration is 6.0 mM. (13) Prior to step a), the pH of the preparation is between about 7.3 and about 8.5, for example about 7.8 to about 8.2, for example, about 7.9 to about 8.1, for example, about 8.0. The method according to any one of claims 1 to 12. (14) Prior to step a), the initial oxygen saturation percentage of the preparation is compared at 25° C. At least about 80% or at least about The method according to any one of (1) to (13) above, wherein the content of the ionic liquid is 60%. (15) The reducing mixture further contains EDTA, and the EDTA in the reducing mixture The concentration of is about 1.3 mM to about 0.8 mM, for example, about 1.1 to about 0.9 mM, for example, about The method according to any one of (1) to (14) above, wherein the concentration is 1.0 mM. (16) Between steps a) and b), the reducing mixture is heated to about 32° C. to about 42° C. For example, the mixture is heated to a temperature between about 35° C. and about 39° C., for example, to about 37° C. The method according to any one of (1) to (15) above. (17) The reducing mixture is stirred for about 45 to about 90 minutes, for example, for about 45 to about 75 minutes, e.g. The method according to (16) above, wherein the heating is performed for about 60 minutes. (18) kLa* in the system during heating is ≦about 0.69 h-1, and a* is calculated by fitting the saturation curve to the dissolved oxygen curve, The method described. (19) The reducing mixture is heated to a temperature between about 20° C. and about 42° C. according to step b). For example, about 32°C to about 42°C, for example, about 35°C to about 39°C, for example, about 37°C. The method according to any one of (1) to (18), wherein the culture medium is incubated. (20) The reducing mixture is subjected to step b) for about 210 to about 420 minutes, e.g. For example, about 210 to about 330 minutes, for example, about 240 to about 300 minutes, for example, about 250 minutes The method according to (7) above, wherein the mixture is incubated. (21) During step b), the reducing mixture is stirred for ≦about 15 minutes per hour, for example The mixture is stirred for ≦about 5 minutes per hour, for example, for ≦about 2 minutes per hour, 20). The method according to any one of claims 1 to 20. (22) The level of intact IL-17 antibody in the reducing mixture after step a) is , as measured by sodium dodecyl sulfate capillary electrophoresis (CE-SDS) The method according to any one of (1) to (21) above, wherein the concentration of the ionizing radiation is reduced by at least about 80%. (23) c) The mixture resulting from step b) is cooled to room temperature, e.g., at about 16°C to Cool to approximately 28°C. The method according to any one of (1) to (22) above, further comprising: (24) The mixture is stirred according to step c) for about 45 to about 90 minutes, for example for about 45 to about 90 minutes. The method according to (23) above, wherein the mixture is cooled for about 75 minutes, for example, about 60 minutes. (25) kLa* in the system in step c) is ≦about 0.69 h-1; The kLa* is calculated by fitting a saturation curve to a dissolved oxygen curve. 23). The method according to claim 23. (26) d) adjusting the pH of the mixture resulting from step c) to about 5.1 to about 5.3. The method according to (23), further comprising a step of adjusting, for example, to 5.2 during the step. (27) The preparation step d) comprises adding o-phosphoric acid to the mixture resulting from step c). The method according to (26) above, comprising adding to the mixture. (28) of intact IL-17 antibody in the mixture resulting from step d). The level is at least about 90% as measured by CE-SDS. ) The method according to claim 1. (29) Activity level of IL-17 antibody in the mixture resulting from step d). However, the cystamine cation exchange chromatography (cystamine-CEX) The method according to (26), wherein the rate is at least about 90%. (30) The oxidized form of the reducing agent, e.g., cystine or cystamine, is added to the reducing mixture. The method according to any one of (1) to (29), wherein the compound is added to the (31) The method of the present invention, wherein no denaturing agent, such as guanidine hydrochloride, is added to the reducing mixture. The method according to any one of (1) to (30). (32) CysL in preparations of IL-17 antibodies recombinantly produced in mammalian cells A method for selectively reducing 97, comprising the steps of: a) said preparation being selected from cysteine / cystine and cysteine / cystamine with a set of oxidation / reduction reagents to form a reducing mixture; b) incubating the reducing mixture under anaerobic conditions at a temperature of about 37° C. for at least 4 hours; or incubate the reducing mixture at a temperature of about 18-24 degrees for about 16-24 hours. Steps to bet Including, Each of the IL-17 antibodies comprises three complementary determining regions (CDRs) of VH as set forth in SEQ ID NO:8. ) and VL as set forth in SEQ ID NO: 10. and an immunoglobulin light chain variable domain (VL) comprising the CDRs of: (33) The molar ratio of the oxidizing / reducing reagents in the reducing mixture is between about 4:1 and about 80:1. The method according to (32) above, (34) The molar ratio of the oxidizing / reducing reagent in the reducing mixture is from about 26:1 to about 80:1. The method according to (33) above, wherein (35) The above (33), wherein the set of oxidizing agent / reducing agent is cysteine / cystine. The method according to (36) The above-mentioned (33), wherein the set of oxidizing agent / reducing agent is cysteine / cystamine. ) The method according to claim 1. (37) The molar ratio of cysteine / IL-17 antibody in the reducing mixture is about 21:1 to The method according to (32), wherein the ratio is between about 296:1. (38) The three CDRs of the VH described in SEQ ID NO: 8 are, in order, and 3, or the amino acid sequences set forth in SEQ ID NOs: 11, 12 and 13. and The three CDRs of the VL described in SEQ ID NO: 10 are, in order, those described in SEQ ID NOs: 4, 5 and 6. The method according to any one of (1) to (37) above, wherein the amino acid sequence is as described above. (39) Each of the IL-17 antibodies is i) a VL set as set forth in SEQ ID NO: 10 and a VH set as set forth in SEQ ID NO: 8, or ii) a full-length light chain as set forth in SEQ ID NO: 14, with or without a C-terminal lysine; and Full length heavy chain as set forth in SEQ ID NO:15 The method according to any one of (1) to (38) above, (40) The method according to (1), wherein the IL-17 antibody is a human antibody of the IgG1 isotype. The method according to any one of (39) to (39). (41) Any of the above (1) to (40), wherein the IL-17 antibody is a secukinumab antibody. Any method according to the present invention. (42) The activity level of the IL-17 antibody in the preparation is increased by cystamine-CEX. at least about 10 percentage points within about 60 minutes after step b), , at least about 15 percentage points, at least about 20 percentage points, or at least about 25 percentage points, or at least about 30 percentage points, The method according to any one of (1) to (41) above. (43) Cys in preparations of secukinumab antibodies recombinantly produced in mammalian cells A method for selectively reducing L97, comprising the steps of: g) adjusting the concentration of secukinumab in the preparation to between about 4 mg / ml and about 19.4 mg / ml , for example, about 10 mg / ml to about 19.4 mg / ml, for example, about 10 to about 15.4, e.g. For example, the concentration is adjusted to about 12 mg / ml to about 15 mg / ml, for example, about 13.5 mg / ml. Step, h) increasing the oxygen saturation percentage of the preparation to at least about 60%, for example at least about Steps to adjust to 80% i) adjusting the pH of the preparation to about 7.4 to about 8.5, for example, about 7.8 to about 8.2, for example , about 7.9 to about 8.1, for example, about 8.0; j) contacting said preparation with cysteine ​​in a vessel to form a reducing mixture. and the concentration of cysteine ​​in the reducing mixture is about 4.0 mM to about 8.0 mM, for example: About 4.8 mM to about 8.0 mM, for example, about 5.5 mM to about 6.7 mM, for example, about 6.0 mM, step; k) The reducing mixture is heated to a temperature between about 32° C. and about 42° C., for example, about 35° C. to about 39° C. The heating is continued for about 45 to about 90 minutes, for example, for about 45 to about 90 minutes. 75 minutes, for example about 60 minutes, l) The reducing mixture from step e) is heated at a temperature between about 20° C. and about 42° C., for example at about 32° C. Incubate at a temperature of about 35° C. to about 42° C., for example, about 35° C. to about 39° C., for example, about 37° C. The incubation is for about 210 to about 420 minutes, for example, about The reaction is carried out for 210 to about 330 minutes, for example, for about 240 to about 300 minutes, for example, for 250 minutes. During this time, a volumetric oxygen mass transfer coefficient (KLa*) of ≦0.37 h-1 is maintained in the vessel, Step 2, where La* is calculated by fitting the saturation curve to the dissolved oxygen curve; g) cooling the resulting mixture from step f to a temperature of about 16° C. to about 28° C. The cooling is performed for about 45 to about 90 minutes, for example, about 45 to about 75 minutes, for example, about 60 minutes. and h) adjusting the pH of the mixture resulting from step g) to about 5.1 to about 5. The method includes adjusting during 5.3, e.g., to 5.2. (44) A purified IL-17 antibody produced by any one of (1) to (43) above. The preparation was (45) The purification method according to (44), wherein the IL-17 antibody is a secukinumab antibody. The preparation was (46) A purified preparation of secukinumab, comprising: Numab levels were measured by sodium dodecyl sulfate capillary electrophoresis (CE-SDS). and the activity level of secukinumab in said preparation is at least about 90%, as measured by The purified product is at least about 90% pure as measured by cystamine-CEX. Preparations.

Claims

1. A method for preparing a secukinumab composition, comprising: The method further comprising: i. forming a reaction mixture comprising a plurality of secukinumab molecules comprising a light chain of SEQ ID NO: 14 and a heavy chain of SEQ ID NO: 15, with or without a C-terminal lysine, and a reducing agent; the plurality of secukinumab molecules 1. A conserved disulfide bond formed by a conserved cysteine ​​residue, and 2. A masked cysteine ​​at position L97 of the light chain; The steps include: ii. incubating the reaction mixture under conditions in which the conserved cysteine ​​residues are reduced; and iii. Incubating the secukinumab molecules obtained in (ii) under conditions sufficient to form a reaction mixture comprising a plurality of secukinumab molecules, 1. (ii) The multiple conserved cysteine ​​residues reduced in (ii) are reoxidized to form conserved disulfide bonds; 2. At least 90% of the light chains of the secukinumab molecules contain a reduced cysteine ​​at position L97; 3. At least 90% of the secukinumab molecules are intact as measured by sodium dodecyl sulfate capillary electrophoresis (CE-SDS) under non-reducing conditions. A method comprising:

2. A method for preparing the secukinumab composition described in claim 1, wherein at least 90% of the light chains of secukinumab molecules containing a reduced cysteine ​​at position L97 described in (iii) (2) are measured by cystamine-CEX.

3. A method for preparing the secukinumab composition of claim 2, wherein the reaction mixture described in (iii) contains at least 2 moles of sulfhydryl groups per mole of secukinumab molecule, as measured by Ellman's test.

4. A method for preparing the secukinumab composition of claim 1, wherein the reaction mixture described in (iii) contains a plurality of secukinumab molecules, at least 97% of which are intact as measured by non-reducing CE-SDS. (iii) A method for preparing the secukinumab composition of claim 4, comprising at least 90% of a light chain of a secukinumab molecule containing a reduced cysteine ​​at position L97 as described in (2), as measured by cystamine CEX.

6. A method for preparing the secukinumab composition of claim 5, wherein the reaction mixture described in (iii) contains at least 2 moles of sulfhydryl groups per mole of secukinumab molecule, as measured by Ellman's test.

7. A preparation comprising secukinumab in a reduced state with a cysteine ​​at position 97 (CysL97) of the light chain and in a reoxidized state of the conserved cysteine, a heavy chain of SEQ ID NO: 15, with or without a C-terminal lysine, and a light chain of SEQ ID NO: 14; said secukinumab being recombinantly produced in CHO cells; and The preparation of secukinumab comprises at least 90% intact secukinumab as measured by non-reducing CE-SDS, and a preparation in which the conserved disulfide bonds have been reformed such that the preparation exhibits at least 90% activity, but one or more conserved cysteines have not been oxidized to form disulfide bonds.

8. A preparation of secukinumab as described in claim 7, wherein the preparation exhibits at least 90% activity as measured by cystamine-CEX.

9. A preparation of secukinumab as described in claim 7, wherein the preparation exhibits at least 90% activity as measured by a cell-based assay.

10. The preparation of secukinumab described in claim 9, wherein the cell-based assay comprises an assay for evaluating inhibition of IL-17-dependent IL-6 release.

11. A preparation of secukinumab as described in claim 7, wherein the preparation exhibits at least 97% intact secukinumab and at least 95% activity as measured by non-reducing CE-SDS.

12. A preparation of secukinumab as described in claim 11, wherein the preparation exhibits at least 95% activity as measured by cystamine-CEX.

13. A preparation comprising secukinumab in a reduced light chain cysteine ​​97 (CysL97) and in a reoxidized state of the conserved cysteine, comprising: comprising a heavy chain of SEQ ID NO: 15, with or without a C-terminal lysine, and a light chain of SEQ ID NO: 14, the secukinumab is recombinantly produced in a mammalian cell; and The secukinumab preparation has been re-equilibrated to exhibit at least 90% intact secukinumab and at least 90% activity as measured by non-reducing CE-SDS.

14. A preparation of secukinumab as described in claim 13, wherein the preparation exhibits at least 90% activity as measured by cystamine-CEX.

15. A preparation of secukinumab as described in claim 14, wherein the preparation exhibits at least 90% activity as measured by a cell-based assay.

16. A preparation of secukinumab as described in claim 15, wherein the preparation comprises a cell-based assay comprising an assay to evaluate inhibition of IL-17-dependent IL-6 release.

17. A preparation of secukinumab as described in claim 16, wherein the preparation exhibits at least 97% intact secukinumab and at least 95% activity as measured by non-reducing CE-SDS.

18. A preparation of secukinumab as described in claim 17, wherein the preparation exhibits at least 95% activity as measured by cystamine-CEX.

19. A preparation of secukinumab as described in claim 18, wherein the preparation contains at least 2 moles of sulfhydryl groups per mole of secukinumab molecule as measured by Ellman's test.

20. The preparation of secukinumab described in claim 19, wherein the mammalian cells are CHO cells.

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