JP2018508465A - Methods for producing long acting CTP-modified growth hormone polypeptides - Google Patents

Abstract

Disclosed herein is a method of producing recombinant human growth hormone (hGH) by addition of CTP in a mammalian cell culture system. [Selection] Figure 6

Description

  The present invention provides a method for producing recombinant human growth hormone (hGH) by addition of CTP in a mammalian cell culture system.

  Growth hormone (somatotropin) has been in clinical use for over 50 years, primarily to treat growth hormone secretion deficits in children with dwarfism. In 1985, human growth hormone (hGH) derived from recombinant DNA replaced hGH from the cadaveric pituitary, which had previously been the only available source of material. hGH replacement therapy has become the standard therapy for tens of thousands of patients and has proven safety and efficacy. The need to maintain hGH blood levels within the effective therapeutic range requires a subcutaneous or intramuscular injection once a day or once every two days. Most of the commercial hGH is produced in bacterial systems.

  Growth hormone (GH) is a 191 pituitary protein that stimulates the production of insulin-like growth factor-1 (IGF-1) in the liver and release into the systemic circulation. Most of the currently available GH formulations require administration once a day and therefore their compliance can be a problem, especially in adolescents. In adult GH deficiency (GHD), once-daily administration and associated side effects (eg injection site discomfort, temporary edema and joint pain) limit the utility of existing formulations. The long-acting form of GH reduces discomfort by requiring fewer injections and minimizing adverse events associated with peak and trough plasma concentrations that occur with a single daily injection Has potential.

The methods disclosed herein include the generation of long acting hGH (MOD-4023; FIG. 1) that obviates the need for numerous injections currently required for the treatment of GHD. This technology is based on the use of the C-terminal peptide (CTP) of the beta chain of the natural peptide human chorionic gonadotropin (hCG), which provides hCG with the long life required for maintenance of pregnancy ( Initial T _1/2 approximately 10 hours, final T _1/2 approximately 37 hours). CTP has 28 amino acids and 4-6 O-linked sugar chains, all of which are bound to a serine residue. The beta chain of luteinizing hormone (LH), the reproductive hormone that causes ovulation, is almost identical to hCG but does not contain CTP. As a result, LH has a remarkably short half-life in blood (initial T _1/2 approximately 1 hour, final T _1/2 approximately 10 hours).

  MOD-4023 is an hGH molecule fused to three copies of CTP, one fused at the N-terminus and two at the C-terminus (CTP-hGH-CTP-CTP described in SEQ ID NO: 7). (FIG. 1). MOD-4023 is a single chain protein with 275 amino acids and 12-18 O-linked carbohydrates. As demonstrated in an animal model (weight increase in hypophysectomized rats), MOD-4023 is injected once a week to once every two weeks, similar to a single daily injection of hGH. It has the potential to bring about clinical effects.

  In one aspect, a method of producing a human chorionic gonadotropin carboxy-terminal peptide (CTP) modified human growth hormone (hGH) polypeptide, comprising: (a) encoding the CTP modified human growth hormone in a predetermined number of cells. Stably transfecting an expression vector comprising a coding portion, wherein the transfected cells express and secrete the CTP modified hGH, and (b) overexpress the CTP modified hGH. Obtaining a cell clone; (c) growing the clone in solution to a predetermined scale; (d) collecting the solution containing the clone; and (e) containing the clone. Filtering the solution to obtain a purified collection solution; and (f) purifying the purified collection solution. Obtaining a purified protein solution having a desired concentration of CTP-modified hGH, and producing a human chorionic gonadotropin peptide (CTP) -modified human growth hormone (hGH) polypeptide, and the amino acid sequence of the CTP-modified hGH Disclosed herein is a method wherein is set forth in SEQ ID NO: 7. In a related embodiment, the produced CTP modified hGH is highly glycosylated. In a related embodiment, the produced CTP modified hGH is highly sialic acidated. In another related aspect, the glycosylation pattern of the produced CTP-modified hGH comprises glycosylation of at least 4 O-linked sites per CTP.

  In one aspect, human chorionic gonadotropin carboxy terminal peptide (CTP) modified human growth hormone (hGH) polypeptides produced by the methods disclosed herein are disclosed. In a related embodiment, the glycosylation of CTP modified hGH comprises more than 4 O-linked glycans per CTP. In a related embodiment, glycosylation comprises at least 4-6 O-glycans per CTP unit.

  Other features and advantages will be apparent from the following detailed description, examples, and drawings. However, while the detailed description and specific examples illustrate the method of manufacture and the resulting products, various changes and modifications may be made within the principles and scope of the methods and products disclosed herein. It should be understood that these are provided for illustrative purposes only, as they will be apparent to those skilled in the art from this detailed description.

  Recombinant long-acting human chorionic gonadotropin carboxy-terminal peptide (CTP) modified human growth hormone (hGH) polypeptide (CTP modified hGH) and the subject matter regarded as a method for producing the Pointed out and clearly claimed. However, the CTP modified hGH and the method of manufacturing it, together with its objects, features, and advantages, as well as its construction and method of operation, are best described with reference to the following detailed description when read in conjunction with the accompanying drawings. Can be understood.

A schematic diagram illustrating MOD-4023 (CTP-hGH-CTP-CTP) is presented. A map of the MOD-4023 pCI-dhfr plasmid is shown. FIG. 6 shows an upstream process flow generation chart of a CTP modified polypeptide, such as MOD-4023. 2 shows a flow generation chart of a CTP modified polypeptide such as MOD-4023. A flow chart overview of the MOD-4023 pharmaceutical (DP) manufacturing process is presented. Presents Coomassie-stained SDS-PAGE, showing purification of MOD-4023 high glycosylation form by first ion exchange chromatography (IEX) column. Lane (1) Collected total protein, step 8 of FIG. 3; Lane (2) Ultrafiltration / diafiltration, step 1 of FIG. 4 (UFDF1) concentrated and diafiltered total protein; Lane (3) IEX flow through 4, step 3; lane (4) IEX wash material, step 3 of FIG. 4; and lane (5) IEX elution, step 3 of FIG. Data presenting the robust O-glycan content of MOD-4023 from different batches is presented. O-glycan molar content per mole of MOD-4023 was determined for different batches of MOD-4023 drug substance (DS) and drug product (DP). A batch-by-batch comparison between individual O-glycosylated peptides based on mass spectrometry (MS) responses is presented. Note in the column is the amino acid sequence where “O” is an abbreviation for O-linked glycan. The MS response of the first peptide was used for data normalization and was set to 100%. 3 presents the reverse phase high performance liquid chromatography (RP-HPLC) profile of MOD-4023. Peak 4 corresponds to the main peak of MOD-4023. There are also four minor related forms (peaks 1, 2, 3, and 5). 1 presents a size exclusion high performance liquid chromatography (SEC-HPLC) profile of MOD-4023. Peak 3 corresponds to MOD-4023 monomer. Peaks 1 and 2 correspond to MOD-4023 dimer and polymer, respectively. MOD-4023 reference standard SR-929SI., Analyzed by RP-HPLC at 220 nm. A comparison between 1 (top profile) and MOD-4023 I15-PP (UFDF1 sample) (bottom profile) is presented. Peak 1 corresponds to the MOD-4023 glycosylated type, and peak 2 corresponds to the non-glycosylated type. MOD-4023 reference standard SR-929SI. Analyzed by RP-HPLC at 220 nm. A comparison between 1 (top profile) and MOD-4023 C10-PP (Elution DEAE Sepharose) is presented. At this point in the production process, only the glycosylation peak was observed in the MOD-4023 sample. FIG. 6 shows hGH receptor activation by MOD-4023 in stably transfected cells. Typical dose response activation curve with MOD-4023. MOD-4023 activation of hGH receptors on Baf cells that stably express hGH receptors on their cell surface. Individuals obtained in virus safety assessment studies using Ebelson murine leukemia virus (A-MuLV), murine microvirus (MVM), reovirus type 3 (Reo-3), and pseudorabies virus (PrV) Present the process step and overall process clearance factor. The theoretical viral load per dose was calculated with a maximum dose of 15 mg / dose. O-linked glycan structures and their abundance in the two MOD-4023 products measured by 2AB labeling of the removed glycans separated by HPLC using a normal phase (NP) column. Show. 1,2-Diamino-4,5-methylenedioxybenzene of removed sialic acid, separated by ultra high performance liquid chromatography (UPLC) and specified against commercial standards. The richness of the sialic acid form in the two MOD-4023 product samples measured by 2HCl (DMB) labeling is shown. 3 shows an overlay of three total ion current chromatograms obtained from analysis of trypsin digests of three MOD-4023 batches by online liquid chromatograph / electrospray mass spectrometry (LC / ES-MS). Obtained from analysis of trypsin digestion of desialylated, reduced, and carboxymethylated MOD-4023 protein batch (as shown in FIG. 16 and described in Example 2) by online LC / ES-MS Signal evaluation and focus on signals modified with at least one HexNAc-Hex residue. Amino acid sequence 1-30 of SEQ ID NO: 7 of MOD-4023 is shown, and O-glycosylation is performed on serine (S) residues at positions 10, 13, 15, and 21 (shown in red). Done. All of these serines (S) follow a proline (P) residue in the sequence. At least two of the S residues at positions 1-4 (1-4) are occupied by O-glycosylation sites (shown in purple). The amino acid sequence 211-275 of SEQ ID NO: 7 of MOD-4023 is shown, and O-glycosylation is performed on the serine (S) residue shown in red. All of these serines (S) follow a proline (P) residue in the sequence. In addition, in the region where serine is continuous, at least two of the S residues are occupied by O-glycosylation sites (shown in purple). Deconvoluted mass spectrum obtained during elution of UV absorbing components at tR 19.8 min obtained from analysis of MOD4023 lot 648-01-10-014A sample by online LC / ES-MS after desialation Indicates. FIG. 3 shows MOD-4023 (DS) purity assessed using reverse phase (RP) -HPLC. Figure 3 shows the MOD-4023 (DS) purity assessed using size exclusion chromatograph (SEC) -HPLC. Indication of efficacy. The results of impurity analysis are shown for host cell protein (HCP), DNA, methotrexate (MTX), propylene glycol (PG), triton, insulin, DMSO, and bioburden.

  It will be understood that for simplicity and clarity of illustration, the elements shown in the figures are not necessarily drawn to scale. For example, some dimensions of the elements may be emphasized relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the drawings to refer to corresponding or analogous elements.

  In the following detailed description, recombinant long-acting human chorionic gonadotropin carboxy-terminal peptide (CTP) modified human growth hormone (hGH) polypeptide (CTP modified hGH; MOD-4023; SEQ ID NO: 7) and method for producing the same Numerous specific details are set forth in order to provide a thorough understanding of. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the CTP-modified hGH polypeptide and the method of producing it.

  In one embodiment, the long acting CTP-modified hGH polypeptide disclosed herein comprises the carboxy terminal peptide (CTP) of human chorionic gonadotropin (hCG). In another embodiment, CTP acts as a protective agent against degradation of the protein or peptide derived therefrom. In another embodiment, CTP increases the circulating half-life of the protein or peptide derived therefrom. In some embodiments, CTP enhances the potency of the protein or peptide derived therefrom.

  One skilled in the art will understand that the terms “CTP peptide”, “carboxy terminal peptide”, and “CTP sequence” can be used interchangeably. In one embodiment, the carboxy terminal peptide is full length CTP. In another embodiment, the carboxy terminal peptide is a missing CTP.

  One skilled in the art will appreciate that the terms “signal sequence” and “signal peptide” can be used interchangeably. In addition, one of skill in the art will understand that the term “sequence”, when associated with a polynucleotide, can encompass the coding portion of a polynucleotide sequence.

  Those skilled in the art will understand that the terms “polypeptide”, “peptide”, “peptide of interest”, “polypeptide of interest”, and “polypeptide sequence of interest” can be used interchangeably. I will. In one embodiment, the peptide of interest is a full-length protein. In another embodiment, the peptide of interest is growth hormone. In another embodiment, the peptide of interest is human growth hormone. In another embodiment, the peptide of interest is a protein fragment of human growth hormone.

  In another embodiment, growth hormone, a single human chorionic gonadotropin carboxy terminal peptide (CTP) attached to the human growth hormone (hGH) amino terminus, and two human chorionic gonadotropin carboxy attached to the carboxy terminus of GH. A polypeptide consisting of a terminal peptide (CTP) is disclosed herein, the polypeptide lacking a signal peptide, and the CTP-modified hGH polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 7. In another embodiment, a CTP comprising GH, a single CTP attached to the amino terminus of GH, two CTPs attached to the carboxy terminus of GH, and a signal peptide attached to the amino terminus of the amino terminus CTP. A modified hGH polypeptide is disclosed herein, and the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4. One skilled in the art will appreciate that mature secreted polypeptides lack a signal peptide.

  In one embodiment, a method of producing a human chorionic gonadotropin carboxy-terminal peptide (CTP) modified human growth hormone (hGH) polypeptide (CTP modified hGH) comprising: (a) adding a predetermined number of cells to the CTP modified stably transfecting an expression vector comprising a coding portion encoding hGH, wherein the transfected cells express and secrete the CTP modified hGH; and (b) the Obtaining a cell clone overexpressing CTP-modified hGH; (c) growing the clone in solution to a predetermined scale; (d) collecting the solution containing the clone; e) filtering the solution containing the clones to obtain a clarified collection solution; (f Purifying the clarified collection solution to obtain a purified protein solution having a desired concentration of CTP modified hGH, and human chorionic gonadotropin carboxy terminal peptide (CTP) modified human growth hormone (hGH) poly Disclosed herein are methods for producing a peptide, wherein the amino acid sequence of a CTP-modified hGH polypeptide is set forth in SEQ ID NO: 7.

  In another embodiment, the method of manufacture comprises a clone that expresses and secretes CTP-modified hGH having the amino acid sequence set forth in SEQ ID NO: 7. In another embodiment, disclosed herein is a method of producing a CTP-modified hGH polypeptide having an increased glycosylation content as disclosed herein. In another embodiment, a CTP-modified hGH polypeptide disclosed herein produced by a method disclosed herein has an increased number of glycosylated O-linked glycosylation sites. Have One skilled in the art will appreciate that the phrase “increased number of glycosylated O-linked glycosylation sites” can also be described as “increased O-glycan occupancy”.

  Human chorionic gonadotropin peptide (CTP) modified polypeptide

  In one embodiment, long acting GH polypeptides and methods for their production or manufacture and use are disclosed herein. In another embodiment, the long acting polypeptide comprises a carboxy terminal peptide (CTP) of human chorionic gonadotropin (hCG). In another embodiment, CTP acts as a protective agent against degradation of the protein or peptide derived therefrom. In another embodiment, CTP increases the circulating half-life of the protein or peptide derived therefrom. In some embodiments, CTP enhances the potency of the protein or peptide derived therefrom.

  One skilled in the art will recognize that the terms “CTP peptide”, “CTP”, “human chorionic gonadotropin carboxy-terminal peptide”, “carboxy-terminal peptide”, and “CTP sequence” can be used interchangeably herein. Will understand. In one embodiment, the carboxy terminal peptide is full length CTP. In another embodiment, the carboxy terminal peptide is a missing CTP.

  In another embodiment, a poly consisting of GH, a single CTP attached to the amino terminus of GH, two CTPs attached to the carboxy terminus of GH, and a signal peptide attached to the amino terminus of N-terminal CTP. A peptide is disclosed herein, and the polypeptide has the amino acid sequence set forth in SEQ ID NO: 4. One skilled in the art will appreciate that mature secreted polypeptides can lack a signal peptide. Thus, in yet another embodiment, a polypeptide consisting of GH, a single CTP attached to the amino terminus of GH, and two CTPs attached to the carboxy terminus of GH, without a signal peptide, is provided herein. Disclosed, the polypeptide has the amino acid sequence set forth in SEQ ID NO: 7.

  In another embodiment, a poly consisting of GH, a single CTP attached to the amino terminus of GH, two CTPs attached to the carboxy terminus of GH, and a signal peptide attached to the amino terminus of the amino terminus CTP. A method for producing a peptide is disclosed herein, wherein the polypeptide has the amino acid sequence set forth in SEQ ID NO: 4. In yet another embodiment, a method for producing a polypeptide comprising GH, a single CTP attached to the amino terminus of GH, and two CTPs attached to the carboxy terminus of GH, and not including a signal peptide is provided herein. The polypeptide has the amino acid sequence set forth in SEQ ID NO: 7.

  In another embodiment, a GH comprising CTP as set forth in SEQ ID NO: 7 has enhanced in vivo biological activity compared to the same GH without CTP.

  In another embodiment, the subject is a human subject. In another embodiment, the subject is a pet animal. In another embodiment, the subject is a mammal. In another embodiment, the subject is livestock. In another embodiment, the subject is a dog. In another embodiment, the subject is a cat. In another embodiment, the subject is a monkey. In another embodiment, the subject is a horse. In another embodiment, the subject is a cow. In another embodiment, the subject is a mouse. In another embodiment, the subject is a rat. In one embodiment, the subject is male. In another embodiment, the subject is a female.

  In one embodiment, the carboxy-terminal peptide (CTP) sequence comprises the amino acid sequence set forth in SEQ ID NO: 1: DPRFQDSSSKAPPPSLPSPSRLPGPSDTPILQ (SEQ ID NO: 1).

  In some embodiments, CTP sequences at both the amino terminus of the polypeptide and the carboxy terminus of the polypeptide provide enhanced protection against protein degradation. In some embodiments, CTP sequences at both the amino terminus of the polypeptide and the carboxy terminus of the polypeptide provide an extended half-life for the protein to which it is attached.

  In some embodiments, the CTP-modified hGH polypeptides disclosed herein have enhanced protection against polypeptide degradation. In some embodiments, the CTP-modified hGH polypeptides disclosed herein have an extended half-life of the protein that adheres. In some embodiments, the CTP-modified hGH polypeptides disclosed herein have enhanced hGH activity.

In another embodiment, the CTP-modified hGH polypeptides disclosed herein have an extended clearance time. In another embodiment, the CTP-modified hGH polypeptides disclosed herein have an enhanced C _max for hGH. In another embodiment, the CTP-modified hGH polypeptide disclosed herein has an enhanced T _max of hGH. In another embodiment, the CTP-modified hGH polypeptides disclosed herein have an enhanced T1 _{/ 2} of hGH. In some embodiments, the CTP-modified hGH polypeptides disclosed herein provide an extended half-life of hGH.

  In another embodiment, a carboxy terminal peptide (CTP) peptide disclosed herein comprises the amino acid sequence of amino acids 112-145 of human chorionic gonadotropin, as set forth in SEQ ID NO: 1. In another embodiment, the CTP sequence disclosed herein comprises the amino acid sequence of amino acids 118-145 of human chorionic gonadotropin, as set forth in SEQ ID NO: 2: SSSKAPPPPSLSPSRLPGPSDTPILPQ. In another embodiment, the CTP sequence also starts at any position between positions 112-118 of human chorionic gonadotropin and ends at position 145. In some embodiments, the CTP sequence peptide is 28, 29, 30, 31, 32, 33, or 34 amino acids in length and 112, 113, 114, 115, 116, 117 of the CTP amino acid sequence. Or start from 118th place.

  In another embodiment, the CTP peptide is a variant of chorionic gonadotropin CTP that differs from native CTP by 1 to 5 conservative amino acid substitutions, as described in US Pat. No. 5,712,122. . In another embodiment, the CTP peptide is a variant of chorionic gonadotropin CTP that differs from natural CTP by one conservative amino acid substitution. In another embodiment, the CTP peptide is a variant of chorionic gonadotropin CTP that differs from native CTP by two conservative amino acid substitutions. In another embodiment, the CTP peptide is a variant of chorionic gonadotropin CTP that differs from native CTP by 3 conservative amino acid substitutions. In another embodiment, the CTP peptide is a variant of chorionic gonadotropin CTP that differs from native CTP by 4 conservative amino acid substitutions. In another embodiment, the CTP peptide is a variant of chorionic gonadotropin CTP that differs from native CTP by 5 conservative amino acid substitutions. In another embodiment, the CTP peptide amino acid sequence disclosed herein is at least 70% homologous to the native CTP amino acid sequence or a peptide thereof. In another embodiment, the CTP peptide amino acid sequence disclosed herein is at least 80% homologous to the native CTP amino acid sequence or a peptide thereof. In another embodiment, the CTP peptide amino acid sequence disclosed herein is at least 90% homologous to the native CTP amino acid sequence or a peptide thereof. In another embodiment, the CTP peptide amino acid sequence disclosed herein is at least 95% homologous to the native CTP amino acid sequence or a peptide thereof.

  In another embodiment, a CTP peptide DNA sequence disclosed herein is at least 70% homologous to a native CTP DNA sequence or a peptide thereof. In another embodiment, the CTP peptide DNA sequences disclosed herein are at least 80% homologous to the native CTP DNA sequence or its peptides. In another embodiment, the CTP peptide DNA sequences disclosed herein are at least 90% homologous to the native CTP DNA sequence or peptides thereof. In another embodiment, a CTP peptide DNA sequence disclosed herein is at least 95% homologous to a native CTP DNA sequence or a peptide thereof.

  In one embodiment, at least one of the chorionic gonadotropin CTP amino acid sequences is missing. In another embodiment, both chorionic gonadotropin CTP amino acid sequences are missing. In another embodiment, two of the chorionic gonadotropin CTP amino acid sequences are missing. In another embodiment, two or more of the chorionic gonadotropin CTP amino acid sequences are missing. In another embodiment, the entire chorionic gonadotropin CTP amino acid sequence is missing. In one embodiment, the missing CTP comprises the first 10 amino acids of SEQ ID NO: 3: SSSKAPPPSLP. In one embodiment, the missing CTP comprises the first 11 amino acids of SEQ ID NO: 3. In one embodiment, the missing CTP comprises the amino acid of SEQ ID NO: 3.

  In one embodiment, at least one of the chorionic gonadotropin CTP amino acid sequences is glycosylated. In another embodiment, both chorionic gonadotropin CTP amino acid sequences are glycosylated. In another embodiment, two of the chorionic gonadotropin CTP amino acid sequences are glycosylated. In another embodiment, two or more of the chorionic gonadotropin CTP amino acid sequences are glycosylated. In another embodiment, all of the chorionic gonadotropin CTP amino acid sequence is glycosylated. In one embodiment, the CTP sequences disclosed herein comprise at least one glycosylation site. In another embodiment, the CTP sequences disclosed herein contain two glycosylation sites. In another embodiment, the CTP sequence disclosed herein comprises three glycosylation sites. In another embodiment, the CTP sequence disclosed herein comprises 4 glycosylation sites. In another embodiment, the CTP sequence disclosed herein comprises 5 glycosylation sites. In another embodiment, the CTP sequence disclosed herein comprises 6 glycosylation sites. In another embodiment, the CTP sequence disclosed herein comprises 7 glycosylation sites. In another embodiment, the CTP sequences disclosed herein comprise 8 glycosylation sites. In another embodiment, the glycosylation site is an O-glycosylation site. In another embodiment, the glycosylation is at a serine residue. In another embodiment, the glycosylation is at a threonine residue.

  In one embodiment, the methods disclosed herein produce a human chorionic gonadotropin carboxy-terminal peptide (CTP) modified polypeptide, which is highly glycosylated. In another embodiment, the methods described herein produce a CTP modified polypeptide human growth hormone (CTP-hGH), wherein the CTP modified hGH is highly glycosylated. In another embodiment, the methods disclosed herein produce CTP-modified hGH, which is highly sialic acid-added. Sialic acid addition is important because the higher the sialic acid addition, the longer the half-life can be extended.

  In another embodiment, the methods disclosed herein produce CTP-modified hGH, wherein at least one of the chorionic gonadotropin CTP amino acid sequences is glycosylated. In another embodiment, both chorionic gonadotropin CTP amino acid sequences are glycosylated. In another embodiment, two of the chorionic gonadotropin CTP amino acid sequences are glycosylated. In another embodiment, the methods disclosed herein produce CTP-modified hGH, wherein three of the chorionic gonadotropin CTP amino acid sequences are glycosylated. In another embodiment, three or more of the chorionic gonadotropin CTP amino acid sequences are glycosylated. In another embodiment, all of the chorionic gonadotropin CTP amino acid sequence is glycosylated.

  In one embodiment, each CTP sequence of the CTP modified hGH comprises glycosylation at at least one glycosylation site. In another embodiment, each CTP sequence of the CTP modified hGH comprises glycosylation at at least two glycosylation sites. In another embodiment, each CTP sequence of a CTP modified hGH comprises glycosylation at at least 3 glycosylation sites. In another embodiment, each CTP sequence of the CTP modified hGH comprises glycosylation at at least 4 glycosylation sites. In another embodiment, each CTP sequence of the CTP modified hGH comprises glycosylation at at least 5 glycosylation sites. In another embodiment, each CTP sequence of the CTP modified hGH comprises glycosylation at at least 6 glycosylation sites. In another embodiment, each CTP sequence of the CTP modified hGH comprises glycosylation at at least 7 glycosylation sites. In another embodiment, each CTP sequence of the CTP modified hGH comprises glycosylation at at least 8 glycosylation sites. In another embodiment, the glycosylation site is an O-glycosylation site. In another embodiment, the glycosylation is at a serine residue. In another embodiment, the glycosylation is at a threonine residue.

  In one embodiment, the produced CTP modified hGH of SEQ ID NO: 7 consists of two CTPs attached to the carboxy terminus of hGH and one CTP attached to the amino terminus of hGH, wherein the CTP modified hGH is 12 and Includes glycosylation at between 18 glycosylation sites. In another embodiment, the CTP modified hGH comprises glycosylation at between 12-21 glycosylation sites. In another embodiment, the CTP modified hGH comprises glycosylation at between 12-24 glycosylation sites. In another embodiment, 13 and 18, 14 and 18, 15 and 18, 16 and 18, 17 and 18, 13-21, 14-21, 15-21, 16-21, 17-21, 18-21, 19-21, 20-21, 13-24, 14-24, 15-24, 16-24, 17-24, 18-24, 19-24, 20-24, 21-24, 22-24, or 23 Includes glycosylation at between ~ 24 glycosylation sites. In another embodiment, the CTP modified hGH has 12 glycosylation sites, 13 glycosylation sites, 14 glycosylation sites, 15 glycosylation sites, 16 glycosylation sites. 17 glycosylation sites, 18 glycosylation sites, 19 glycosylation sites, 20 glycosylation sites, 21 glycosylation sites, 21 glycosylation sites, 22 Including glycosylation at one glycosylation site, 23 glycosylation sites, or 24 glycosylation sites. Each possibility represents a separate embodiment.

  In another embodiment, the amino acid sequence of CTP modified hGH is set forth in SEQ ID NO: 7, and O-linked glycosylation occurs at an available serine (S) residue present within each CTP unit. In another embodiment, each CTP contains 4-6 O-linked glycans and glycosylation is to the serine (S) residue present in each CTP unit. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 12-18 O-linked sugar chains. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 12 to 21 O-linked sugar chains. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 12 to 24 O-linked sugar chains. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 12 O-linked sugar chains. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 13 O-linked sugar chains. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 14 O-linked sugar chains. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 15 O-linked sugar chains. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 16 O-linked sugar chains. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 17 O-linked sugar chains. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 18 O-linked sugar chains. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 19 O-linked sugar chains. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 20 O-linked sugar chains. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 21 O-linked sugar chains. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 22 O-linked sugar chains. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 23 O-linked sugar chains. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 24 O-linked sugar chains. In another embodiment, no O-linked sugar chain is present on the hGH sequence of the CTP-modified hGH (SEQ ID NO: 8).

  In another embodiment, the CTP-modified hGH polypeptides disclosed herein comprise at least 4 O-glycans per CTP. In another embodiment, the CTP modified hGH polypeptides disclosed herein comprise at least 5 O-glycans per CTP. In another embodiment, the CTP-modified hGH polypeptides disclosed herein comprise at least 6 O-glycans per CTP. In another embodiment, the CTP-modified hGH polypeptides disclosed herein comprise at least 7 O-glycans per CTP. In another embodiment, the CTP-modified hGH polypeptides disclosed herein comprise at least 8 O-glycans per CTP. One skilled in the art will appreciate that O-glycan occupancy can vary for each CTP present in the CTP-modified hGH polypeptide.

  In another embodiment, a CTP-hGH-CTP-CTP polypeptide disclosed herein comprises at least one CTP comprising more than 4 O-glycan occupancy. In another embodiment, a CTP-hGH-CTP-CTP polypeptide disclosed herein comprises at least one CTP comprising more than 5 O-glycan occupancy. In another embodiment, a CTP-hGH-CTP-CTP polypeptide disclosed herein comprises at least one CTP comprising more than 6 O-glycan occupancy. In another embodiment, a CTP-hGH-CTP-CTP polypeptide disclosed herein comprises at least one CTP comprising more than 7 O-glycan occupancy.

  One skilled in the art will appreciate that the term “homology” can encompass deletions, insertions, or substitution mutations, including amino acid substitutions thereof, and biologically active polypeptide fragments thereof. In one embodiment, the substitution mutation is one in which glutamine at position 65 of hGH is replaced with valine [Gellerfors et al. , J Pharm Biomed Anal 1989, 7: 173-83].

  In one embodiment, “peptide” or “peptide fragment” includes compounds in which a plurality of amino acids are linked by peptide bonds. Here, when a non-amino acid is contained, the bond between the non-amino acid and the adjacent amino acid may not be a peptide bond. However, the compounds in this case are also collectively referred to as peptides or peptide fragments.

  One skilled in the art will recognize that the term “protected peptide fragment” can result in undesirable side reactions during peptide fragment preparation or peptide fragment condensation reactions, amino acid or non-amino acid side chain hydroxy groups, amino groups of the peptide fragment. A peptide fragment in which one or more reactive substituents selected from the group consisting of guanidino group, imidazolyl group, indolyl group, mercapto group, and carboxyl group are protected with a protecting group. You will understand. Hereinafter, this is abbreviated as “protected peptide fragment” in the present specification.

  In some embodiments, human growth hormone (hGH) is utilized in accordance with the teachings disclosed herein. In some embodiments, adhesion of the CTP sequence (s) to both the amino and carboxy termini of the hGH protein results in increased efficacy. In some embodiments, attachment of CTP sequences to both the amino and carboxy termini of hGH proteins results in prolonged in vivo activity.

  In one embodiment, the CTP modified hGH precursor polypeptide disclosed herein is set forth in SEQ ID NO: 4.

  SEQ ID NO: 4: MATGSRTSLLLAFGLLCLPWLQEGSASSSSKAPPPSLPSPSRLPGPSDTPILPQFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQ.

  One skilled in the art will recognize that the phrase “human growth hormone” (hGH) includes a signal peptide that expresses hGH activity (ie, stimulation of growth) as described in Genbank Accession No. P01241 (SEQ ID NO: 5). It will be appreciated that body polypeptides may be included. SEQ ID NO: 5: MATGSRTSLLLAFGLLCLPWLQEGSAFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF. In another embodiment, mature hGH disclosed herein, SEQ ID NO: 8: having the amino acid sequence set forth in EfuPitiaiPieruesuarueruefudienueiemueruarueieichiaruerueichikyuerueiefuditiwaikyuiefuiieiwaiaiPikeiikyukeiwaiesuefuerukyuenuPikyutiesuerushiefuesuiesuaiPitiPiesuenuaruiitikyukyukeiesuenueruierueruaruaiesueruerueruaikyuesudaburyueruiPibuikyuefueruaruesubuiefueienuesuerubuiwaijieiesudiesuenubuiwaidieruerukeidieruiijiaikyutieruemujiarueruidijiesuPiarutijikyuaiefukeikyutiwaiesukeiefuditienuesueichienudidieieruerukeienuwaijierueruwaishiefuRKDMDKVETFLRIVQCRSVEGSCGF.

  One skilled in the art will appreciate that “hGH” disclosed herein may encompass homologues. In another embodiment, the GH amino acid sequences of the methods and compositions disclosed herein are as determined herein using National Biotechnology Information Center (NCBI) BlastP software using default parameters. Is at least 50% homologous to the described hGH sequence. In another embodiment, the percent homology is 60%. In another embodiment, the percent homology is 70%. In another embodiment, the percent homology is 80%. In another embodiment, the percent homology is 90%. In another embodiment, the percent homology is 95%. In another embodiment, the percent homology is greater than 95%.

  In one embodiment, following expression and secretion of the CTP-modified hGH polypeptide disclosed herein, the signal peptide is cleaved from the precursor protein to become a mature protein. For example, in SEQ ID NO: 4, amino acids 1 to 26, MATGSRTSLLLAFGLLCLPWLQEGSA (SEQ ID NO: 6) represents a signal peptide CTP modified hGH polypeptides, amino acids SSSSKAPPPSLPSPSRLPGPSDTPILPQFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFSSSSKAPPPSLPSPSRLPGPSDTPI PikyuesuesuesuSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 7) represents the engineered mature CTP modified hGH polypeptides lacking a signal peptide (MOD-4023). In one embodiment, the amino acid sequence of CTP modified hGH without a signal peptide is set forth in SEQ ID NO: 7. In another embodiment, the signal peptide of CTP modified hGH is set forth in SEQ ID NO: 6. In another embodiment, amino acids 29-219 of the CTP modified hGH sequence set forth in SEQ ID NO: 7 represents hGH. In another embodiment, amino acids 1-28, 220-247, and 248-275 of the CTP modified hGH sequence set forth in SEQ ID NO: 7 are each CTP unit of CTP modified hGH, one N-terminus to hGH, and Represents the two C-termini to hGH.

  In one embodiment, MOD-4023 (SEQ ID NO: 7) comprises two disulfide (S—S) bridges, both of which are in the hGH molecule. In another embodiment, one disulfide bridge is between cysteine residue 81 and cysteine residue 193 of SEQ ID NO: 7, and the second disulfide bridge is cysteine residue 210 and cysteine residue 217 of SEQ ID NO: 7 Between.

  In another embodiment, the methods disclosed herein provide CTP-modified hGH polypeptides that stimulate muscle growth.

  In one embodiment, the methods of manufacture disclosed herein generate the amino acid sequence of a mature CTP modified hGH polypeptide that lacks the signal peptide disclosed herein. In another embodiment, the methods of manufacture disclosed herein generate the amino acid sequence of the mature CTP modified hGH polypeptide set forth in SEQ ID NO: 7.

  In another embodiment, the methods disclosed herein provide a CTP-modified hGH polypeptide set forth in SEQ ID NO: 7 that stimulates muscle growth.

  In another embodiment, the methods disclosed herein include the use of nucleic acid sequences encoding CTP-modified hGH polypeptides disclosed herein. In one embodiment, the method disclosed herein comprises the nucleic acid of SEQ ID NO: 9 encoding an hGH peptide having one CTP amino acid peptide on the N-terminus and two CTP amino acid peptides on the C-terminus. Including use. SEQ ID NO: 9: ATGGCCACCGGCAGCAGGACCAGCCTGCTGCTGGCCTTCGGCCTGCTGTGCCTGCCATGGCTGCAGGAGGGCAGCGCCAGCTCTTCTTCTAAGGCTCCACCCCCATCTCTGCCCAGCCCCAGCAGACTGCCGGGCCCCAGCGACACACCCATTCTGCCCCAGTTCCCCACCATCCCCCTGAGCAGGCTGTTCGACAACGCCATGCTGAGGGCTCACAGGCTGCACCAGCTGGCCTTTGACACCTACCAGGAGTTCGAGGAAGCCTACATCCCCAAGGAGCAGAAGTACAGCTTCCTGCAGAACCCCCAGACCTCCCTGTGCTTCAGC AGAGCATCCCCACCCCCAGCAACAGAGAGGAGACCCAGCAGAAGAGCAACCTGGAGCTGCTGAGGATCTCCCTGCTGCTGATCCAGAGCTGGCTGGAGCCCGTGCAGTTCCTGAGAAGCGTGTTCGCCAACAGCCTGGTGTACGGCGCCAGCGACAGCAACGTGTACGACCTGCTGAAGGACCTGGAGGAGGGCATCCAGACCCTGATGGGCCGGCTGGAGGACGGCAGCCCCAGGACCGGCCAGATCTTCAAGCAGACCTACAGCAAGTTCGACACCAACAGCCACAACGACGACGCCCTGCTGAAGAACTACGGGCTGCTGTACTGCTTC GAAAGGACATGGACAAGGTGGAGACCTTCCTGAGGATCGTGCAGTGCAGAAGCGTGGAGGGCAGCTGCGGCTTCAGCTCCAGCAGCAAGGCCCCTCCCCCGAGCCTGCCCTCCCCAAGCAGGCTGCCTGGGCCCTCCGACACACCAATCCTGCCACAGAGCAGCTCCTCTAAGGCCCCTCCTCCATCCCTGCCATCCCCCTCCCGGCTGCCTGGCCCCTCTGACACCCCTATCCTGCCTCAG.

  In one embodiment, the method includes the use of a nucleic acid sequence comprising a coding portion that encodes a CTP modified hGH disclosed herein. In another embodiment, the method disclosed herein comprises the use of the nucleic acid sequence set forth in SEQ ID NO: 9. One skilled in the art will appreciate that the nucleic acid sequence can be part of an expression vector that includes a coding portion that encodes a CTP-modified hGH disclosed herein.

  In another embodiment, the methods disclosed herein provide CTP for therapeutic use in a number of indications as provided in US Pat. No. 8,946,155, incorporated herein by reference. Nucleic acid sequences encoding modified hGH peptides are provided. In addition, such CTP-modified hGH polypeptides are fully described in U.S. Patent Application Publication No. US-2014-0113860-A1, U.S. Patent No. 8,450,269, and U.S. Patent No. 8,304,386, All of which are incorporated herein by reference in their entirety.

  In some embodiments, the polypeptides disclosed herein are utilized in therapy that require the polypeptide to be in a soluble form.

  In some embodiments, the polypeptides disclosed herein are synthesized biochemically using standard solid phase methods. In some embodiments, these biochemical production methods include exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, or classical solution synthesis. In some embodiments, these methods are used when the polypeptide is relatively short (about 5-15 kDa) and / or cannot be produced by recombinant techniques (ie, not encoded by a nucleic acid sequence). And therefore requires a different chemistry.

  In some embodiments, solid phase polypeptide synthesis procedures are known to those of skill in the art and are described in John Morrow Stewart and Janis Dillaha Young, Solid Phase Polypeptide Syntheses (2nd Ed., Pierce Chemical Co., 198). . In some embodiments, the synthetic polypeptide is purified by preparative high performance liquid chromatography [Creighton T. et al. (1983) Proteins, structures and molecular principals. WH Freeman and Co. N. Y. The composition can be confirmed via the amino acid sequence by methods known to those skilled in the art.

  In some embodiments, recombinant protein technology is used to produce the polypeptides disclosed herein. In some embodiments, recombinant protein technology is used to produce relatively long polypeptides (eg, longer than 18-25 amino acids). In some embodiments, recombinant protein technology is used to produce large quantities of the polypeptides disclosed herein. In some embodiments, recombinant techniques are performed by Bitter et al. , (1987) Methods in Enzymol. 153: 516-544, Study et al. (1990) Methods in Enzymol. 185: 60-89, Brisson et al. (1984) Nature 310: 511-514, Takamatsu et al. (1987) EMBO J. et al. 6: 307-311, Coruzzi et al. (1984) EMBO J. et al. 3: 1671-1680, and Brogli et al, (1984) Science 224: 838-843, Gurley et al. (1986) Mol. Cell. Biol. 6: 559-565, and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp421-463.

  In one embodiment, the polypeptides disclosed herein are synthesized using a polynucleotide that encodes a polypeptide disclosed herein. In some embodiments, a polynucleotide encoding a polypeptide disclosed herein is linked to an expression vector that includes transcriptional control of a cis-regulatory sequence (eg, a promoter sequence). In some embodiments, cis regulatory sequences are suitable for directing constitutive expression of the polypeptides disclosed herein. In some embodiments, cis-regulatory sequences are suitable for directing tissue specific expression of the polypeptides disclosed herein. In some embodiments, cis-regulatory sequences are suitable for directing inducible expression of a polypeptide disclosed herein.

  In some embodiments, the polynucleotide that expresses the polypeptide disclosed herein comprises the nucleotide sequence set forth in SEQ ID NO: 9.

  In some embodiments, tissue specific promoters suitable for use in conjunction with the polynucleotide sequences disclosed herein include sequences that are functional in specific cell populations, such as liver specific Albumin [Pinkert et al. (1987) Genes Dev. 1: 268-277], a lymphocyte-specific promoter [Calam et al. , (1988) Adv. Immunol. 43: 235-275]; in particular, the promoter of the T cell receptor [Winoto et al. (1989) EMBO J. et al. 8: 729-733] and immunoglobulins; [Banerji et al. (1983) Cell 33729-740], neuron-specific promoters, such as the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci. USA 86: 5473-5477], pancreas specific promoter [Edrunch et al. (1985) Science 230: 912-916], or mammary gland specific promoters, such as, but not limited to, whey promoters (US Pat. No. 4,873,316 and European Published Application 264,166). Not. Suitable inducible promoters for use in the methods disclosed herein include, for example, tetracycline inducible promoters (Srour, MA, et al., 2003. Thromb. Haemost. 90: 398-405). It is done.

  One skilled in the art will recognize that the phrase “polynucleotide” is isolated and in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence, and / or a composite polynucleotide sequence (eg, a combination of the above). It will be appreciated that the provided single stranded or double stranded nucleic acid sequence may be included.

  One skilled in the art will appreciate that the phrase “complementary polynucleotide sequence” can encompass sequences resulting from reverse transcription of messenger RNA using reverse transcriptase or any other RNA-dependent DNA polymerase. . In one embodiment, the sequence can then be amplified in vivo or in vitro using DNA polymerase.

  One skilled in the art will understand that a “genomic polynucleotide sequence” can include a sequence that is derived (isolated) from a chromosome, and thus represents an adjacent portion of a chromosome.

  One skilled in the art will appreciate that the phrase “composite polynucleotide sequence” can encompass sequences that are at least partially complementary and at least partially genomic. In one embodiment, the composite sequence may include several exon sequences required to encode the polypeptides disclosed herein, as well as several intron sequences sandwiched therebetween. In one embodiment, intron sequences may be from any source, including other genes, and will typically include conserved splicing signal sequences. In one embodiment, the intron sequence comprises a cis activity expression regulatory element.

  In one embodiment, the polynucleotide disclosed herein further comprises a signal sequence encoding a signal peptide for secretion of the polypeptide disclosed herein. In some embodiments, the signal sequence includes, but is not limited to, the endogenous signal sequence of hGH set forth in SEQ ID NO: 6. In another embodiment, the signal sequence is a sequence from the N-terminus to the CTP sequence, which in turn is a sequence from the N-terminus to the polypeptide sequence of interest, eg, the sequence is (a) signal sequence- (b) CTP- (c) sequence of interest- (d) optionally one or more additional CTP sequences. In another embodiment, one or more CTP sequences are inserted between the signal sequence of the polypeptide sequence of interest and the polypeptide of interest itself, thereby blocking the wild-type sequence of interest.

  In another embodiment, the polypeptides disclosed herein and methods for producing the same provide mature CTP-modified GH lacking a signal peptide as set forth in SEQ ID NO: 7.

  In some embodiments, the polynucleotides disclosed herein are prepared using PCR techniques, or any other method or procedure known to those skilled in the art. In some embodiments, the procedure involves ligation of two different DNA sequences (see, eg, “Current Protocols in Molecular Biology”, eds. Ausubel et al., John Wiley & Sons, 1992).

  Biological activity and use

  In one embodiment, a method for reducing the number of doses of GH to a subject comprising hGH, one chorionic gonadotropin carboxy terminal peptide (CTP) attached to the amino terminus of the GH, and the carboxy terminus of the GH. Administering to the subject a therapeutically effective amount of a polypeptide comprising two attached chorionic gonadotropin CTPs, the polypeptide lacking a signal peptide, the amino acid sequence of the polypeptide being SEQ ID NO: 7 A method for reducing the number of doses of GH to a subject is disclosed herein.

  In another embodiment, a method for improving the area under the curve (AUC) of GH to a subject comprising hGH and one chorionic gonadotropin carboxy terminal peptide (CTP) attached to the amino terminus of the hGH, administering to the subject a therapeutically effective amount of a polypeptide consisting of two chorionic gonadotropin CTPs attached to the carboxy terminus of hGH, the polypeptide lacking a signal peptide, and the amino acids of the polypeptide The sequence is set forth in SEQ ID NO: 7, and a method for improving the area under the curve (AUC) of GH to a subject is disclosed herein.

  In one embodiment, a method of treating a subject in need of GH treatment comprising hGH, one chorionic gonadotropin carboxy terminal peptide (CTP) attached to the amino terminus of the hGH, and the carboxy terminus of the hGH. Administering to the subject a therapeutically effective amount of a polypeptide comprising two attached chorionic gonadotropin CTPs, the polypeptide lacking a signal peptide, the amino acid sequence of the polypeptide being SEQ ID NO: 7 Disclosed herein are methods for treating said subject in need of GH in therapy.

  In another embodiment, a method of increasing insulin-like growth factor (IGF-1) levels in a subject comprising hGH and one chorionic gonadotropin carboxy terminal peptide (CTP) attached to the amino terminus of the hGH; Administering to the subject a therapeutically effective amount of a polypeptide consisting of two chorionic gonadotropin CTPs attached to the carboxy terminus of the hGH, the polypeptide lacking a signal peptide, The amino acid sequence is set forth in SEQ ID NO: 7, and a method is disclosed herein for increasing insulin-like growth factor (IGF-1) levels in a subject.

  In another embodiment, a method of maintaining insulin-like growth factor (IGF-I) levels in a subject comprising hGH and one chorionic gonadotropin carboxy terminal peptide (CTP) attached to the amino terminus of the hGH; Administering to the subject a therapeutically effective amount of a polypeptide consisting of two chorionic gonadotropin CTPs attached to the carboxy terminus of the hGH, the polypeptide lacking a signal peptide, The amino acid sequence is set forth in SEQ ID NO: 7, thereby disclosing herein a method for maintaining a subject's insulin-like growth factor (IGF-I) levels. In another embodiment, IGF-I is maintained within a defined range, as further disclosed herein.

  In another embodiment, a method of increasing and maintaining insulin-like growth factor (IGF-I) levels in a subject comprising hGH and one chorionic gonadotropin carboxy terminal peptide (CTP) attached to the amino terminus of the hGH. And administering to the subject a therapeutically effective amount of a polypeptide consisting of two chorionic gonadotropin CTPs attached to the carboxy terminus of the hGH, the polypeptide lacking a signal peptide, The amino acid sequence of the peptide is set forth in SEQ ID NO: 7, thereby disclosing herein a method for increasing and maintaining a subject's insulin-like growth factor (IGF-I) levels within a defined range.

  In another embodiment, the defined range is the therapeutic dose range achieved by administration of the CTP modified hGH disclosed herein. In another embodiment, the defined range is such that the CGF and C trough of the sinusoidal action of IGF-I is maintained after continuous administration of the CTP modified hGH disclosed herein. In another embodiment, the defined range is a therapeutic dose range for continuous administration of CTP-modified hGH disclosed herein with excellent responsiveness in the subject and the need for minimal dose changes. is there. In another embodiment, the defined range is equivalent to IGF-I levels in individuals considered normal. In another embodiment, the defined range is the normal range of IGF-I levels / values in normal solids. In yet another embodiment, the defined range is within the normal range when the IGF-I SDS value is ± 2 SDS.

  In another embodiment, the methods disclosed herein provide a CTP-modified hGH polypeptide described herein that stimulates bone growth.

  In another embodiment, the methods disclosed herein provide a nucleic acid sequence encoding a CTP-modified hGH polypeptide described herein that stimulates bone growth.

  In another embodiment, the CTP modified hGH disclosed herein is used in the same manner as unmodified GH. In another embodiment, the CTP modified hGH disclosed herein has increased circulating half-life and plasma residence time, decreased clearance, and increased clinical activity in vivo. In another embodiment, due to the improved properties of CTP modified hGH described herein, the conjugate is administered less frequently than unmodified GH. In another embodiment, the CTP-modified GH described herein is administered from once a week to once every two weeks. In another embodiment, the CTP-modified GH described herein is administered from once every two weeks to once every three weeks. In another embodiment, the CTP-modified GH described herein is administered from once daily to 3 times per week. In another embodiment, the reduced dosing frequency results in improved patient compliance, leading to improved treatment outcomes as well as improved patient quality of life. In another embodiment, GH CTP conjugates having the molecular weight and linker structure of the conjugates disclosed herein have improved efficacy compared to conventional GH conjugates conjugated to poly (ethylene glycol). It has been discovered to have improved stability, increased AUC levels, and improved circulatory half-life. In another embodiment, a GH having the molecular weight and linker structure of a conjugate disclosed herein has improved potency, improved compared to a conventional GH conjugate conjugated to poly (ethylene glycol). It was discovered to have stability, increased AUC levels, and improved circulatory half-life. In another embodiment, the therapeutically effective amount of CTP-modified hGH is the amount of conjugate required for a measurable expected biological activity in vivo. In another embodiment, GH utilized in accordance with the teachings disclosed herein exhibits increased efficacy. In some embodiments, adhesion of CTP sequences to both the amino and carboxy termini of GH results in prolonged in vivo activity.

  In another embodiment, the therapeutically effective amount of CTP-modified hGH is determined by factors such as the exact type of illness being treated, the condition of the patient being treated, and other ingredients in the composition. In another embodiment, the therapeutically effective amount of CTP-modified hGH is 0.01-10 μg / kg body weight administered once a week. In another embodiment, the therapeutically effective amount of CTP-modified hGH is 0.1-1 μg / kg body weight administered once a week. In another embodiment, the therapeutically effective amount of CTP-modified hGH is 1 μg / kg body weight to 1 mg / kg body weight administered once a week. In another embodiment, the therapeutically effective amount of CTP-modified hGH is 1 mg / kg body weight administered once a week. In another embodiment, the therapeutically effective amount comprising CTP-modified hGH for patients with adult growth hormone deficiency is 1 mg to 15 mg administered once a week. In another embodiment, the therapeutically effective amount comprising CTP-modified hGH for patients with adult growth hormone deficiency is greater than 1 mg administered once a week. In another embodiment, the therapeutically effective amount comprising CTP-modified hGH for a patient with pediatric growth hormone deficiency is between 1 μg / kg body weight / kg body weight administered once a week. In another embodiment, the therapeutically effective amount comprising CTP-modified hGH to a patient with pediatric growth hormone deficiency is up to 600 μg / kg body weight administered once a week. In another embodiment, the therapeutically effective amount comprising CTP-modified hGH for pediatric growth hormone deficiency patients is up to 700 μg / kg body weight administered once a week. In another embodiment, the therapeutically effective amount comprising CTP-modified hGH to a patient with pediatric growth hormone deficiency is between about 600 μg to 700 μg / kg body weight administered once a week. In another embodiment, a pharmaceutical composition comprising CTP-modified hGH is prepared with a strength effective for administration to a human patient by various means.

  In one embodiment, the methods, polynucleotides, and polypeptides disclosed herein are used in veterinary medicine. In one embodiment, nourished as a human companion (eg, dog, cat, horse), has significant commercial value (eg, dairy cattle, beef cattle, competition animals), has significant scientific value (eg, endangered species) Disclosed herein are treatments of captive mammals that are trapped or free species) or otherwise have value.

  In one embodiment, a polypeptide or polynucleotide disclosed herein is an animal (eg, mouse, rat, rabbit, hamster, guinea pig, pig, micropig, chicken, camel, goat, horse, cow, sheep. , Dogs, cats, non-human primates, and humans). In one embodiment, the cited applications can be used in a wide variety of hosts. In some embodiments, such hosts include humans, mice, rabbits, goats, guinea pigs, camels, horses, mice, rats, hamsters, pigs, micropigs, chickens, chickens, goats, cows, This includes but is not limited to sheep, dogs, cats, or non-human primates.

  In one embodiment, livestock are treated with the methods disclosed herein. In one embodiment, livestock includes pigs, beef cattle, dairy cows, horses, goats, sheep, chickens, turkeys, cancers, ducks, and related species. In one embodiment, experimental animals are treated with the methods disclosed herein. In one embodiment, experimental animals include rats, mice, guinea pigs, rabbits, goats, monkeys, dogs, cats, and others. In one embodiment, zoo animals are treated with the methods disclosed herein. In one embodiment, zoo animals include all vertebrates bred at the zoo. In one embodiment, the aquatic animal is treated with the methods disclosed herein. In one embodiment, aquatic animals include fish, eels, turtles, seals, penguins, sharks, whales, and related species. In one embodiment, domestic animals are treated with the methods disclosed herein. In one embodiment, domestic animals include any pet, such as cats and dogs, or animals raised by humans, such as horses, cows, pigs, goats, rabbits, chickens, turkeys, cancers, ducks, etc. .

  One skilled in the art will appreciate that the term “pigs” includes pigs, piglets, meat pigs, heifers, castrated pigs, breed pigs, and farmed pigs. Similarly, those skilled in the art will understand that the term “cow” includes calves, cows, dairy cows, heifers, steers, and bulls.

  In some embodiments, CTP sequence modifications are advantageous in that they allow the use of lower dosages.

  Manufacturing

  In one embodiment, a method of producing a human chorionic gonadotropin carboxy-terminal peptide (CTP) modified human growth hormone (hGH) polypeptide comprising: (a) encoding a predetermined number of cells with the CTP modified (hGH). Stably transfecting an expression vector comprising a coding portion that transfects, wherein the transfected cells express and secrete the CTP modified hGH, and (b) the CTP modified hGH. Obtaining a cell clone that overexpresses, (c) growing the clone in solution to a predetermined scale, (d) collecting the solution containing the clone, Filtering the solution containing the clones to obtain a clarified collection solution; (f) the clarified yield. Purifying the solution to obtain a purified protein solution having a desired concentration of CTP-modified hGH, wherein the amino acid sequence of the produced CTP-modified hGH is set forth in SEQ ID NO: 7, whereby human villi Disclosed herein are methods for producing sex gonadotropin carboxy-terminal peptide (CTP) modified human growth hormone (hGH) polypeptides. In an alternative embodiment, in step (e), cells and debris are removed by centrifugation. In the production of CTP modified hGH, transfection is performed at an early stage. In another embodiment, the method of manufacture comprises a clone that expresses and secretes the CTP-modified hGH. Once the final high-expressing clone is selected, each generation includes working cell bank (WCB) thawing, propagation, collection, and purification (eg, steps 1-8 of FIG. 3 describing clonal propagation through collection; See steps 8-16 of FIG. 4 illustrating the purification). In an alternative embodiment, once the final high-expressing clone is selected, each generation includes thawing, growing, collecting, and purifying the master cell bank (MCB) (eg, illustrating clonal growth through collection). 3 steps 1-8; see steps 8-16 of FIG. 4 illustrating the purification).

  In one embodiment, the polynucleotide disclosed herein is inserted into an expression vector (ie, a nucleic acid construct) to allow expression of the recombinant polypeptide. In one embodiment, the expression vectors disclosed herein contain additional sequences that make the vector suitable for replication and integration in prokaryotes. In one embodiment, the expression vectors disclosed herein contain additional sequences that make the vector suitable for replication and integration in eukaryotes. In one embodiment, the expression vectors disclosed herein include shuttle vectors that make this vector suitable for replication and integration in both prokaryotes and eukaryotes. In some embodiments, cloning vectors include transcription and translation initiation sequences (eg, promoters, enhancers) and transcription and translation terminators (eg, polyadenylation signals).

  In one embodiment, a method for producing a CTP-modified polypeptide, eg, CTP-modified hGH, includes the step of using an expression vector, the expression vector comprising a promoter, a coding sequence for the CTP-modified polypeptide, and polyadenyl. Including a sequence. In another embodiment, the coding sequence is set forth in SEQ ID NO: 9. In another embodiment, the polyadenylation sequence is a simian virus (SV) 40 polyadenylation sequence.

  In one embodiment, a variety of eukaryotic cells can be used as a host expression system to express a polypeptide disclosed herein. In some embodiments, these include microorganisms, eg, yeast transformed with a recombinant yeast expression vector containing a polypeptide coding sequence; recombinant virus expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, Plant cell lines infected with TMV) or transformed with a recombinant plasmid vector such as a Ti plasmid containing a polypeptide coding sequence include, but are not limited to.

  In one embodiment, non-bacterial expression systems (eg, mammalian expression systems such as CHO cells or cells derived from CHO cells) are used to express the polypeptides disclosed herein. In one embodiment, the expression vector used to express a polynucleotide disclosed herein in mammalian cells is a pCI-DHFR vector comprising a CMV promoter and a neomycin resistance gene. In another embodiment, the cells are grown as adherent cell cultures. In another embodiment, it is grown in suspension culture.

  In one embodiment, the expression vectors disclosed herein include additional polynucleotide sequences that allow translation of several proteins from a single mRNA, such as, for example, an intrasequence ribosome entry site (IRES), And a sequence for genomic integration of the promoter-chimeric polypeptide.

  In some embodiments, mammalian expression vectors include pcDNA3, pcDNA3.1 (+/−), pGL3, pZeoSV2 (+/−), pSecTag2, pDisplay, pEF / myc / cyto, pCMV, available from Invitrogen. / Myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81, pCI available from Promega, pMbac available from Strategene, pPbac, pBK-RSV, and available from pBK-CMV, Clonch PTRES, as well as derivatives thereof, but are not limited to these.

In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses can be found in the vectors disclosed herein. SV40 vectors include pSVT7 and pMT2. In some embodiments, the vector derived from bovine papilloma virus includes pBV-1MTHA, and the vector derived from Epstein Barr virus includes pHEBO and p2O5. Other representative ^{examples, pMSG, pAV009 / A +,} pMTO10 / A +, pMAMneo-5, baculovirus pDSVE, and SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Any other vector that allows for expression of the protein under the direction of the Rous sarcoma virus promoter, polyhedrin promoter, or other promoter shown to be effective for expression in eukaryotic cells.

  In one embodiment, various methods can be used to introduce an expression vector encoding a CTP-modified hGH disclosed herein into a cell. “Transfection” of eukaryotic host cells with polynucleotides or expression vectors resulting in genetically modified cells or transgenic cells is known in the art and is described, for example, in Sambrook et al. , Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), Ausubel et al. , Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al. , Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al. Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988), and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] can be performed, including, for example, stable or transient transfection, and electroporation. In addition, see US Pat. Nos. 5,464,764 and 5,487,992 for positive and negative selection methods. Transfection methods include liposome-mediated transfection, calcium phosphate coprecipitation, electroporation, polycation (DEAE-dextran, etc.) mediated transfection, protoplast fusion, viral infection (including recombinant viral infection), and microinjection. For example, but not limited to. Preferably, the transfection is a stable transfection. A transfection method that provides optimal transfection frequency and expression in a particular host cell line and type of a heterologous gene encoding a peptide of interest disclosed herein is recommended. Suitable methods can be determined by routine methods. For stable transfection, the construct is integrated into the genome or artificial chromosome / minichromosome of the host cell or is positioned episomally so as to be stably maintained in the host cell.

The methods disclosed herein employ conventional techniques such as cell biology, molecular biology, cell culture, immunology, and the like in the art, unless otherwise indicated. These techniques are fully disclosed in the latest literature. For example, Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2 ^nd Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N .; Y. (1989), Ausubel et al. , Current Protocols in Molecular Biology (1987, updated); Brown ed. , Essential Molecular Biology, IRL Press (1991), Goeddel ed. Gene Expression Technology, Academic Press (1991), Bothwell et al., Gene Expression Technology. eds. , Methods for Cloning and Analysis of Eukaryotic Genes, Bartlett Publ. (1990), Wu et al. , Eds. , Recombinant DNA Methodology, Academic Press (1989), Kriegler, Gene Transfer and Expression, Stockton Press (1990), McPherson et al. , PCR: A Practical Approach, IRL Press at Oxford University Press (1991), Gait ed. , Oligonucleotide Synthesis (1984), Miller & Calos eds. Gene Transfer Vectors for Mammalian Cells (1987), Butler ed. , Mammalian Cell Biotechnology (1991), Pollard et al. , Eds. , Animal Cell Culture, Humana Press (1990), Freshney et al. , Eds. , Culture of Animal Cells, Alan R .; Liss (1987), Studzinski, ed. , Cell Growth and Apoptosis, A Practical Approach, IRL Press at Oxford University Press (1995), Melamed et al. , Eds. , Flow Cytometry and Sorting, Wiley-Liss (1990), Current Protocols in Cytometry, John Wiley & Sons, Inc. (Updated), Whith & Hauser, Genetic Engineering of Animals Cells, in: Biotechnology Vol. 2, Puhler ed. , VCH, Weinheim 663-744, the series Methods of Enzymology (Academic Press, Inc.), and Harlow et al. , Eds. , Antibodies: A Laboratory Manual (1987).

  The heterologous gene of interest encoding the CTP modified hGH disclosed herein may be introduced into the cells disclosed herein by a variety of methods, for example, by viral transformation. The desired heterologous gene may be introduced into the cell as linear DNA or as part of an expression vector. Many eukaryotic expression vectors that provide multiple cloning sites for the insertion of one or more heterologous genes and their expression are known. Commercial suppliers include, among others, Stratagene, La Jolla, Calif. USA, Invitrogen, Carlsbad, Calif. USA, Promega, Madison, Wis. , USA, or BD Biosciences Clontech, Palo Alto, Calif. , USA. Transfection of cells with DNA or an expression vector encoding one or more genes of interest is described, for example, in Sambrook et al. , 1989 or Ausubel et al. 1994, 1994. Suitable methods of transfection include, for example, liposome-mediated transfection, calcium phosphate coprecipitation, electroporation, polycation (eg, DEAE dextran) mediated transfection, protoplast fusion, microinjection, and viral infection. . Preferably, stable transfection is performed while the DNA molecule is integrated into the genome or artificial chromosome / minichromosome of the host cell or is episomally contained in the host cell in a stable manner. Preferred are transfection methods that provide optimal transfection frequency and expression of one or more heterologous genes of interest in the host cell line of interest. In one embodiment, the gene of interest encodes a CTP modified GH disclosed herein.

  In some embodiments, introduction of nucleic acids by viral infection has several advantages over other methods such as lipofection and electroporation because higher transfection efficiency is obtained due to the infectious nature of the virus. provide.

  In one embodiment, the polypeptides disclosed herein can also be expressed from nucleic acid constructs administered to an individual using any suitable method of administration described above (ie, in vivo gene therapy). Will be understood. In one embodiment, the nucleic acid construct is introduced into a suitable cell via an appropriate gene delivery vehicle / method (transfection, transduction, heterologous recombination, etc.) and optionally an expression system and then modified. The cells are grown in culture and returned to the individual (ie, ex vivo gene therapy).

  The heterologous gene of interest is usually functionally linked to a promoter that allows transcription of the gene of interest and other regulatory elements that allow transcription or translation (expression) of the gene of interest or increase its efficiency. Join.

  One skilled in the art will appreciate that the term “promoter” can encompass polynucleotide sequences that permit and control transcription of a gene or sequence operably linked thereto. The promoter contains a recognition sequence for binding RNA polymerase and an initiation site for transcription (transcription initiation site). In order to express the desired sequence in a particular cell type or host cell, a suitable functional promoter must be selected. Those skilled in the art will know a variety of promoters from a variety of sources, including constitutive, inducible and repressible promoters. These are deposited in a data bank such as GenBank and can be obtained as separate elements or as elements cloned in polynucleotide sequences from commercial or individual sources. For inducible promoters, the activity of the promoter can be reduced or increased in response to the signal. An example of an inducible promoter is the tetracycline (tet) promoter. It contains a tetracycline operator sequence (tetO) that can be induced by a tetracycline-regulated transactivator protein (tTA). In the presence of tetracycline, tTA binding to tetO is inhibited. Examples of other inducible promoters are the jun, fos, metallothionein, and heat shock promoters (Sambrook, J., Fritsch, EF & Maniatis, T., Molecular Cloning: A Laboratory Manual Cold Spring Harbor See also Spring Harbor, NY, 1989, Gossen, M. et al., Curr Opi Biotech 1994, 5, 516-520). Among promoters that are particularly suitable for high expression in eukaryotes, for example, hamster ubiquitin / S27a promoter (WO 97/15664), SV40 early promoter, adenovirus tumor late promoter, mouse metallothionein-1 promoter, us sarcoma There are viral terminal repeat regions and human cytomegalovirus. Examples of other heterologous mammalian promoters are actin, immunoglobulins, or heat shock promoter (s).

  For example, a promoter can be operably linked to an enhancer sequence to increase transcriptional activity. For this purpose, several copies of one or more enhancer and / or enhancer sequences can be used, for example CMV or SV40 enhancers. For example, a promoter can be operably linked to an enhancer sequence to increase transcriptional activity. For this purpose, several copies of one or more enhancer and / or enhancer sequences can be used, for example CMV or SV40 enhancers.

  One skilled in the art will understand that the term “enhancer” refers to a polynucleotide sequence that acts on the activity of a promoter at the cis position, thereby stimulating transcription of a gene that is operably linked to this promoter. Let's go. Unlike promoters, enhancer effects are independent of position and location, and therefore they may be located in front of or behind the transcription unit, in introns, or even in coding regions. Enhancers can be located both in the immediate vicinity of the transcription unit and at a considerable distance from the promoter. It is also possible to have physical and functional overlap with the promoter. Those skilled in the art will recognize that many enhancers from a variety of sources are available as independent elements or elements cloned within a polynucleotide sequence (eg, deposited at ATCC or derived from commercial sources or individual sources). (And, for example, SV40 enhancer, CMV enhancer, polyoma enhancer, adenovirus enhancer) deposited in data banks such as GenBank. Many promoter sequences also contain enhancer sequences such as the frequent CMV promoter. The human CMV enhancer is one of the strongest enhancers identified so far. An example of an inducible enhancer is a metallothionein enhancer that can be stimulated by glucocorticoids or heavy metals.

  Basically, regulatory elements include promoters, enhancers, termination of polyadenylation signals, and other expression control elements. Both inducible and structural types of regulatory sequences are known for various cell types. “Transcriptional regulatory elements” generally include a promoter upstream of the expressed gene sequence, transcription initiation and termination sites, and polyadenylation signals.

  One skilled in the art will appreciate that the term “transcription start site” can encompass a nucleic acid in a construct that corresponds to a primary transcript, ie, a first nucleic acid that is incorporated into an mRNA precursor. The transcription start position can overlap with the promoter sequence.

  One skilled in the art will appreciate that the term “transcription termination site” is usually at the 3 ′ end of the gene of interest or the section of the gene to be transcribed, and may include nucleotide sequences that result in termination of transcription by RNA polymerase. I will.

  One skilled in the art will recognize that the term “polyadenylation signal” refers to cleavage at a specific site at the 3 ′ end of eukaryotic mRNA, and a sequence of about 100-200 adenine nucleotides at the 3 ′ end that is cleaved (polyA). It will be understood that a signal sequence that causes post-translational introduction of tail) may be included. The polyadenylation signal comprises the sequence AATAAA 10-30 nucleotides upstream of the cleavage site and the sequence located downstream. Various polyadenylation elements are known, such as tk polyA, SV40 late and early polyA, or BGH polyA (for example, described in US Pat. No. 5,122,458).

  One skilled in the art will appreciate that the term “translational regulatory element” can include a translation initiation site (AUG), a stop codon, and a polyA signal for each polypeptide expressed. In order to remove any potentially inappropriate additional translation initiation codons or other sequences that could affect expression at the transcriptional or expression level in optimal expression, 5′- and / or of the expressed nucleic acid sequence Or it may be sensible to remove, add or change 3'-untranslated regions. In order to facilitate expression, a ribosome consensus binding site may alternatively be inserted immediately upstream of the start codon. To produce a secreted polypeptide, the gene of interest usually contains a signal sequence that encodes a signal precursor peptide that transports the synthetic polypeptide to and through the ER membrane. The signal sequence is not always, but is often located at the amino terminus of the secreted protein and is cleaved by signal peptidase after the protein is filtered through the ER membrane. A gene sequence, usually but not necessarily, contains a unique signal sequence. In the absence of the native signal sequence, the heterologous signal sequence may be introduced in a known manner. A number of similar signal sequences are known to those skilled in the art and are deposited in sequence data banks such as GenBank and EMBL.

  One skilled in the art will recognize that “polypeptides”, “polypeptides”, or synonyms thereof, are used for amino acid sequences or proteins and can encompass polymers of amino acids of any length. You will understand. The term also includes proteins that are post-translationally modified by reactions such as glycosylation, phosphorylation, acetylation, or protein processing. The structure of a polypeptide may be modified, for example, by amino acid substitutions, deletions, or insertions, and fusions with other proteins while retaining its biological activity.

  In order to produce one or more gene products of interest in the cell, the cells may be grown in serum-free medium and in suspension culture under conditions that allow expression of the gene of interest. For example, when the gene of interest is under the control of a structural promoter, it is not necessary to add a special inducer. If the expression of the gene of interest is under the control of an inducible promoter, for example, the corresponding inducer must be added to the cell culture medium at a sufficient but non-toxic concentration. Cells can be grown as desired by multiple subcultures and transferred to a suitable cell culture vessel. The gene product (s) can be produced as either cellular, membrane bound, or secreted products.

  In one embodiment, the step of producing a CTP modified hGH disclosed herein stabilizes an expression vector comprising a coding portion encoding a CTP modified hGH disclosed herein in a predetermined number of cells. Transfecting. In another embodiment, the nucleotide sequence of the coding portion is set forth in SEQ ID NO: 9. In another embodiment, the step of producing CTP-modified hGH comprises transfecting a cell with an expression vector comprising a coding portion that encodes the CTP-modified hGH. In one embodiment, the cell is a CHO cell. In one embodiment, the cell is a DG44 cell. In another embodiment, the cell is any cell known in the art suitable for expression and secretion of CTP-modified hGH. In another embodiment, the expression vector is a pCI-dhfr-MOD-23 expression vector (Figure 2). In another embodiment, the transfected cell expresses a CTP-modified hGH disclosed herein. In another embodiment, the CTP-modified hGH produced and expressed comprises two chorionic gonadotropin carboxy terminal peptides attached to the carboxy terminus of the hGH and one chorionic gonadotropin carboxy terminal peptide attached to the amino terminus of the hGH. And the amino acid sequence of the expressed immature CTP modified hGH is set forth in SEQ ID NO: 4. In another embodiment, the mature, CTP-modified hGH produced, expressed, and secreted comprises two chorionic gonadotropin carboxy terminal peptides attached to the carboxy terminus of the hGH and one chorionicity attached to the amino terminus of the hGH. The amino acid sequence of mature CTP-modified hGH consisting of, and expressed and secreted from, a gonadotropin carboxy terminal peptide is set forth in SEQ ID NO: 7. In other embodiments, the expression of CTP modified hGH is at a high expression level. In another embodiment, the CTP modified hGH is highly glycosylated. In another embodiment, the CTP modified hGH is highly sialylated. As described in detail above, CTP-modified hGH can have different glycosylation contents and patterns. The CTP-modified hGH produced by the method disclosed herein may include any of the glycosylation patterns and contents disclosed above. In general, the production methods presented herein provide CTP-modified hGH having a high glycosylation content and a high percentage of glycosylated glycosylation sites.

  In one embodiment, the step of producing CTP-modified hGH comprises obtaining a cell clone that overexpresses CTP-modified hGH. In another embodiment, the expression of CTP modified hGH is optimal. In one embodiment, the level of expression is at least 200 mg / L. In another embodiment, the level of expression is at least 300 mg / L. In another embodiment, the level of expression is at least 400 mg / L. In another embodiment, the level of expression is at least 500 mg / L. In another embodiment, the level of expression is at least 600 mg / L. In another embodiment, the level of expression is at least 700 mg / L. In another embodiment, the level of expression is at least 800 mg / L. In another embodiment, the level of expression is at least 900 mg / L. In another embodiment, the level of expression is at least 1 gm / L. In another embodiment, the level of expression is at least 1.2 gm / L. In another embodiment, the level of expression is at least 1.4 gm / L. In another embodiment, the level of expression is at least 1.6 gm / L. In another embodiment, the level of expression is at least 1.8 gm / L. In another embodiment, the level of expression is at least 2 gm / L. In another embodiment, the level of expression is at least 2.2 gm / L. In another embodiment, the level of expression is at least 2.4 gm / L. In another embodiment, the level of expression is at least 2.6 gm / L. In another embodiment, the level of expression is at least 2.8 gm / L. In another embodiment, the level of expression is at least 3 gm / L. In another embodiment, the clones of step (b) are propagated in media to form a master cell bank (MCB) and a working cell bank (WCB). In one embodiment, the clone of step (c) is obtained from the MCB. In another embodiment, the clone of step (c) is obtained from WCB.

  The mature CTP modified hGH polypeptide product disclosed herein is obtained from cell culture media as a secreted gene product. One skilled in the art will appreciate that the secreted gene product will be included in the full-length translation product and will lack the signal peptide encoded by the nucleic acid sequence. However, if the protein or polypeptide is expressed without a secretion signal, the gene product can also be isolated from the cell lysate. Conventional purification procedures have been performed to obtain pure heterologous products that are substantially free of other recombinant proteins and host cell proteins. First, cells and cell debris are frequently removed from the culture medium or lysate. The desired gene product is then analyzed by, for example, immunoaffinity and ion exchange column preparative, ethanol precipitation, Sephadex, hydroxyapatite, silica, or reverse phase HPLC or chromatography on cation exchange resins, such as DEAE. Separated from mixed soluble proteins, polypeptides, and nucleic acids (see examples herein). Methods known in the art to effect the purification of heterologous proteins expressed by recombinant host cells are known to those skilled in the art and are described in the literature, eg, Harris et al. (Harris et al., Protein Purification: A Practical Approach, Pickwood and Hames, eds., IRL Press, Oxford, 1995) and Scopes (Scopes, R., Protein Purification, Springer Verlag, 1988). These methods may be used in whole or in part in the methods disclosed herein. Based on the methodology provided herein, one of ordinary skill in the art will be able to understand, adapt, and modify the use of a particular chromatography methodology based on the needs and knowledge of the art. For example, those skilled in the art may select alternative cation exchange columns instead of DEAE columns as long as the purpose of cation exchange separation is maintained. In the art for preparative, immunoaffinity, protein precipitation, nucleic acid precipitation, ethanol precipitation, reverse phase HPLC, or chromatography on Sephadex, hydroxyapatite, silica, anion exchange or cation exchange resins. Those skilled in the art will understand similar known alternatives.

  In another embodiment, a method of preparing one or more products in mammalian cells under serum-free conditions, wherein (i) the mammalian cell comprises a CTP-modified hGH polypeptide disclosed herein. (Ii) the mammalian cells are grown under serum-free conditions that allow replication of the mammalian cells, and (iii) in each case these mammalian cells At least one of the (s) is deposited in a cell culture container under serum-free conditions; and (iv) suitably deposited mammalian cells are replicated under serum-free conditions; (V) replicated cells are cultured under serum-free conditions in which the gene is expressed and CTP-modified hGH is secreted; and (vi) the gene product is then isolated and purified from the supernatant of the medium. And Wherein are disclosed herein (see Examples herein). In another embodiment, the method of manufacture comprises a clone that expresses and secretes the CTP-modified hGH polypeptide. In another embodiment of this process, the mammalian cell is a transfected mammalian cell into which a gene of interest has been introduced. Accordingly, in the method for preparing a recombinant gene product disclosed herein, prior to step (i) of the above process, a mammalian cell is transfected with a nucleic acid encoding at least the gene of interest. Can be characterized. Stable transfection of corresponding mammalian cells is preferred.

  Examples of serum-free, protein-free, or known composition media include, for example, commercially available media Ham F12 (Sigma, Deisenhofen, DE), RPMI 1640 (Sigma), Dulbecco's Modified Eagle Medium (DMEM; Sigma). ), Minimal essential medium (MEM; Sigma), Iskov modified Dulbecco medium (IMDM; Sigma), CDCHO (Invitrogen, Carlsbad, Calif., USA), CHOS-SFMII (Invitrogen), serum-free CHO medium (Sigma), CD- Examples include PowerCHO2 medium (Lonza) and protein-free CHO medium (Sigma). Each of these media can contain, if desired, hormones and / or other growth factors (eg, insulin, transferrin, epidermal growth factor, insulin-like growth factor), salts (eg, sodium chloride, calcium, magnesium, Phosphate), buffers (eg, HEPES), nucleosides (eg, adenosine, thymidine), glutamine, glucose or equivalent nutrients, antibiotics and / or trace elements, or commercially available Power Feed A ( Lonza) or Cell Boost 6 (HyClone) feeds can be supplemented with various compounds. Where the replicable cell is a recombinant cell that expresses one or more selectable markers, one or more suitable selection agents such as antibiotics may also be added to the medium.

  In addition to containing the elements necessary for transcription and translation of the inserted coding sequence (encoding the polypeptide), the expression constructs disclosed herein provide stability, production, purification, Sequences engineered to optimize yield or activity may also be included.

  In some embodiments, the transformed cells are cultured under effective conditions that allow a high degree of expression of the recombinant polypeptide. In some embodiments, effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH, and oxygen conditions that allow protein production. In one embodiment, effective media can include any media in which cells are cultured to produce the recombinant polypeptides disclosed herein. In some embodiments, the medium typically includes an aqueous solution having an absorbable carbon, nitrogen, and phosphate source, and appropriate salts, minerals, metals, and other nutrients, such as vitamins. In some embodiments, the cells disclosed herein may be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri dishes. In some embodiments, culturing is performed at a temperature, pH, and oxygen content appropriate for the recombinant cell. In some embodiments, the culture conditions are based on the expertise of one skilled in the art.

  In one embodiment, the culture conditions include a dissolved oxygen (DO) content of about 20-80%. In another embodiment, the DO content is about 20-30%. In another embodiment, the DO content is about 30-40%. In another embodiment, the DO content is about 40-50%. In another embodiment, the DO content is about 50-60%. In another embodiment, the DO content is about 60-70%. In another embodiment, the DO content is about 70-80%.

  In one embodiment, the culture conditions include a pH that starts at one temperature and transitions to another temperature during manufacture. In another embodiment, the pH starts at about 7.3 and shifts to about 6.7 during incubation in the bioreactor. In another embodiment, the pH starts at about 7.3, about 7.2, or about 7.1 and is about 6.7, about 6.8, about 6.9, or during incubation in the bioreactor. Move to about 7.0. Each possibility represents an embodiment disclosed herein.

  In some embodiments, depending on the vector and host system used for production, the resulting polypeptide described herein remains in the recombinant cell or is secreted into the medium.

  In one embodiment, the recombinant polypeptide is recovered after a predetermined time in culture.

  One skilled in the art will understand that the phrase “recovering the recombinant polypeptide” can include collecting the entire medium containing the polypeptide and can involve additional steps of separation or purification. .

  In one embodiment, the polypeptides disclosed herein are affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin. Purified using a variety of standard protein purification techniques including, but not limited to, A chromatography, hydroxyapatite chromatography, isoelectric focusing and differential solubilization.

  In one embodiment, each column is run under controlled or uncontrolled temperature.

  In one embodiment, the expressed coding sequence may be engineered to encode a polypeptide disclosed herein and fused to a cleavable moiety to facilitate recovery. In one embodiment, the fusion protein is designed such that the polypeptide can be easily isolated by affinity chromatography, for example by immobilization on a column specific for the cleavable moiety. In one embodiment, the cleavage site is engineered between a polypeptide and a cleavable site, and the cleavable site and polypeptide are chromatographed by treatment with an appropriate enzyme or agent that specifically cleaves the fusion protein at this site. Can be released from the graph column [see, eg, Booth et al. , Immunol. Lett. 19: 65-70 (1988), and Gardella et al. , J .; Biol. Chem. 265: 15854-15859 (1990)].

  In one embodiment, the polypeptides disclosed herein are removed in “substantially pure” form.

  One skilled in the art will appreciate that the phrase “substantially pure” may encompass a purity that allows for the effective use of the protein in the applications described herein. Such forms also include highly glycosylated and highly sialic acid-added forms that are also disclosed herein.

  In one embodiment, the polypeptides disclosed herein are synthesized using an in vitro expression system. In one embodiment, in vitro synthesis methods are known in the art and the components of the system are commercially available.

  In one embodiment, the production of CTP modified GH is performed using recombinant DNA technology.

  In some embodiments, recombinant polypeptides are synthesized and purified; their therapeutic effect can be assayed either in vivo or in vitro. In one embodiment, the binding activity of recombinant GH modified with CTP disclosed herein can be confirmed using various assays.

  In one embodiment, a method of producing a CTP-modified hGH disclosed herein comprises obtaining a clone from the WCB that optimally expresses and secretes the CTP-modified hGH and propagating the clone. In another embodiment, a method for producing CTP-modified hGH comprises obtaining a clone that optimally expresses the CTP-modified hGH from the MCB and propagating the clone. In another embodiment, the method of production comprises a clone that optimally expresses and secretes CTP-modified hGH. In another embodiment, cell clones are grown in solution through a series of subculture steps to the production bioreactor level. In another embodiment, the solution containing the subcultured clone is seeded in a bioreactor. In another embodiment, the bioreactor is a disposable bioreactor. In another embodiment, the bioreactor comprises a stainless steel bioreactor, a rocking motion bioreactor such as the GE Wave system, a perfusion bioreactor, or any other bioreactor system known in the art. In one embodiment, removal of cells from the bioreactor is achieved through the use of a disposable filtration system. If large scale manufacturing is performed, continuous centrifugation can be used prior to use of the filtration system.

  In one embodiment, cell clones are further expanded or expanded by sequentially culturing the cells in larger bioreactors until the desired scale is achieved. In another embodiment, the bioreactor is operated in fed-batch mode. In another embodiment, the bioreactor is operated in batch mode. In another embodiment, the bioreactor is operated in a repeated batch mode. In another embodiment, the bioreactor is operated in a perfusion mode. Each of the above possibilities is another embodiment.

The peak of viable cell density depends on the type of bioreactor used. In one embodiment, the peak of viable cell density of the bioreactor used in the manufacturing methods disclosed herein is about 0.2 × 10 ^{6 to} 1.4 × 10 ⁶ cells / ml. . In another embodiment, the viable cell density peak of the bioreactor used in the manufacturing methods disclosed herein is about 0.05 × 10 ^{6 to} 100 × 10 ⁶ . In another embodiment, the bioreactor live cell density peak is between about 0.05 × 10 ^{6 and} 0.5 × 10 ⁶ . In another embodiment, the bioreactor live cell density peak is about 0.5 × 10 ^{6 to} 5 × 10 ⁶ . In another embodiment, the bioreactor live cell density peak is about 5.0 × 10 ⁶ to 50 × 10 ⁶ . In another embodiment, the bioreactor live cell density peak is about 50 × 10 ^{6 to} 100 × 10 ⁶ . Each possibility represents a separate embodiment disclosed herein.

  The feed scheme in bioactor utilization can vary, for example, from a particular day, whether it is fed once a day or fixed for several days, in addition, the percentage of feed added can be several percent Can vary by up to -50%. Each possibility is an embodiment disclosed herein.

  DMSO may be added to the bioreactor at different concentrations known in the art. In one embodiment, 0.1-3% DMSO is added to the bioreactor during its use. In another embodiment, 0.1-0.5% DMSO is added. In another embodiment, 0.5-1.0% DMSO is added. In another embodiment, 1.0-1.5% DMSO is added. In another embodiment, 1.5-2.0% DMSO is added. In another embodiment, 2.0-2.5% DMSO is added. In another embodiment, 2.5-3.0% DMSO is added.

  In one embodiment, the method for producing CTP-modified hGH includes purifying the clarified collection solution to obtain a purified protein solution. In another embodiment, the purified protein solution produced using the methods presented herein comprises at least 40% CTP modified hGH. In another embodiment, the purified protein solution comprises at least 50% CTP modified hGH. In another embodiment, the purified protein solution comprises at least 60% CTP modified hGH. In another embodiment, the purified protein solution comprises at least 70% CTP modified hGH. In another embodiment, the purified protein solution comprises at least 80% CTP modified hGH. In another embodiment, the purified protein solution comprises at least 90% CTP modified hGH.

  In one embodiment, the clarified collection is held at 2-25 ° C. for up to 24 hours. In another embodiment, the clarified collection is stored at 5 ° C. for up to 1 month.

  In one embodiment, the purified collection obtained in step (e) is bioburden, bacterial endotoxin, specific protein content, residual DNA, virus, virus-like particle, and / or mycoplasma, or these Any combination is tested.

  In one embodiment, the purification of the clarified collection in step (f) is the step of (g) concentrating, diafiltering, and purifying the clarified collection solution, wherein the concentration, diafiltration, and Purification is achieved by a hollow fiber cassette or a tangential flow cassette that sequentially passes the purified collection solution through an anion exchange column and a hydrophobic interaction column, and step (h) following the step Obtaining a collection; (i) inactivating the presence in the clarified collection by incubating in a solution that is toxic to the virus; and (j) decontaminating the clarified collection solution. Concentrating, diafiltering, and purifying the clarified collection solution obtained from (h), wherein the concentration, diafiltration, and purification comprises hydrolyzing the clarified collection solution. A step of sequentially passing through an apatite mixed mode column and a cation exchange column; and (j) following step (i), obtaining the purified collection solution and applying the purified collection solution by nanofiltration Physically removing from the virus; and (k) obtaining the clarified collection solution following step (j) and maximally purified highly glycosylated form of CTP-modified polypeptide Concentrating, diafiltrating, and purifying the clarified collection solution until a clarified collection containing is achieved is achieved by sequentially performing steps comprising: In another embodiment, the methods disclosed herein include a centrifugation step prior to final filtration. One skilled in the art will understand that both centrifugation and / or filtration are methods of purifying the solution.

  In one embodiment, ultrafiltration and diafiltration to concentrate and filter the clarified collection can be performed using a hollow fiber cartridge or equivalent TTF-based UFDF step. The nominal molecular weight cut-off size of the cartridge is 10,000 kDa. In another embodiment, the membrane cartridge may comprise a PES / PS / RC membrane having a 3 kDa to 30 kDa cutoff.

  In another embodiment, the anion exchange column of step (g) is a DEAE-Sepharose Fast Flow column. In another embodiment, the DEAE column purifies CTP-modified hGH disclosed herein in a highly glycosylated form. In one embodiment, the higher the glycosylation, the better the pharmacodynamics of CTP modified hGH. In another embodiment, the anion exchange column may comprise other anion exchange columns known in the art, such as other resins such as Capto DEAE anion exchange column or Eshmuno Q.

  In one embodiment, the hydrophobic column of step (g) is phenyl hydrophobic interaction chromatography (HIC). The number of cycles used for phenyl HIC ranges between about 1-10. In one embodiment, 1 to 3 cycles are performed. In another embodiment, 1 to 5 cycles are performed. In another embodiment, 1 to 6 cycles are performed. In another embodiment, 1 to 7 cycles are performed. In another embodiment, 1 to 8 cycles are performed. In another embodiment, 1 to 9 cycles are performed. In another embodiment, 1 to 10 cycles are performed. In another embodiment, buffers known in the art are used for washing and elution. In one embodiment, the elution buffer comprises ammonium sulfate containing propylene glycol. In one embodiment, the elution buffer comprises ammonium sulfate containing ethylene glycol.

  In one embodiment, the hydroxyapatite mixed mode column comprises a ceramic hydroxyapatite mixed mode column (CHT). The number of cycles used for CHT ranges between about 1-10. In one embodiment, 1 to 3 cycles are performed. In another embodiment, 1 to 5 cycles are performed. In another embodiment, 1 to 6 cycles are performed. In another embodiment, 1 to 7 cycles are performed. In another embodiment, 1 to 8 cycles are performed. In another embodiment, 1 to 9 cycles are performed. In another embodiment, 1 to 10 cycles are performed. Elution from the CHT column can be performed between about 3-10 column volumes (CV). In one embodiment, the elution is performed at about 3 CV. In another embodiment, elution is performed at about 4 CV. In another embodiment, the elution is performed at about 5 CV. In another embodiment, the elution is performed at about 6 CV. In another embodiment, the elution is performed at about 7 CV. In another embodiment, the elution is performed at about 8 CV. In another embodiment, the elution is performed at about 9 CV. In another embodiment, the elution is performed at about 10 CV.

  In one embodiment, viruses that may be present in a clarified collection due to contamination are inactivated in the clarified collection. In another embodiment, the virus is inactivated using a 1% Triton-X100 solution. In another embodiment, the virus is inactivated using a 0.1-2% Triton-X100 solution. In another embodiment, the virus is inactivated using a 0.2% Triton-X100 solution. In another embodiment, the virus is inactivated using a 0.5% Triton-X100 solution. In another embodiment, the virus is inactivated using 1-4% Triton-X100 solution. In another embodiment, the virus is inactivated using a 0.2-0.5% Triton-X100 solution. In another embodiment, the virus is inactivated using a 0.5-1.0% Triton-X100 solution. In another embodiment, the virus is inactivated using a 2% Triton-X100 solution. In another embodiment, the virus is inactivated using a 3% Triton-X100 solution. In another embodiment, the virus is inactivated using a 4% Triton-X100 solution. In another embodiment, the virus is inactivated using 5-10% Triton-X100 solution. In another embodiment, virus inactivation in Triton-X100 solution ranges from about 0.5 to 24 hours. In another embodiment, virus inactivation in Triton-X100 solution ranges from about 0.5 to 1 hour. In another embodiment, virus inactivation in the Triton-X100 solution ranges from about 1-2 hours. In another embodiment, virus inactivation in the Triton-X100 solution ranges from about 2-3 hours. In another embodiment, virus inactivation in the Triton-X100 solution ranges from about 3 to 4 hours. In another embodiment, virus inactivation in Triton-X100 solution ranges from about 4 to 6 hours. In another embodiment, virus inactivation in Triton-X100 solution ranges from about 6-8 hours. In another embodiment, virus inactivation in Triton-X100 solution ranges from about 8 to 10 hours. In another embodiment, virus inactivation in Triton-X100 solution ranges from about 10-12 hours. In another embodiment, virus inactivation in Triton-X100 solution ranges from about 12-24 hours.

  Other concentrates or other solutions that are available in the art and are toxic to these viruses, including but not limited to sodium cholate and Tween 80, may be used in the methods disclosed herein. This will be understood by those skilled in the art. In another embodiment, a mixture of tri-n-butyl phosphate (TNBP) and polysorbate 80 (Tween 80) is used to inactivate the virus in step (h). In another embodiment, the method of virus inactivation comprises lowering the pH. One skilled in the art will appreciate that virus inactivation can include altering the conditions of the solution as is known in the art.

In one embodiment, the virus is physically removed by using nanofiltration. One skilled in the art will appreciate that any filter for virus removal known in the art can be applied to the methods disclosed herein. In another embodiment, nanofiltration is performed using a Planova or Planova type filtration cartridge (1-60 mm ² ). Following such methods is confirmation of viral clearance from the clarified collection using methods known in the art.

  In one embodiment, the cation exchange column of step (i) herein is an SP-Sepharose Fast Flow column. In another embodiment, the cation exchange column comprises CM sepharose Capto S. In another embodiment, the cation exchange column comprises any known in the art for purposes herein.

  In one embodiment, the methods disclosed herein achieve at least 20% recovery of highly glycosylated CTP-modified hGH. In another embodiment, the method comprises at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least of highly glycosylated CTP-modified hGH. A recovery of 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9% is achieved.

  In one embodiment, following purification of the highly glycosylated CTP modified hGH disclosed herein, the method disclosed herein further comprises characterizing the CTP modified polypeptide. . In another embodiment, the purity of CTP modified hGH is determined. In another embodiment, the glycosylation content is determined. In another embodiment, glycosylation site occupancy is determined. In one embodiment, purity, glycosylation content, and glycosylation site occupancy are determined in the produced CTP-modified hGH.

  In another embodiment, cell clones utilized in the methods disclosed herein are stored in a frozen cell bank. In another embodiment, cell clones are stored in a lyophilized cell bank.

  In another embodiment, the cell bank of the methods and compositions disclosed herein is a master cell bank. In another embodiment, the cell bank is a working cell bank. In another embodiment, the cell bank is a Good Manufacturing Practice (GMP) cell bank. In another embodiment, the cell bank is intended for the production of clinical grade material. In another embodiment, the cell bank complies with regulatory practices for human use. In another embodiment, the cell bank is any other type of cell bank known in the art.

  The “Proper Manufacturing Code” is, in another embodiment, defined by US Federal Regulations (21 CFR 210-211). In another embodiment, a “good manufacturing practice” is defined by other standards for the production of clinical grade material or consumption by humans, such as standards from countries other than the United States.

  In another embodiment, the medium used to propagate the cells contains methotrexate (MXT). In another embodiment, the medium is a methotrexate-free medium. In another embodiment, the concentration of MXT present in the medium is about 0.1-2 uM. In another embodiment, the concentration of MXT present in the medium is about 0.1-0.5 uM. In another embodiment, the concentration of MXT present in the medium is about 0.5-1.0 uM. In another embodiment, the concentration of MXT present in the medium is about 1.0-1.5 uM. In another embodiment, the concentration of MXT present in the medium is about 1.5-2.0 uM. It will be understood that the term “medium” can encompass a liquid or gel or powder suitable for the growth or culture of cells containing the CTP-modified hGH disclosed herein. Such media are alternatively referred to as “growth media” or “culture media” and may include, but are not limited to, nutrient media, enriched media, minimal media, confirmation media, transport media, or selective media. In further embodiments, the selective media may be suitable for selecting a particular group of cells during the manufacturing process.

  In one embodiment, the purified protein solution contains at least 70% CTP modified hGH. In another embodiment, the purified protein solution contains at least 80% CTP modified hGH. In another embodiment, the purified solution contains at least 90-95% CTP modified hGH. In another embodiment, the purified solution contains 95.1-99.9% CTP modified hGH. In another embodiment, the purified solution contains 100% CTP modified hGH.

  In one embodiment, the CTP modified hGH disclosed herein is highly glycosylated. When the term “highly glycosylated” relates to the collected CTP-modified hGH polypeptide present in the collection, about 60-80% of the total CTP-modified hGH polypeptide in the purified solution. It will be appreciated by those skilled in the art (see, for example, those measured by reverse phase HPLC in FIG. 11). In another embodiment, a collection comprising highly glycosylated CTP modified hGH has a glycosylation level of at least 50% of the CTP modified hGH polypeptide in the purified solution. In another embodiment, a collection comprising highly glycosylated CTP modified hGH has a glycosylation level of at least 60% of the CTP modified hGH polypeptide in purified solution. In another embodiment, a collection comprising highly glycosylated CTP modified hGH has a glycosylation level of at least 70% of the CTP modified hGH polypeptide. In another embodiment, the highly glycosylated CTP modified hGH has a glycosylation level of at least 80% of the CTP modified hGH polypeptide in the purified solution. In another embodiment, the collection comprises about 81-90% glycosylation levels of total CTP-modified hGH polypeptide in purified solution. In another embodiment, the highly glycosylated CTP modified hGH has a glycosylation level of at least 90% of the CTP modified hGH polypeptide in the purified solution. In another embodiment, the collection comprises about 91-95% glycosylation level of total CTP modified hGH polypeptide in purified solution. In another embodiment, the collection comprises about 95.1-99% glycosylation level of total CTP-modified hGH polypeptide in the purified solution. In another embodiment, the collection comprises 100% glycosylation level of total CTP modified hGH polypeptide in purified solution.

  In another embodiment, a purified CTP modified hGH solution (API (DS)) comprises a solution in which at least 90% of the molecules are highly glycosylated. In another embodiment, the purified CTP-modified hGH solution (API (DS)) comprises a solution in which at least 95% of the molecules are highly glycosylated. In another embodiment, a purified CTP-modified hGH solution (API (DS)) comprises a solution in which 100% of the molecules are highly glycosylated. One skilled in the art will appreciate that purified CTP-modified hGH drug substance (DS) can include CTP-modified hGH with each molecule containing 12-18 O-glycan occupancy.

  One skilled in the art will appreciate that the term “Drug Substance” (DS) can include or be equivalent to an active ingredient (API). In one embodiment, the CTP-modified hGH polypeptide drug substance (DS) set forth in SEQ ID NO: 7 comprises a bulk purified drug. One skilled in the art will also understand that the term “pharmaceutical” (DP) can include a drug that is formulated under sterile conditions into a final container, such as a vial, and finally formulated. In one embodiment, the CTP-modified hGH polypeptide drug substance (DP) set forth in SEQ ID NO: 7 comprises the final formulated CTP-modified hGH.

  Highly glycosylated CTP-modified hGH polypeptides support long-acting hGH (eg, MOD-4023) that aids in reduced dose frequency, eg, once a week injection, in patients with growth hormone secretion deficiency. ) Can have beneficial properties in the method of use. High glycosylation levels contribute to the hydrodynamic volume of CTP modified hGH, eg, CTP modified hGH, which is significantly increased compared to recombinant hGH. This can result in an extended circulation time of CTP modified hGH.

  In one embodiment, the number of O-glycans per CTP is at least 4-6. In another embodiment, the number of O-glycans per CTP is between 4-6. In another embodiment, the number of O-glycans per CTP is at least 4-8. In another embodiment, the number of O-glycans per CTP is between 4-8. In one embodiment, the number of O-glycans per CTP is at least 6-8. In one embodiment, the number of O-glycans per CTP is between 6-8. In another embodiment, the number of O-glycans per CTP is 4, 5, 6, 7, or 8. Each possibility represents another embodiment of CTP O-linked glycosylation.

  In one embodiment, the number of O-glycans per CTP modified hGH polypeptide with one CTP attached is at least 4-6. In another embodiment, the number of O-glycans per CTP-modified hGH polypeptide having one CTP attached is at least 6-8. In another embodiment, the number of O-glycans per CTP modified hGH polypeptide having one CTP attached is at least 4-8. In another embodiment, the number of O-glycans per CTP modified hGH polypeptide having two CTPs attached is at least 8-12. In another embodiment, the number of O-glycans per CTP modified hGH polypeptide having two CTPs attached is at least 12-16. In another embodiment, the number of O-glycans per CTP modified hGH polypeptide having two CTPs attached is at least 8-16. In another embodiment, the number of O-glycans per CTP modified hGH polypeptide having 3 CTPs attached is at least 12-18. In another embodiment, the number of O-glycans per CTP modified hGH polypeptide having 3 CTPs attached is at least 18-24. In another embodiment, the number of O-glycans per CTP modified hGH polypeptide having 3 CTPs attached is at least 12-24. In another embodiment, the number of O-glycans per CTP-modified hGH polypeptide having 4 CTPs attached is at least 16-24. In another embodiment, the number of O-glycans per CTP-modified hGH polypeptide having 4 CTPs attached is at least 24-32. In another embodiment, the number of O-glycans per CTP modified hGH polypeptide having 4 CTPs attached is at least 16-32. In another embodiment, the number of O-glycans per CTP-modified hGH polypeptide having 5 CTPs attached is at least 20-30. In another embodiment, the number of O-glycans per CTP-modified hGH polypeptide having 5 CTPs attached is at least 30-40. In another embodiment, the number of O-glycans per CTP-modified hGH polypeptide having 5 CTPs attached is at least 20-40.

  In another embodiment, each CTP-hGH-CTP-CTP (also known as “MOD-4023”) has at least 12-18 O-glycans. In another embodiment, the number of O-glycans per MOD-4023 is at least 18-24. In another embodiment, the number of O-glycans per MOD-4023 is at least 12-24. In another embodiment, the number of O-glycans per MOD-4023 is at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. Each possibility represents another embodiment of CTP O-linked glycosylation.

  In one embodiment, the O-glycan occupancy per CTP is at least 70%. In another embodiment, the O-glycan occupancy per CTP is at least 80%. In another embodiment, the O-glycan occupancy per CTP is at least 90%. In another embodiment, the O-glycan occupancy per CTP is 100%.

  In one embodiment, the CTP modified hGH is highly sialylated. When the term “highly sialylated” relates to CTP-modified hGH, it can encompass a sialic acid addition level of about 60-80% of the CTP-modified hGH polypeptide in purified solution, Those skilled in the art will appreciate. In another embodiment, a sialic acid addition level of about 60-70% of the total CTP-modified hGH polypeptide in purified solution may be included. In another embodiment, a sialic acid addition level of about 70-80% of the total CTP-modified hGH polypeptide in purified solution may be included. In another embodiment, a sialic acid addition level of about 80-90% of the total CTP-modified hGH polypeptide in the purified solution. In another embodiment, a sialic acid addition level of about 90-95% of the total CTP-modified hGH polypeptide in the purified solution. In another embodiment, a sialic acid addition level of about 95.1-99% of the total CTP-modified hGH polypeptide in the purified solution. In another embodiment, 100% sialic acid addition level of total CTP-modified hGH polypeptide in purified solution. In another embodiment, the O-glycan linkage in CTP modified hGH comprises mono-sialicated core 1.

  In one embodiment, an expression vector comprising a coding portion encoding a CTP modified human GH disclosed herein also comprises a promoter, a coding sequence for a CTP modified polypeptide, and a polyadenylation sequence. In one embodiment, the polyadenylation sequence is a simian virus (SV) 40 polyadenylation sequence.

  In one embodiment, the CTP modified hGH is expressed at a level of at least 600 mg / L. In another embodiment, the CTP modified hGH is expressed at a level of at least 600-700 mg / L. In another embodiment, CTP modified hGH is expressed at a level of at least 701-800 mg / L. In another embodiment, CTP modified hGH is expressed at a level of at least 801-900 mg / L. In another embodiment, CTP modified hGH is expressed at a level of at least 901-1000 mg / L. In another embodiment, CTP modified hGH is expressed at a level of at least 1001-1100 mg / L. In another embodiment, CTP modified hGH is expressed at a level of at least 1101-1200 mg / L. In another embodiment, CTP modified hGH is expressed at a level of at least 1201-1300 mg / L. In another embodiment, CTP modified hGH is expressed at a level of at least 1301-1400 mg / L. In another embodiment, CTP modified hGH is expressed at a level of at least 1401-1500 mg / L. In another embodiment, CTP modified hGH is expressed at a level of at least 1501-1600 mg / L. In another embodiment, CTP modified hGH is expressed at a level of at least 1601-1700 mg / L. In another embodiment, CTP modified hGH is expressed at a level of at least 1701-1800 mg / L. In another embodiment, CTP modified hGH is expressed at a level of at least 1801-1900 mg / L. In another embodiment, CTP modified hGH is expressed at a level of at least 1901-2000 mg / L. In another embodiment, CTP modified hGH is expressed at a level of at least 2001-2100 mg / L. In another embodiment, CTP-modified hGH is expressed at a level of at least 2101-2200 mg / L. In another embodiment, CTP modified hGH is expressed at a level of at least 2201-2300 mg / L. In another embodiment, CTP modified hGH is expressed at a level of at least 2301-2400 mg / L. In another embodiment, CTP modified hGH is expressed at a level of at least 2401-2500 mg / L. In another embodiment, CTP modified hGH is expressed at a level of at least 2501-2600 mg / L. In another embodiment, CTP modified hGH is expressed at a level of at least 2601-2700 mg / L. In another embodiment, CTP modified hGH is expressed at a level of at least 2701-2800 mg / L. In another embodiment, CTP modified hGH is expressed at a level of at least 2801-2900 mg / L. In another embodiment, CTP modified hGH is expressed at a level of at least 2901-3000 mg / L.

  One skilled in the art will appreciate that the term “expression” may include transcription and / or translation of a heterologous nucleic acid sequence in a host cell. The level of expression of the desired product / protein of interest in the host cell depends on the amount of corresponding mRNA or cDNA present in the cell, or the desired sequence encoded as in the examples of the invention. Or the amount of the protein of interest. For example, mRNA transcribed from selected sequences can be detected by Northern blot hybridization, ribonuclease RNA protection, in situ hybridization to intracellular RNA, or by PCR (Sambrook et al., 1989, Ausubel et al.,. 1987 updated). Proteins encoded by selected sequences can be analyzed by FACS analysis following protein immunostaining (Sambrook et al.) By assaying for protein biological activity in a variety of ways, eg, by ELISA, by Western blotting, by radioprecipitation. al., 1989, Ausubel et al., 1987 updated) or by heterogeneous time-resolved fluorescence (HTRF) assay. In one embodiment, quantification of CTP modified hGH includes the use of reverse phase high performance liquid chromatography (RP-HPLC). In another embodiment, RP-HPLC includes a C-18 column. In another embodiment, the RP-HPLC includes a C-8 column. In another embodiment, the method disclosed herein quantifies CTP-modified hGH in the collection using RP-HPLC (see steps 3-8 of the examples). In another embodiment, the methods disclosed herein use RP-HPLC to quantify CTP-modified hGH in the first step of purification (see steps 9-12 of the examples). ).

  In another embodiment, a cell bank or frozen stock disclosed herein exhibits a viability of greater than 90% upon thawing. In another embodiment, the storage spans an indefinite amount of time. Each possibility represents a separate embodiment disclosed herein.

  In another embodiment, the storage lasts 2 weeks. In another embodiment, the storage lasts for 3 weeks. In another embodiment, storage lasts for a month. In another embodiment, storage lasts for two months. In another embodiment, storage lasts for 3 months. In another embodiment, storage lasts for 5 months. In another embodiment, storage lasts for 6 months. In another embodiment, storage lasts for 9 months. In another embodiment, storage lasts for a year.

In another embodiment, a cell bank or frozen stock disclosed herein grows a cell culture in the defined media disclosed herein and the culture in a solution containing glycerol. Cryopreserved by a method comprising freezing and storing the cell clones at less than -20 degrees Celsius. In another embodiment, the temperature is about -70 degrees Celsius. In another embodiment, the temperature is degrees Celsius to about ^- 80 ° ^- 70. In another embodiment, any defined medium disclosed herein may be used in this method. Each defined medium represents a separate embodiment disclosed herein.

  In another embodiment of the methods and compositions disclosed herein, the culture medium is planted from a cell bank. In another embodiment, the culture medium is planted from a frozen stock. In another embodiment, the culture medium is planted from a seed culture. In another embodiment, the culture medium is planted from a cell population. In another embodiment, the culture is planted in the mid-log growth phase. In another embodiment, the culture is planted at approximately mid-log growth phase. In another embodiment, the culture is planted in another growth phase.

  In another embodiment of the methods and compositions disclosed herein, the solution used for freezing contains DMSO in an amount of 2-20%. In another embodiment, the amount is 2%. In another embodiment, the amount is 20%. In another embodiment, the amount is 1%. In another embodiment, the amount is 1.5%. In another embodiment, the amount is 3%. In another embodiment, the amount is 4%. In another embodiment, the amount is 5%. In another embodiment, the amount is 2%. In another embodiment, the amount is 2%. In another embodiment, the amount is 7%. In another embodiment, the amount is 7.5%. In another embodiment, the amount is 9%. In another embodiment, the amount is 10%. In another embodiment, the amount is 12%. In another embodiment, the amount is 14%. In another embodiment, the amount is 16%. In another embodiment, the amount is 18%. In another embodiment, the amount is 22%. In another embodiment, the amount is 25%. In another embodiment, the amount is 30%. In another embodiment, the amount is 35%. In another embodiment, the amount is 40%.

  In another embodiment, the additive is sucrose. In another embodiment, the additive is any other consistent additive known in the art or an additive with antifreeze properties. Each possibility represents a separate embodiment disclosed herein.

  In one embodiment, the frozen solution for the composition used in the method and disclosed herein comprises a culture supernatant and DMSO. In one embodiment, the frozen solution for the composition used in the method and disclosed herein comprises about 46.255% culture supernatant and 7.5% DMSO.

  In one embodiment, the cell culture medium is grown by techniques commonly used in the art. In another embodiment, a constant pH is maintained during cell culture growth. In another embodiment, the pH is maintained at about 7.0. In another embodiment, the pH is about 6. In another embodiment, the pH is about 6.5. In another embodiment, the pH is about 7.5. In another embodiment, the pH is about 8. In another embodiment, the pH is 6.5-7.5. In another embodiment, the pH is 6-8. In another embodiment, the pH is 6-7. In another embodiment, the pH is 7-8. Each possibility represents a separate embodiment disclosed herein.

  In another embodiment, a constant temperature is maintained during cell culture growth. In another embodiment, the temperature is maintained at about 37 ° C. In another embodiment, the temperature is 37 ° C. In another embodiment, the temperature is 25 ° C. In another embodiment, the temperature is 27 ° C. In another embodiment, the temperature is 28 ° C. In another embodiment, the temperature is 30 ° C. In another embodiment, the temperature is 32 ° C. In another embodiment, the temperature is 34 ° C. In another embodiment, the temperature is 35 ° C. In another embodiment, the temperature is 36 ° C. In another embodiment, the temperature is 38 ° C. In another embodiment, the temperature is 39 ° C.

  In another embodiment, a constant dissolved oxygen concentration is maintained during cell culture growth. In another embodiment, the dissolved oxygen concentration is maintained at 20% of saturation. In another embodiment, the concentration is 15% of saturation. In another embodiment, the concentration is 16% of saturation. In another embodiment, the concentration is 18% of saturation. In another embodiment, the concentration is 22% of saturation. In another embodiment, the concentration is 25% of saturation. In another embodiment, the concentration is 30% of saturation. In another embodiment, the concentration is 35% of saturation. In another embodiment, the concentration is 40% of saturation. In another embodiment, the concentration is 45% of saturation. In another embodiment, the concentration is 50% of saturation. In another embodiment, the concentration is 55% of saturation. In another embodiment, the concentration is 60% of saturation. In another embodiment, the concentration is 65% of saturation. In another embodiment, the concentration is 70% of saturation. In another embodiment, the concentration is 75% of saturation. In another embodiment, the concentration is 80% of saturation. In another embodiment, the concentration is 85% of saturation. In another embodiment, the concentration is 90% of saturation. In another embodiment, the concentration is 95% of saturation. In another embodiment, the concentration is 100% of saturation. In another embodiment, the concentration is close to 100% of saturation.

  In another embodiment of the methods and compositions disclosed herein, the culture is grown in a medium having a maximum volume of 2 liters (L) per vessel. In another embodiment, the media has a maximum volume of 200 ml per container. In another embodiment, the media has a maximum volume of 300 ml per container. In another embodiment, the media has a maximum volume of 500 ml per container. In another embodiment, the media has a maximum capacity of 750 ml per container. In another embodiment, the media has a maximum capacity of 1 L per container. In another embodiment, the media has a maximum capacity of 1.5 L per container. In another embodiment, the media has a maximum volume of 2.5 L per container. In another embodiment, the media has a volume of 3 L per container. In another embodiment, the media has a volume of 5 L per container. In another embodiment, the media has a capacity of at least 5 L per container. In another embodiment, the media has a capacity of at least 10 L per container.

  In another embodiment, the media has a minimum volume of 2 L per container. In another embodiment, the media has a minimum volume of 500 ml per container. In another embodiment, the media has a minimum volume of 750 ml per container. In another embodiment, the media has a minimum volume of 1 L per container. In another embodiment, the media has a minimum volume of 1.5 L per container. In another embodiment, the media has a minimum volume of 2.5 L per container. In another embodiment, the media has a minimum volume of 3 L per container. In another embodiment, the media has a minimum volume of 4 L per container. In another embodiment, the media has a minimum volume of 5 L per container. In another embodiment, the media has a minimum volume of 6 L per container. In another embodiment, the media has a minimum volume of 8 L per container. In another embodiment, the media has a minimum volume of 10 L per container.

In another embodiment, the freezing step is performed when the culture has a density of 1 × 10 ⁶ viable cells (VC) / ml. In another embodiment, the biomass is 1.5 × 10 ⁶ VC / ml. In another embodiment, the biomass is 1.5 × 10 ⁶ VC / ml. In another embodiment, the biomass is 2 × 10 ⁶ VC / ml. In another embodiment, the biomass is 3 × 10 ⁶ VC / ml. In another embodiment, the biomass is 4 × 10 ⁶ VC / ml. In another embodiment, the biomass is 5 × 10 ⁶ VC / ml. In another embodiment, the biomass is 7 × 10 ⁶ VC / ml. In another embodiment, the biomass is 9 × 10 ⁶ VC / ml. In another embodiment, the biomass is 10 × 10 ⁶ VC / ml. In another embodiment, the biomass is 12 × 10 ⁶ VC / ml. In another embodiment, the biomass is 15 × 10 ⁶ VC / ml. In another embodiment, the biomass is 20 × 10 ⁷ VC / ml. In another embodiment, the biomass is 25 × 10 ⁶ VC / ml. In another embodiment, the biomass is 30 × 10 ⁷ VC / ml. In another embodiment, the biomass is 33 × 10 ⁶ VC / ml. In another embodiment, the biomass is 40 × 10 ⁶ VC / ml. In another embodiment, the biomass is 50 × 10 ⁶ VC / ml. In another embodiment, the biomass is greater than 50 × 10 ⁶ VC / ml.

  In another embodiment of the methods and compositions disclosed herein, the cell culture fluid is snap frozen in liquid nitrogen followed by storage at the final freezing temperature. In another embodiment, the culture is frozen in a more gradual manner, for example by placing it in a culture vial at the final freezing temperature. In another embodiment, the culture broth is frozen by any other method known in the art for freezing cell culture broth.

  The terms “cell culture” and “tissue culture” are used interchangeably and refer to cells in vitro, in suspension culture in liquid medium, or on the surface of glass, plastic or agar culture medium supplied with liquid medium. It will be understood by those skilled in the art to mean maintenance. In general, “cell culture” requires a buffered medium to maintain a constant and suitable pH. The medium used for cell culture is generally prepared to contain an appropriate supply of the necessary nutrients and is osmotic to the particular cells being maintained while controlling the temperature and gas phase within suitable limits. Can be adapted. Cell culture techniques are known in the art. For example, Morgan et al. 1993 Animal Cell Culture, BIOS Scientific Publishers, Oxford, UK, and Adams, R .; L. P. See 1990 Cell Culture for Biochemistry, Second Edition, Elsevier.

  It will be appreciated by those skilled in the art that the term “passage” can encompass the effect of subculturing a cell population. “Passaging” can include cell culture established by inoculation of sterile medium with a sample from a previous culture, which in one embodiment is fresh sterile medium.

  It will also be understood by those skilled in the art that the term “cell strain” can encompass a population of cells derived from a primary culture using subculture techniques. Thus, the primary culture can be subcultured into two or more new cultures, and the subculture is repeated on a regular basis over several months to maintain the cell line. Subculturing under the methods disclosed herein can be performed using established cell culture techniques.

  In one embodiment, subcultured cell lines and immortalized cell lines can be characterized by the expression of their specific functional markers, such as keratins, hormones and growth factor receptors.

  In some embodiments, culturing may be performed in a serum-free defined medium containing added growth factors. In other embodiments, the media contains serum with or without added growth factors. Such changes can be determined empirically by those skilled in the art to optimize cell growth.

  It will be appreciated by those skilled in the art that the term “cell line” can encompass a population of cells derived from a single explant that is characterized by having infinite growth potential in vitro. Will be understood. As disclosed herein, cell lines can be isolated from primary cultures based on their viability and the ability to continue to grow in culture. Although not all tumor cell populations have the ability to grow indefinitely in vitro, originally cell lines derived from tumor tissue may have been transformed in vivo. In addition, cell lines generally retain their differentiated attributes through multiple rounds of differentiation.

  Suitable cell culture substrates are generally containers that can be sterilized, do not leach toxic factors, and do not distort microscopic images. Thus, plates formed from glass and plastic are suitable substrates for use in the methods disclosed herein. The plastic container is further treated to promote cell adhesion using techniques known in the art (Ramsey et al. 1984 In vitro 20: 802). Suitable tissue culture media generally consist of isotonic, buffered, basal nutrient media that provide a source of energy with the addition of inorganic salts, amino acids, vitamins, and various supplements. Adjuvants include serum (eg, fetal bovine serum), various antibiotics to prevent contamination, or to provide selective conditions, adhesion, and growth factors. Many media formulations are known in the art, such as, but not limited to, Rosewell Park Memory Institute (RPMI) 1640 basal medium (MEM) or Dulbecco's modified Eagle medium (DMEM). Suitable tissue culture conditions are also known in the art. For example, Morgan et al. 1993 Animal Cell Culture, BIOS Scientific Publishers Ltd. , Oxford, UK, and Adams, R .; L. P. See 1990 Cell Culture for Biochemistry, Second Edition, Elsevier. In another embodiment disclosed herein, the method of producing CTP-modified hGH is a serum-free process. In another embodiment disclosed herein, the method of producing CTP-modified hGH is a process that does not include those of animal origin.

In another embodiment of the methods and compositions disclosed herein, the storage temperature of the culture, Celsius ^- is between ⁸⁰ degrees (° C.) -. 20 to. In another embodiment, the temperature is ^- significantly lower than 20 ° C.. In another embodiment, the temperature is ^- 70 not higher than ° C.. In another embodiment, the temperature is ^- 70 ° C.. In another embodiment, the temperature is about ^- is 70 ° C.. In another embodiment, the temperature is ^- 20 ° C.. In another embodiment, the temperature is about ^- is 20 ° C.. In another embodiment, the temperature is ^- 30 ° C.. In another embodiment, the temperature is ^- 40 ° C.. In another embodiment, the temperature is ^- 50 ° C.. In another embodiment, the temperature is ^- 60 ° C.. In another embodiment, the temperature is ^- 80 ° C.. In another embodiment, the temperature ^is - 70 ° C. ^{-. 30 to.} In another embodiment, the temperature ^is - 70 ° C. - ^{40 to.} In another embodiment, the temperature ^is - 70 ° C. - ^{50 to.} In another embodiment, the temperature ^is - 70 ° C. - ^{60 to.} In another embodiment, the temperature ^is - 80 ° C. ^{-. 30 to.} In another embodiment, the temperature ^is - 80 ° C. - ^{40 to.} In another embodiment, the temperature ^is - 80 ° C. - ^{50 to.} In another embodiment, the temperature ^is - 80 ° C. - ^{60 to.} In another embodiment, the temperature ^is - 80 ° C. - ^{70 to.} In another embodiment, the temperature is ^- 70 less than ° C.. In another embodiment, the temperature is ^- 80 less than ° C..

  In another embodiment, for cryopreservation, the cells are slowly frozen in a medium containing a cryoprotectant until they reach a temperature below −70 ° C. and then maintain them at a temperature below −130 ° C. To do so, the vial is transferred to a liquid nitrogen freezer.

  In another embodiment of the methods and compositions disclosed herein, cryopreservation or cryopreservation extends for up to 24 hours. In another embodiment, the cryopreservation or cryopreservation extends for up to 2 days. In another embodiment, the cryopreservation or cryopreservation extends for up to 3 days. In another embodiment, the cryopreservation or cryopreservation extends for up to 4 days. In another embodiment, the cryopreservation or cryopreservation extends for up to a week. In another embodiment, the cryopreservation or cryopreservation extends for up to 2 weeks. In another embodiment, the cryopreservation or cryopreservation extends for up to 3 weeks. In another embodiment, the cryopreservation or cryopreservation extends for up to a month. In another embodiment, the cryopreservation or cryopreservation extends for up to 2 months. In another embodiment, the cryopreservation or cryopreservation extends for up to 3 months. In another embodiment, the cryopreservation or cryopreservation extends for up to 5 months. In another embodiment, the cryopreservation or cryopreservation extends for up to 6 months. In another embodiment, the cryopreservation or cryopreservation extends for up to 9 months. In another embodiment, the cryopreservation or cryopreservation extends for up to one year.

  In another embodiment, the cryopreservation or cryopreservation extends for a minimum of one week. In another embodiment, the cryopreservation or cryopreservation extends for a minimum of 2 weeks. In another embodiment, the cryopreservation or cryopreservation extends for a minimum of 3 weeks. In another embodiment, the cryopreservation or cryopreservation extends for a minimum of one month. In another embodiment, the cryopreservation or cryopreservation extends over a minimum of 2 months. In another embodiment, the cryopreservation or cryopreservation extends over a minimum of 3 months. In another embodiment, the cryopreservation or cryopreservation extends for a minimum of 5 months. In another embodiment, the cryopreservation or cryopreservation extends over a minimum of 6 months. In another embodiment, the cryopreservation or cryopreservation lasts for a minimum of 9 months. In another embodiment, the cryopreservation or cryopreservation extends for a minimum of one year. In another embodiment, the cryopreservation or cryopreservation extends for a minimum of 1.5 years. In another embodiment, the cryopreservation or cryopreservation extends for a minimum of 2 years. In another embodiment, the cryopreservation or cryopreservation extends for a minimum of 3 years. In another embodiment, the cryopreservation or cryopreservation extends for a minimum of 5 years. In another embodiment, the cryopreservation or cryopreservation extends for a minimum of 7 years. In another embodiment, the cryopreservation or cryopreservation extends for a minimum of 10 years. In another embodiment, the cryopreservation or cryopreservation extends for more than 10 years.

  In another embodiment of the methods and compositions disclosed herein, the cells exhibit growth after thawing following prolonged cryopreservation or cryopreservation. In another embodiment, the cells show growth within about 15-22 hours after inoculating cells from a cell bank or seed culture in fresh media. In another embodiment, the cells exhibit growth within about 12-20 hours after inoculating cells from a cell bank or seed culture in fresh media. In one embodiment, cell lines must be maintained in exponential growth phase (via periodic subculture) to ensure viability, genetic stability, and phenotypic stability.

  One skilled in the art will understand that the term “long term” of cryopreservation or cryopreservation can encompass one month. In another embodiment, the period is 2 months. In another embodiment, the period is 3 months. In another embodiment, the period is 5 months. In another embodiment, the period is 6 months. In another embodiment, the period is 9 months. In another embodiment, the period is one year. In another embodiment, the period is 1.5 years. In another embodiment, the period is 2 years. In another embodiment, the period is 2-7 years. In another embodiment, the period is at least 7 years. In another embodiment, the period is at least 10 years.

  In another embodiment, the cells of the methods and compositions disclosed herein retain a viability of greater than 90% after thawing following cryopreservation. In another embodiment, the survival rate upon thawing following a cryopreservation period is close to 100%. In another embodiment, the survival rate upon thawing is close to 90%. In another embodiment, the survival rate upon thawing is at least 90%. In another embodiment, the survival rate upon thawing is greater than 80%.

  In another embodiment, the vaccine-administered cell bank, frozen stock, or batch disclosed herein is grown in defined cell culture media. Such media are known in the art and include Dulbecco's Modified Eagle Medium (DMEM) (ATCC® No. 30-2002), Iskov's Modified Dulbecco Medium (IMDM) (ATCC® No. 30-2005), Hybri-Care medium (ATCC (registered trademark) No. 46-X), McCoy 5A and RPMI-1640 (ATCC (registered trademark) No. 30-2007), Ham nutrition mixture (ATCC (registered trademark)) CCL-61 ™), PowerCHO ™ known composition serum-free CHO medium (Lonza Cat. No. 12-771Q), or any other medium known in the art, including but not limited to . In another embodiment, these media may be supplemented with antibiotics or animal serum as determined empirically by one skilled in the art.

  In one embodiment, a bioreactor and method are disclosed herein that allow for culturing mammalian cells in large quantities. Furthermore, in yet another embodiment, the bioreactor and method allows for culturing mammalian cells under optimal conditions even when grown in large quantities, and thus is a process independent of bioreactor size. Allows performance and production quality. Changing the scale and bioreactor system may change the duration of incubation in the bioreactor, for example, the duration may be between 8-9 days, or between 15-16 days. In another embodiment, the duration of incubation in the bioreactor is about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days. Days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days or more. In another embodiment, if a perfusion bioreactor is used, the incubation period can range from 7 to 120 days. Each possibility listed above is an embodiment disclosed herein.

  In another embodiment, different types of process parameters such as pH, dissolved oxygen partial pressure (DOT), and temperature are maintained while maintaining a well-mixed cell suspension in the bioreactor and mixing the nutrient feeds. Disclosed herein is a large-scale bioreactor that enables the cultivation of mammalian cells in the environment. In another embodiment, the bioreactor is a disposable bioreactor.

  The methods of production disclosed herein are the technical underlying the method of producing CTP-modified hGH polypeptides by providing bioreactors, bioreactor systems, and in particular methods for culturing eukaryotic cells of mammalian cells. Solve a problem.

  In one embodiment, the bioreactor has a capacity of at least 250 liters (L). In another embodiment, the bioreactor has a capacity of at least 500L. In another embodiment, the capacity is at least 1000L, at least 2000L, at least 5,000L, at least 10,000L, at least 12,000L, or at least 15,000L.

  In another embodiment, the cells are subcultured in increasingly larger volume bioreactors (see the examples herein).

  Composition

  In some embodiments, a CTP-modified human growth hormone (hGH) polypeptide disclosed herein and produced using the methods disclosed herein is used to relate to growth and weight. Symptoms such as growth deficiency disorder, AIDS exhaustion, aging, immune dysfunction in HIV-infected subjects, degradation disease, postoperative recovery, congestive cardiomyopathy, liver transplantation, liver regeneration after hepatectomy, chronic renal failure, kidney Chronic inflammatory or nutritional disorders such as osteogenesis dystrophy, osteoporosis, achondroplasia / hypocartilage, skeletal dysplasia, Crohn's disease, short bowel syndrome, juvenile rheumatoid arthritis, cystic fibrosis, Subjects with male infertility, X-linked hypophosphatemic rickets, Down's syndrome, spinal rupture, Noonan syndrome, obesity, muscle weakness, and fibromyalgia can be treated. In one embodiment, the polypeptides disclosed herein can be provided to the individual itself. In one embodiment, a polypeptide disclosed herein can be provided to an individual as part of a pharmaceutical composition where it is mixed with a pharmaceutically acceptable carrier.

  One of ordinary skill in the art would find a formulation of one or more active ingredients as described herein where the term “pharmaceutical composition” is combined with other chemical ingredients such as physiologically suitable carriers and excipients. It will be understood that it can be included. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

  The modified peptides disclosed herein may be administered orally or parenterally to mammals (eg, cows, horses, pigs, sheep, humans), mixed with carriers, diluents, etc. known per se It can be formulated into suitable pharmaceutical preparations such as capsules and injections.

  One skilled in the art will appreciate that the term “active ingredient” can encompass a polypeptide sequence of interest that can explain a biological effect.

  In some embodiments, any of the compositions disclosed herein will comprise at least two CTP sequences bound to the protein of interest in any form. In one embodiment, a combination formulation is disclosed herein. The person skilled in the art will understand that the term “combination formulation” means that the combination partner as defined above is used independently, or by the use of different fixed combinations in significant amounts of the combination partner, ie simultaneously. It will be understood that “part kits” can be included in the sense that they can be dosed simultaneously, separately or sequentially. In some embodiments, the parts of the parts kit can then be administered offset over time, for example, at the same time, or at different times for any part of the parts kit, and at equal or different time intervals. In some embodiments, a ratio of the total amount of combination partners can be administered to the combination formulation. In one embodiment, the combination formulation is a single patient that may differ in the need for, for example, the patient subpopulation to be treated, or the need for a particular disease, disease severity, age, gender, or weight To meet this need, which can be easily made by one skilled in the art.

  Those skilled in the art will recognize that the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” that can be used interchangeably do not cause significant irritation to the organism, and that the biological activity of the administered compound and It will be understood that carriers or diluents that do not inhibit the properties may be included. Adjuvant is included in these phrases. In one embodiment, one of the ingredients contained in the pharmaceutically acceptable carrier is, for example, polyethylene glycol (PEG), a biocompatible polymer having a wide range of solubility in both organic and water soluble media. (Mutter et al. (1979)).

  One of ordinary skill in the art will understand that the term “excipient” can include an inert substance added to the pharmaceutical composition to further facilitate administration of the active ingredient. In one embodiment, excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

  Drug formulation and administration techniques are described in “Remington's Pharmaceutical Sciences” Mack Publishing Co. , Easton, PA, latest edition, which is incorporated herein by reference.

  In one embodiment, suitable routes of administration include, for example, oral, rectal, transmucosal, nasal, intestinal, or intramuscular, subcutaneous, and intramedullary injection, and intrathecal, intraventricular, intravenous, Direct parenteral delivery includes intraperitoneal, intranasal, or intraocular injection.

  In one embodiment, the formulation is administered in a local rather than systemic manner, for example, via injection of the formulation directly into a specific area of the patient's body.

  In another embodiment, the CTP-modified hGH polypeptide disclosed herein is administered at a dose of 1-90 micrograms in a 0.1-5 ml solution. In another embodiment, the CTP modified hGH polypeptide is administered at a dose of 1-50 micrograms in a 0.1-5 ml solution. In another embodiment, the CTP modified hGH polypeptide is administered at a dose of 1-25 micrograms in a 0.1-5 ml solution. In another embodiment, the CTP modified hGH polypeptide is administered at a dose of 50-90 micrograms in a 0.1-5 ml solution. In another embodiment, the CTP modified hGH polypeptide is administered at a dose of 10-50 micrograms in a 0.1-5 ml solution.

  In another embodiment, the CTP-modified hGH polypeptide is in a 0.1-5 ml solution by intramuscular (IM), subcutaneous (SC), or intravenous (IV) injection once a week. It is administered at a dose of 1-90 micrograms. In another embodiment, the CTP-modified hGH polypeptide is in 0.1-5 ml solution by intramuscular (IM) injection, subcutaneous (SC) injection, or intravenous (IV) injection twice a week. It is administered at a dose of 1-90 micrograms. In another embodiment, the CTP-modified hGH polypeptide is in 0.1-5 ml solution by intramuscular (IM), subcutaneous (SC), or intravenous (IV) injection three times per week. It is administered at a dose of 1-90 micrograms. In another embodiment, the CTP-modified hGH polypeptide is administered in 0.1-5 ml solution by intramuscular (IM), subcutaneous (SC), or intravenous (IV) injection once every two weeks. In doses of 1 to 90 micrograms. In another embodiment, the CTP-modified hGH polypeptide is administered in 0.1-5 ml solution by intramuscular (IM), subcutaneous (SC), or intravenous (IV) injection once every 17 days. In doses of 1 to 90 micrograms. In another embodiment, the CTP-modified hGH polypeptide is in 0.1-5 ml solution by one intramuscular (IM), subcutaneous (SC), or intravenous (IV) injection every 19 days. In doses of 1 to 90 micrograms.

  Various embodiments of dosage ranges are contemplated. In one embodiment, the dosage of a polypeptide disclosed herein is in the range of 0.05-80 mg / day. In another embodiment, the dosage is in the range of 0.05-50 mg / day. In another embodiment, the dosage is in the range of 0.1-20 mg / day. In another embodiment, the dosage is in the range of 0.1-10 mg / day. In another embodiment, the dosage is in the range of 0.1-5 mg / day. In another embodiment, the dosage is in the range of 0.5-5 mg / day. In another embodiment, the dosage is in the range of 0.5-50 mg / day. In another embodiment, the dosage is in the range of 5-80 mg / day. In another embodiment, the dosage is in the range of 35-65 mg / day. In another embodiment, the dosage is in the range of 35-65 mg / day. In another embodiment, the dosage is in the range of 20-60 mg / day. In another embodiment, the dosage is in the range of 40-60 mg / day. In another embodiment, the dosage is in the range of 45-60 mg / day. In another embodiment, the dosage is in the range of 40-60 mg / day. In another embodiment, the dosage is in the range of 60-120 mg / day. In another embodiment, the dosage is in the range of 120-240 mg / day. In another embodiment, the dosage is in the range of 40-60 mg / day. In another embodiment, the dosage is in the range of 240-400 mg / day. In another embodiment, the dosage is in the range of 45-60 mg / day. In another embodiment, the dosage is in the range of 15-25 mg / day. In another embodiment, the dosage is in the range of 5-10 mg / day. In another embodiment, the dosage is in the range of 55-65 mg / day.

  In one embodiment, the dosage is 20 mg / day. In another embodiment, the dosage is 30 mg / day. In another embodiment, the dosage is 40 mg / day. In another embodiment, the dosage is 50 mg / day. In another embodiment, the dosage is 60 mg / day. In another embodiment, the dosage is 70 mg / day. In another embodiment, the dosage is 80 mg / day. In another embodiment, the dosage is 90 mg / day. In another embodiment, the dosage is 100 mg / day.

  In one embodiment, the dosage of CTP-modified hGH polypeptide disclosed herein is 0.005 to 100 mg / week. In another embodiment, the dosage is in the range of 0.005-5 mg / week. In another embodiment, the dosage is in the range of 0.01-50 mg / week. In another embodiment, the dosage is in the range of 0.1-20 mg / week. In another embodiment, the dosage is in the range of 0.1-10 mg / week. In another embodiment, the dosage is in the range of 0.01-5 mg / week. In another embodiment, the dosage is in the range of 0.001-0.01 mg / week. In another embodiment, the dosage is in the range of 0.001-0.1 mg / week. In another embodiment, the dosage is in the range of 0.1-5 mg / week. In another embodiment, the dosage is in the range of 0.5-50 mg / week. In another embodiment, the dosage is in the range of 0.2-15 mg / week. In another embodiment, the dosage is in the range of 0.8-65 mg / week. In another embodiment, the dosage is in the range of 1-50 mg / week. In another embodiment, the dosage is in the range of 5-10 mg / week. In another embodiment, the dosage is in the range of 8-15 mg / week. In another embodiment, the dosage is in the range of 10-20 mg / week. In another embodiment, the dosage is in the range of 20-40 mg / week. In another embodiment, the dosage is in the range of 60-120 mg / week. In another embodiment, the dosage is in the range of 12-40 mg / week. In another embodiment, the dosage is in the range of 40-60 mg / week. In another embodiment, the dosage is in the range of 50-100 mg / week. In another embodiment, the dosage is in the range of 1-60 mg / week. In another embodiment, the dosage is in the range of 15-25 mg / week. In another embodiment, the dosage is in the range of 5-10 mg / week. In another embodiment, the dosage is in the range of 55-65 mg / week. In another embodiment, the dosage is in the range of 1-5 mg / week.

  In another embodiment, the dosage of CTP-modified hGH polypeptide given to a subject is relative to a reference subject from the same subject population (eg, child, elderly, male, female, GH secretion failure, specific nationality, etc.). 50% of the standard dose given. In another embodiment, the dosage is 30% of the dosage given to subjects from a particular subject population. In another embodiment, the dosage is 45% of the dosage given to subjects from a particular subject population. In another embodiment, the dosage is 100% of the dosage given to subjects from a particular subject population.

  In another embodiment, the dosage is 1-5 mg / week. In another embodiment, the dosage is 2 mg / week. In another embodiment, the dosage is 4 mg / week. In another embodiment, the dosage is 1.2 mg / week. In another embodiment, the dosage is 1.8 mg / week. In another embodiment, the dosage is approximately the dosage described herein.

  In another embodiment, the dosage is 1-5 mg / dose. In another embodiment, the dosage is 2 mg / dose. In another embodiment, the dosage is 4 mg / dose. In another embodiment, the dosage is 1.2 mg / dose. In another embodiment, the dosage is 1.8 mg / dose. In one embodiment, the composition is administered once a week. In another embodiment, the composition is administered once every two weeks. In another embodiment, the composition is administered once a month. In another embodiment, the composition is administered once daily.

  In another embodiment, the CTP-modified hGH polypeptide disclosed herein is formulated in an intranasal dosage form. In another embodiment, the CTP modified hGH polypeptide is formulated into an injectable dosage form. In another embodiment, the CTP modified hGH polypeptide is administered to the subject at a dose ranging from 0.0001 mg to 0.6 mg. In another embodiment, the CTP modified hGH polypeptide is administered to the subject at a dose ranging from 0.001 mg to 0.005 mg. In another embodiment, the CTP modified hGH polypeptide is administered to the subject at a dose ranging from 0.005 mg to 0.01 mg. In another embodiment, the CTP modified hGH polypeptide is administered to the subject at a dose ranging from 0.01 mg to 0.3 mg. In another embodiment, the CTP modified hGH polypeptide is administered to the subject at a dose ranging from 0.2 mg to 0.6 mg.

  In another embodiment, the CTP-modified hGH polypeptide disclosed herein is administered to a subject at a dose in the range of 1-100 micrograms. In another embodiment, the CTP modified hGH polypeptide is administered to the subject at a dose in the range of 10-80 micrograms. In another embodiment, the CTP modified hGH polypeptide is administered to the subject at a dose in the range of 20-60 micrograms. In another embodiment, the CTP modified hGH polypeptide is administered to the subject at a dose in the range of 10-50 micrograms. In another embodiment, the CTP modified hGH polypeptide is administered to the subject at a dose in the range of 40-80 micrograms. In another embodiment, the CTP-modified hGH polypeptide is administered to the subject at a dose ranging from 10-30 micrograms. In another embodiment, the CTP modified hGH polypeptide is administered to the subject at a dose in the range of 30-60 micrograms.

  In another embodiment, CTP modified GH is administered to a subject at a dose ranging from 0.2 mg to 2 mg. In another embodiment, the CTP modified hGH polypeptide is administered to the subject at a dose ranging from 2 mg to 6 mg. In another embodiment, the CTP modified hGH polypeptide is administered to the subject at a dose ranging from 4 mg to 10 mg. In another embodiment, the CTP modified hGH polypeptide is administered to the subject at doses ranging from 5 mg and 15 mg.

  In another embodiment, the CTP-modified hGH polypeptides disclosed herein are injected intramuscularly (intramuscular injection). In another embodiment, the CTP-modified hGH polypeptide is injected under the skin (subcutaneous injection). In another embodiment, the CTP modified hGH polypeptide is injected intramuscularly. In another embodiment, the CTP modified hGH polypeptide is injected under the skin.

  In another embodiment, the methods disclosed herein include improving compliance in the use of GH therapy, and providing a CTP-modified hGH polypeptide disclosed herein to a subject in need thereof And thereby improve compliance in the use of GH therapy.

  In another embodiment, protein agents with a molecular weight of less than 50,000 daltons, such as CTP-modified hGH polypeptides disclosed herein, are generally short-lived species in vivo and have a short circulating half-life of several hours . In another embodiment, the subcutaneous route of administration generally provides a slower release into the circulation. In another embodiment, the CTP-modified polypeptides disclosed herein extend the half-life of a protein drug with a molecular weight of less than 50,000 daltons, such as GH.

  In another embodiment, the immunogenicity of CTP-modified hGH is equal to isolated GH. In another embodiment, the immunogenicity of CTP-modified hGH is equivalent to isolated GH. In another embodiment, modifying GH with a CTP peptide as described herein reduces the immunogenicity of GH. In another embodiment, the CTP modified hGH is as active as isolated GH. In another embodiment, the CTP modified hGH polypeptide is more active than isolated GH. In another embodiment, CTP modified hGH maximizes the ability of GH to protect against degradation while minimizing the reduction in biological activity.

  In another embodiment, the methods disclosed herein include improving the compliance of a subject suffering from a chronic disease in need of GH treatment. In another embodiment, the methods disclosed herein allow for a reduction in GH dosing frequency by modifying GH with CTP as described above. In another embodiment, the term compliance includes adherence. In another embodiment, the methods disclosed herein include improving compliance of patients in need of GH treatment by reducing the frequency of administration of GH. In another embodiment, the reduced frequency of administration of GH is achieved by CTP modification that makes CTP modified GH more stable. In another embodiment, a reduction in the frequency of administration of GH is achieved as a result of increasing T1 / 2 of GH. In another embodiment, a reduction in the frequency of administration of GH is achieved as a result of an increase in GH gliaance time. In another embodiment, a reduction in the frequency of administration of GH is achieved as a result of an increase in the AUC area of GH.

  In another embodiment, the CTP-modified hGH polypeptide disclosed herein is administered to a subject once a day. In another embodiment, the CTP-modified hGH polypeptide is administered to the subject once every two days. In another embodiment, the CTP-modified hGH polypeptide is administered to the subject once every 3 days. In another embodiment, the CTP-modified hGH polypeptide is administered to the subject once every 4 days. In another embodiment, the CTP-modified hGH polypeptide is administered to the subject once every 5 days. In another embodiment, the CTP-modified hGH polypeptide is administered to the subject once every 6 days. In another embodiment, the CTP-modified hGH polypeptide is administered to the subject once a week. In another embodiment, the CTP-modified hGH polypeptide is administered to the subject once every 7-14 days. In another embodiment, the CTP-modified hGH polypeptide is administered to the subject once every 10-20 days. In another embodiment, the CTP-modified hGH polypeptide is administered to the subject once every 5-15 days. In another embodiment, the CTP-modified hGH polypeptide is administered to the subject once every 15-30 days.

  In another embodiment, the dosage is in the range of 50-500 mg / day. In another embodiment, the dosage is in the range of 50-150 mg / day. In another embodiment, the dosage is in the range of 100-200 mg / day. In another embodiment, the dosage is in the range of 150-250 mg / day. In another embodiment, the dosage is in the range of 200-300 mg / day. In another embodiment, the dosage is in the range of 250-400 mg / day. In another embodiment, the dosage is in the range of 300-500 mg / day. In another embodiment, the dosage is in the range of 350-500 mg / day.

  In one embodiment, the dosage is 20 mg / day. In one embodiment, the dosage is 30 mg / day. In one embodiment, the dosage is 40 mg / day. In one embodiment, the dosage is 50 mg / day. In one embodiment, the dosage is 0.01 mg / day. In another embodiment, the dosage is 0.1 mg / day. In another embodiment, the dosage is 1 mg / day. In another embodiment, the dosage is 0.530 mg / day. In another embodiment, the dosage is 0.05 mg / day. In another embodiment, the dosage is 50 mg / day. In another embodiment, the dosage is 10 mg / day. In another embodiment, the dosage is 20-70 mg / day. In another embodiment, the dosage is 5 mg / day.

  In another embodiment, the dosage is 1-90 mg / day. In another embodiment, the dosage is 1-90 mg / 2 days. In another embodiment, the dosage is 1-90 mg / 3 days. In another embodiment, the dosage is 1-90 mg / 4 days. In another embodiment, the dosage is 1-90 mg / 5 days. In another embodiment, the dosage is 1-90 mg / 6 days. In another embodiment, the dosage is 1-90 mg / week. In another embodiment, the dosage is 1-90 mg / 9 days. In another embodiment, the dosage is 1-90 mg / 11 days. In another embodiment, the dosage is 1-90 mg / 14 days.

  In another embodiment, the CTP-modified hGH dosage is 10-50 mg / day. In another embodiment, the dosage is 10-50 mg / 2 days. In another embodiment, the dosage is 10-50 mg / 3 days. In another embodiment, the dosage is 10-50 mg / 4 days. In another embodiment, the dosage is 10-50 micrograms / 5 days. In another embodiment, the dosage is 10-50 mg / 6 days. In another embodiment, the dosage is 10-50 mg / week. In another embodiment, the dosage is 10-50 mg / 9 days. In another embodiment, the dosage is 10-50 mg / 11 days. In another embodiment, the dosage is 10-50 mg / 14 days.

  In one embodiment, oral administration includes unit dosage forms including tablets, capsules, sweetened tablets, chewable tablets, suspensions, emulsions and the like. Such unit dosage forms comprise a safe and effective amount of the desired compound (s), each in one embodiment from about 0.7 or 3.5 mg to about 280 mg / 70 kg, or in other embodiments. About 0.5 or 10 mg to about 210 mg / 70 kg. Pharmaceutically acceptable carriers suitable for the preparation of unit dosage forms for oral administration are known in the art. In some embodiments, the tablets are typically conventional pharmaceutically compatible adjuvants such as inert diluents such as calcium carbonate, sodium carbonate, mannitol, lactose, and cellulose; starch, gelatin, and Binders such as sucrose; disintegrants such as starch, alginic acid, and croscarmellose; lubricants such as magnesium stearate, stearic acid, and talc. In one embodiment, a lubricant such as silicon dioxide can be used to improve the flow characteristics of the powder mixture. In one embodiment, colorants such as FD & C dyes may be added for appearance. Sweeteners and flavoring agents such as aspartame, saccharin, menthol, peppermint, and fruit flavors are useful adjuvants for chewing agents. Capsules typically contain one or more of the above solid diluents. In some embodiments, the choice of carrier component depends on auxiliary considerations such as taste, cost, and shelf life that are not important for the purposes disclosed herein and can be readily made by one skilled in the art. .

  In one embodiment, the oral dosage form includes a predetermined release profile. In one embodiment, the oral dosage forms disclosed herein comprise sustained release tablets, capsules, sweetened tablets, or chewing agents. In one embodiment, the oral dosage forms disclosed herein comprise sustained release tablets, capsules, sweetened tablets, or chewing agents. In one embodiment, the oral dosage forms disclosed herein comprise immediate release tablets, capsules, sweetened tablets, or chewing agents. In one embodiment, the oral dosage form is formulated according to the desired release profile of the pharmaceutically active ingredient, as is known to those skilled in the art.

  In some embodiments, oral compositions include liquid solutions, emulsions, suspensions, and the like. In some embodiments, pharmaceutically acceptable carriers suitable for the preparation of such compositions are known in the art. In some embodiments, the liquid oral composition comprises from about 0.012% to about 0.933%, or in other embodiments, from about 0.033% to 0.7% of the desired compound (s). including.

  In some embodiments, a composition for use in the methods disclosed herein comprises a solution or emulsion, which in some embodiments is disclosed herein in a safe and effective amount. Aqueous solution or emulsion comprising a compound to be administered and optionally other compounds intended for local intranasal administration. In some embodiments, the composition comprises from about 0.01% to about 10.0% w / v, more preferably from about 0.1% to about 2.0, of the subject compound, Used for systemic delivery by intranasal route.

  In another embodiment, the pharmaceutical composition is administered by intravenous, intraarterial, or intramuscular injection of a liquid formulation. In some embodiments, liquid formulations include solutions, dispersions, emulsions, oils, and the like. In one embodiment, the pharmaceutical composition is administered intravenously and is thus formulated into a form suitable for intravenous administration. In another embodiment, the pharmaceutical composition is administered intraarterially and is thus formulated into a form suitable for intraarterial administration. In another embodiment, the pharmaceutical composition is administered intramuscularly and is thus formulated into a form suitable for intramuscular administration.

  In another embodiment, the pharmaceutical composition is administered topically to the body surface and is thus formulated into a form suitable for topical administration. Suitable topical formulations include gels, ointments, creams, lotions, drops, and the like. For topical administration, the compounds disclosed herein were prepared and adapted as solutions, suspensions, or emulsions in physiologically acceptable diluents with or without a pharmaceutical carrier. Combined with additional appropriate therapeutic agent (s).

  In one embodiment, the pharmaceutical compositions disclosed herein can be prepared by processes known in the art, such as conventional mixing, dissolving, granulating, dragee making, wet grinding, emulsifying, encapsulating, Manufactured by encapsulation or lyophilization process.

  In one embodiment, a pharmaceutical composition for use in accordance with the disclosure herein includes one or more physiology comprising excipients and adjuvants that facilitate processing of the pharmaceutically usable active ingredient into a formulation. Formulated in a conventional manner using a commercially acceptable carrier. In one embodiment, the formulation depends on the selected route of administration.

  In one embodiment, the injection disclosed herein is formulated in an aqueous solution. In one embodiment, the injections disclosed herein are formulated in a physiologically compatible buffer, such as Hanks's solution, Ringer's solution, or physiological saline buffer. In some embodiments, for transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art.

  In one embodiment, the formulations described herein are formulated for parenteral administration, eg, by bolus injection or continuous administration. In some embodiments, formulations for injection are presented in unit dosage form, eg, in ampoules or in multiple dose containers, with optionally added preservatives. In some embodiments, the composition is a suspension, solution, or emulsion in an oily or aqueous vehicle and contains a formulation such as a suspending, stabilizing, and / or dispersing agent.

  The composition may also, in some embodiments, be a preservative such as benzalkonium chloride and thimerosal; a chelating agent such as sodium edetate and others; a buffering agent such as phosphate, citrate, acetate; Isotonic agents such as sodium chloride, potassium chloride, glycerin and mannitol; antioxidants such as ascorbic acid, acetylcysteine and sodium disulfite; viscosity modifiers such as polymers containing cellulose and its derivatives; and polyvinyl alcohol, and as required Accordingly, acids and bases are included to adjust the pH of these aqueous compositions. The composition also includes a local anesthetic or other active agent in some embodiments. The composition can be used as a spray, spray, drip, etc.

  In some embodiments, a pharmaceutical composition for parenteral administration comprises an aqueous solution of the activated formulation in water form. In addition, in some embodiments, suspensions of the active ingredients are prepared as appropriate oily or aqueous injection suspensions. Suitable lipophilic solvents or vehicles include, in some embodiments, fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes. Aqueous injection suspensions contain, in some embodiments, substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. In another embodiment, the suspension also contains suitable stabilizers or agents that increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

  In another embodiment, the active compound may be delivered into vesicles, specifically liposomes (Langer, Science 249: 1527-1533 (1990), Treat et al., In Liposomes in the Therapy of Infectious). See Disease and Cancer, Lopez-Berstein and Fiddler (eds.), Liss, New York, pp. 353-365 (1989), Lopez-Berstein, ibid., Pp. 317-327; ).

  In another embodiment, the pharmaceutical composition delivered to the controlled release system is formulated for intravenous infusion, implantable osmotic pump, transdermal patch, liposome, or other mode of administration. In one embodiment, a pump is used (Langer, supra, Sefton, CRC Crit. Ref. Biomed. Eng. 14: 201 (1987), Buchwald et al., Surgery 88: 507 (1980), Saudek et al. N. Engl. J. Med. 321: 574 (1989)). In another embodiment, a polymeric material may be used. In yet another embodiment, the controlled release system may be placed in proximity to the therapeutic target, i.e., in the brain, thereby requiring only a small amount of systemic administration (e.g., Goodson, In Medical Applications of Controlled). Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems are described in the review by Langer (Science 249: 1527-1533 (1990)).

  In some embodiments, the active ingredient is in powder form prior to use for composition with a suitable vehicle, eg, a sterile pyrogen-free aqueous solution. The composition is in some embodiments formulated for nebulization or inhalation administration. In another embodiment, the composition is contained in a container provided with a spraying means.

  In one embodiment, the formulations disclosed herein are formulated into rectal compositions, eg, suppositories or retention enemas, using conventional suppository bases such as cocoa butter or other glycerides. .

  In some embodiments, a pharmaceutical composition suitable for use in the context disclosed herein comprises a composition containing the active ingredient in an amount effective to achieve the intended purpose. Including. In some embodiments, a therapeutically effective amount refers to that amount of active ingredient effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.

  In one embodiment, the determination of a therapeutically effective amount is well within the skill of the artisan.

  The composition also includes preservatives such as benzalkonium chloride and thimerosal; chelating agents such as sodium edetate and others; buffering agents such as phosphate, citrate, acetate; sodium chloride, potassium chloride, glycerin , Isotonic agents such as mannitol; antioxidants such as ascorbic acid, acetylcysteine, sodium disulfite; viscosity modifiers such as polymers containing cellulose and its derivatives; and polyvinyl alcohol, and if necessary, aqueous compositions thereof Acid and base for adjusting the pH of the solution. The composition also includes local anesthetics or other active substances. The composition can be used as a spray, spray, drip, etc.

  Some examples of substances that can act as pharmaceutically acceptable carriers or components thereof include sugars such as lactose, glucose, sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose , And methylcellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants such as stearic acid, magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and theobroma oil; polyols For example, propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as Tween ™ brand emulsifiers; Junzai, such as sodium lauryl sulfate; coloring agents; flavoring agents; tableting, stabilizers; antioxidants; preservatives; pyrogen-free water, isotonic saline; and phosphate buffer is rises. The selection of a pharmaceutically acceptable carrier to be used in conjunction with the compound is basically determined by the method by which the compound is administered. When the compound of interest is injected, in one embodiment, the pharmaceutically acceptable carrier is sterile saline containing a blood-compatible suspending agent, the pH of which is about 7.4. Adjusted.

  In addition, the composition comprises a binder (eg acacia, corn starch, gelatin, carbomer, ethylcellulose, guar gum, hydroxypropylcellulose, hydroxypropylmethylcellulose, povidone), a disintegrant (eg cornstarch, potato starch, alginic acid, silicon dioxide, croscarme). Sodium, crospovidone, guar gum, sodium starch glycolate), buffers of various pH and ionic strength (eg Tris-HCI, acetate, phosphate), additives, eg to prevent absorption on the surface Albumin or gelatin, detergents (eg Tween 20, Tween 80, Pluronic F68, bile salts), protease inhibitors, surfactants (eg sodium lauryl sulfate), permeation enhancers, solubilization (Eg glycerol, preethyleneglycerol) antioxidants (eg ascorbic acid, sodium metabisulfite, butylhydroxyanisole), stabilizers (eg hydroxypropylcellulose, hydroxypropylmethylcellulose), thickeners (eg carbomer, colloidal silicon dioxide) , Ethylcellulose, guar gum), sweeteners (eg, aspartame, citric acid), preservatives (eg, thimerosal, benzyl alcohol, parabens), lubricants (eg, stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow aids Agents (eg colloidal silicon dioxide), plasticizers (eg diethyl phthalate, triethyl citrate), emulsifiers (eg carbomer, hydroxypropylcellulose, lauric) Sodium sulfate), polymer coatings (e.g., poloxamers or poloxamines), further comprising coating or film forming agents (e.g. ethyl cellulose, acrylates, polymethacrylates), and / or adjuvant.

  Typical components of carriers for syrups, elixirs, emulsifiers, and suspensions include ethanol, glycerol, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol, and water. For suspensions, typical suspending agents include methylcellulose, sodium carboxymethylcellulose, cellulose (eg Avicel ™, RC-591), tragacanth, and sodium alginate, and typical wetting agents include Examples include lecithin and polyethylene oxide sorbitan (eg, polysorbate 80). Typical preservatives include methyl paraben and sodium benzoate. In another embodiment, the oral liquid composition also contains one or more ingredients such as the sweetening, flavoring, and coloring agents disclosed above.

  The composition may also be in or on a granular formulation of a polymeric compound such as polylactic acid, porglycolic acid, hydrogel, or in liposomes, microemulsions, micelles, monolayers or multilamellar vesicles, blood cell shadows or spheroplasts Including the incorporation of active materials. Such compositions will affect the physiological state, solubility, stability, rate of in vivo release, and rate of in vivo clearance.

  In another embodiment, the composition is bound to a particulate composition coated with a polymer (eg, poloxamer or poloxamine) and a tissue specific receptor, ligand, or antibody to an antigen or of a tissue specific receptor. Includes compounds bound to a ligand.

  In some embodiments, the compound is modified with a covalent bond of polyethylene glycol, a copolymer of polyethylene glycol, and a water-soluble polymer such as polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, or polyproline. In another embodiment, the modified compound exhibits a substantially longer half-life in blood after intravenous injection than does the corresponding unmodified compound. In one embodiment, the modification also increases the solubility of the compound in aqueous solution, removes aggregation, improves the physical and chemical stability of the compound, and significantly reduces the immunogenicity and reactivity of the compound. . In another embodiment, the desired in vivo biological activity is achieved by administration of such a macromolecular compound and abducts less frequently or at a lower dose than the unmodified compound.

  In some embodiments, the preparation of an effective amount or dose can be predicted initially by in vitro assays. In one embodiment, the dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.

  In one embodiment, the toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, cell culture, or laboratory animals. In one embodiment, these in vitro and cell culture assays and data obtained from animal studies can be used to formulate a range of doses for use in humans. In one embodiment, the dosage varies depending on the dosage form used and the route of administration utilized. In one embodiment, the exact formulation, route of administration, and dosage can be selected by the individual physician in view of the patient's physical condition. [See, eg, Fingl, et al. (1975) “The Pharmaceutical Basis of Therapeutics”, Ch. 1 p. See 1].

  In one embodiment, depending on the severity and responsiveness of the condition being treated, administration may be single or multiple, which may range from days to weeks or result in recovery of this condition. With treatment spanning the period until reduction is achieved.

  In one embodiment, the amount of composition administered will, of course, depend on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the willingness to prescribe, and the like.

  In one embodiment, a composition comprising a formulation disclosed herein formulated in a compatible pharmaceutical carrier is also prepared, placed in a suitable container, and labeled for treatment of an indication. Is done.

  In another embodiment, the CTP modified hGH polypeptide is administered via systemic administration. In another embodiment, the CTP modified hGH described herein is administered by intravenous, intramuscular, or subcutaneous injection. In another embodiment, the CTP-modified hGH polypeptide is a nonionic surfactant (ie, surfactants), various sugars, organic polyols, and / or human serum albumin, etc. A lyophilized (ie, freeze-dried) formulation in combination with complex organic excipients and stabilizers. In another embodiment, the pharmaceutical composition comprises lyophilized GH modified with the described CTP in sterile water for injection. In another embodiment, the pharmaceutical composition comprises a lyophilized CTP modified hGH as described herein in sterile PBS for injection. In another embodiment, the pharmaceutical composition comprises a lyophilized CTP-modified hGH described herein in sterile 0.9% NaCl for injection.

  In another embodiment, the pharmaceutical composition comprises a CTP modified GH as described herein and a complex carrier such as human serum albumin, polyol, sugar, and an anionic surfactant. See, for example, WO 89/10756 (containing Hara et al.-polyol and p-hydroxybenzoate). In another embodiment, the pharmaceutical composition comprises a CTP modified hGH disclosed herein, lactobionic acid, and an acetate / glycine buffer. In another embodiment, the pharmaceutical composition comprises a lyophilized GH modified with CTP as described herein, glycine or human serum albumin (HSA), a buffer (eg, acetic acid), and an isotonic agent. (For example, NaCl). In another embodiment, the pharmaceutical composition comprises lyophilized GH modified with CTP as described herein, a phosphate buffer, glycine, and HSA.

  In another embodiment, a pharmaceutical composition comprising a CTP-modified GH disclosed herein is stable when placed in a buffer solution having a pH between about 4 and 7.2. In another embodiment, a pharmaceutical composition comprising a CTP-modified GH disclosed herein is stabilized with an amino acid, and sometimes a salt (if the amino acid does not contain a loaded side chain) as a stabilizer. To do.

  In another embodiment, a pharmaceutical composition comprising a CTP-modified GH disclosed herein comprises a liquid composition comprising a stabilizer that is between about 0.3% and 5% by weight amino acids. It is.

  In another embodiment, a pharmaceutical composition comprising a CTP modified GH disclosed herein provides dosing accuracy and product safety. In another embodiment, a pharmaceutical composition comprising a CTP modified GH disclosed herein provides a biologically active stable liquid formulation for use in injectable applications . In one embodiment, the compositions disclosed herein comprise a non-viscous liquid formulation. In another embodiment, the pharmaceutical composition comprises non-lyophilized GH modified with CTP as described herein.

  In another embodiment, a pharmaceutical composition comprising a CTP-modified GH disclosed herein is a liquid that permits long-term storage in a liquid state and facilitates storage and transport prior to administration. A formulation is provided.

  In another embodiment, a pharmaceutical composition comprising a CTP modified GH disclosed herein comprises a solid lipid as a matrix material. In another embodiment, an injectable pharmaceutical composition comprising a CTP modified GH described herein comprises a solid lipid as a matrix material. In another embodiment, the production of lipid microparticles by spray coagulation is described in Speiser (Speiser and al., Pharm. Res. 8 (1991) 47-54) followed by lipid nanopellets for oral administration (Speiser). EP0167825 (1990)). In another embodiment, the body is resistant to the lipids used (eg, glycerides consisting of fatty acids present in parenteral nutrition emulsions).

  In another embodiment, a pharmaceutical composition comprising a CTP-modified GH disclosed herein is in the form of a liposome (J. Diederichs and al., Pharm./nd. 56 (1994). 267-275).

  In another embodiment, a pharmaceutical composition comprising GH modified with CTP disclosed herein comprises polymeric microparticles. In another embodiment, an injectable pharmaceutical composition comprising GH modified with CTP disclosed herein comprises polymeric microparticles. In another embodiment, a pharmaceutical composition comprising GH modified with CTP disclosed herein comprises nanoparticles. In another embodiment, a pharmaceutical composition comprising GH modified with CTP disclosed herein comprises a liposome. In another embodiment, a pharmaceutical composition comprising GH modified with CTP disclosed herein comprises a fat emulsion. In another embodiment, a pharmaceutical composition comprising GH modified with CTP disclosed herein comprises microspheres. In another embodiment, a pharmaceutical composition comprising GH modified with CTP disclosed herein comprises lipid nanoparticles. In another embodiment, a pharmaceutical composition comprising GH modified with CTP disclosed herein comprises lipid nanoparticles comprising amphiphilic lipids. In another embodiment, a pharmaceutical composition comprising GH modified with CTP disclosed herein comprises lipid nanoparticles comprising a drug, a lipid matrix, and a surfactant. In another embodiment, the lipid matrix has a monoglyceride content of at least 50% w / w.

  In one embodiment, the compositions disclosed herein are presented in a pack or dispenser device such as an FDA approved kit containing one or more unit dosage forms containing the active ingredients. In one embodiment, the pack includes a plastic foil, such as, for example, a metal or blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser is contained in a notice associated with a container in a form prescribed by a government agency that regulates the manufacture, use, or sale of the medicament, which notice of the composition or human or animal administration. Represents approval by a form agency. Such notification is, in one embodiment, a label approved by the US Food and Drug Administration for prescription drugs or charges for approved products.

  In one embodiment, the CTP-modified GH disclosed herein is provided to an individual with an additional activator to achieve an improved therapeutic effect compared to treatment with each agent alone. It will be understood that this can be done. In another embodiment, means (eg, administration and selection of complementary agents) are taken to minimize side effects associated with combination therapy.

  In another embodiment, disclosed herein is a kit comprising a composition, cell, plasmid, etc. disclosed herein, an applicator, and an instructional material that describes use of the method disclosed herein. Is done. Although model kits are described below, other useful kit contents will be apparent to those of skill in the art in light of this disclosure. Each of these kits represents a separate embodiment disclosed herein.

  Additional objects, advantages, and novel features disclosed herein will become apparent to those skilled in the art upon examination of the following examples, which are not intended to be limiting. In addition, each of the various embodiments and aspects disclosed herein and depicted above and as claimed in the following claims section finds experimental support in the following examples.

  Example 1 Generation of hGH constructs

  Expression of MOD-4023 (CTP-hGH-CTP-CTP)

  Cell transfection (nucleofection): Stable expression was achieved by integration of the MOD-4023 gene into the target cell chromosome: the gene was first introduced into the cell, then into the cell nucleus, and finally into the chromosomal DNA. It was. Following gene transfer, the cells were cultured in a medium containing a selectable marker such as dihydrofolate reductase (DHFR). In brief:

  The mouse dihydrofolate reductase (dhfr) gene from pSV2-dhfr was subcloned into the neomycin site of the pCI neoplasmid. To create a modified expression vector pCI-dhfr (FIG. 2). Thus, the MOD-4023 expression vector contains the cytomegalovirus (CMV) IE (early) enhancer / promoter and the dhfr gene for cell selection. The MOD-4023 gene consists of one copy of the coding sequence for the C-terminal peptide (CTP) preceding the 5 ′ end of the hGH gene and two copies of the CTP coding sequence immediately following the 3 ′ end of the hGH gene. including. The MOD-4023 gene was subcloned into the open reading frame of pCI-dhfr (FIG. 2). Thus, the MOD-4023 expression or set consists of the CMV promoter, the CTP-hGH-CTP-CTP gene sequence, and the SV40 polyadenylation sequence.

  Clone selection—Limited dilution selection of single cells by plating in 96 well plates may be applied to single cell cultures and repeated several times to obtain 100% clonal purity. The most productive clones based on growth curves, clonal characterization, specific productivity and protein profile were propagated in 1 L shake flasks prior to bioreactor planting. In brief:

  The MOD-4023 protein producing cell line was produced by recombinant DNA technology. Medium free of animal elements was used throughout the induction of the master and working cell bank (MCB; WCB). Transfection of dhfr-negative CD DG44 cells (CHO cells) adapted for protein-free medium and suspension growth was performed using FuGENE-6 (Roche Applied Science). Stable clones were isolated by limiting dilution steps during cell culture. The most productive clones were amplified with increasing concentrations of methotrexate (MTX). Based on the clonal population doubling level (PDL), MOD-4023 productivity (picogram per cell per day, PCD), and the highest cell density obtained in the selected medium, the most productive clones were identified. Separately, it is used to prepare the R & D bank, followed by the production of qualified master cell banks (MCB) and working cell banks (WCB).

  Upstream process

  The upstream process of MOD-4023 consists of two types of medium mix, growth medium (medium 1) and production medium (medium 2). Medium 1 contained methotrexate (MTX) and medium 2 was identical to medium 1 except for MTX. Medium 1 is used for cell culture propagation (see steps 1-4 in FIG. 3) before seeding in a 200 liter bioreactor, and medium 2 is the N-2 step (2 before the final product). Used for a 200 liter bioreactor (steps 5-6 in FIG. 3) and a production bioreactor 1000 liters (step 7 in FIG. 3).

  Manufacturing process: MOD-4023

  Clone # 28 was produced on a 1000 liter (L) scale in serum-free medium. The CHO cell line culture was grown in several steps from a single vial of the Working Cell Bank (WCB) to the final product culture volume. Production cell culture supernatants were tested for bioburden, bacterial endotoxin, productivity, and foreign viruses. This process was performed using 50 liter and 200 liter seeded bioreactors and a 1000 L bioreactor for growth. All product contact surfaces are disposable, while non-disposable product contact equipment is dedicated to the product. The parts of these instruments were cleaned and disinfected between batches. For growth, cultures were grown in 50L and 200L seeded bioreactors prior to planting in 1000L bioreactors. Final growth and fed-batch bioreactor production was performed in a 1000 L disposable bioreactor. Cell removal was achieved using a disposable filtration system (Millipore depth filter).

One or two vials of WCB were thawed at 37 ° C. The cells were centrifuged and the cell pellet was resuspended to the target concentration in a 10 mL fresh medium low volume shake flask and incubated with 37 ± 0.5 ° C., 5 ± 1% CO ₂ . After two passages, the cell culture with higher viability was further expanded and the other cell culture was discarded. Four subculture steps in increasing volumes of shake flasks were performed using predefined step parameters such as seeding concentration and working volume. The volume followed two seeding bioreactor steps for a typical 1000 L bioreactor run.

  50 liter and 200 liter seeding bioreactors were used as seeding bioreactors. Prior to planting, the 200 L bioreactor was filled with approximately 50 L PowerCHO 2CD medium 2 (w / o MTX). Process control was set to the following parameters: pH 7, temperature 37 ° C., DO 50%. These parameters apply to media preparation and seed train culture processes.

  When the process parameters are controlled within a predetermined range, inoculum transfer is started. During cell growth in 50L and 200L seeded bioreactors, no feed additions to the process were applied. The expected culture time in the seeded bioreactor was 3-4 days before the cells were transferred to the 1000 L production bioreactor. Samples were taken daily for in-process control (IPC).

  Cell propagation in a 1000L bioreactor for production (step 7 in FIG. 3)

Cultures are incubated in the bioreactor for 11 days (depending on cell viability) at 37 ° C., 20% dissolved oxygen (DO), and pH 7.2. On the third day, the pH is shifted to 6.9 until collection. On day 4, the feed (Power Feed A without lipid) is added. The feed volume is equal to 33% of the final bioreactor volume. On day 6, DMSO is added to the bioreactor. A glucose feed was added to the culture to maintain the desired concentration, and a 1M sodium bicarbonate bolus was added to maintain the desired culture concentration (HCO ₃ ). Collection was performed using predefined criteria. Cell cultures were sampled daily for the first 4 days for cell number, viability, and metabolic analysis. From day 5 onwards, cells were sampled twice a day for cell number, viability and metabolic analysis, and after day 9 for specific productivity by reverse phase HPLC. The productivity of MOD-4023 is at least 500 gr / L and the highly glycosylated form consists of at least 70% of the total hGH-CTP protein in the collection.

  While the examples presented herein show manufacturing using fed-batch mode, those skilled in the art will generally be able to develop a perfusion mode using a similar purification scheme. Alternatively, one skilled in the art would be able to develop a perfusion mode where the incubation period can range from 7 to 120 days.

  Cell collection and storage (step 8 in FIG. 3)

  Collection was performed using a disposable filtration process train. To clean the collection, depth filtration and 0.2 μm filtration were performed. Following purification, 0.45 / 0.2 μm filtration was performed. The depth filter was washed away with water and residual moisture was blown out of the system with air. The filtration process was processed at a pump speed of ≦ 15 L / min and maximum preset pressure. The filter was later washed with Tris-HCl buffer and blown with pressurized air to increase product recovery.

  Purified collections were collected from bioburden, bacterial endotoxin, specific protein content for RP-HPLC, SDS-PAGE, Western blot, HPC Elisa assay, residual DNA, in vitro virus assay, virus-like particles, S + L-, And tested by mycoplasma.

  Purification process

  The purification scheme is described in FIG.

  Ultrafiltration and diafiltration 1-UFDF1 (step 8)

  The clarified collection was concentrated and diafiltered using a hollow fiber cartridge or equivalent TFF-based UFDF step. The nominal molecular weight cut-off size of the cartridge was 10,000 kDa. Concentrated and diafiltered collections were tested for specific protein content by RP-HPLC and HCP Elisa.

  Virus inactivation by incubation with 1% Triton-X100 (step 9)

  The material was filtered through a Millipore 1.55 m2 filter followed by a sterile Sartopore 2 XLG 10 "filter into a sterile mixing bag (depth filtration step). The Tris / 10% Triton solution was then added to the final filtration volume and Triton. The concentration was 1% (w / w) After incubation, the product solution was filtered through a 0.2 μm filter unit before loading on the DEAE column.

  DEAE-Sepharose Fast Flow Chromatography (Step 10)

  A DEAE column packed with DEAE Sepharose resin was used for this step. The column was packed to a default bed height. Due to the interference caused by Triton in the assay, the specific protein in the load was determined prior to the addition of Triton. The DEAE column was equilibrated and loaded with the virus inactivation pool and then washed. A second wash is performed and the material is eluted with 20 mM Tris-HCl / 150 mM NaCl at pH 8.2 and then at ambient temperature (18-26 ° C.) for the next day of treatment or 2 for longer periods. Stored at ~ 8 ° C. All chromatographic steps were performed in downflow mode. Alternatively, the step may be performed in upflow mode. The eluate was tested for specific proteins by RP-HPLC (target: main peak eluate as a single peak), HCP Elisa, SDS-PAGE, Western blot, and residual DNA.

  Phenyl hydrophobic interaction chromatography (HIC) column chromatography (step 11)

  HIC phenyl resin was used for this step. The column was packed to a default bed height. HIC chromatography was performed in 1-5 cycles depending on the amount of product. Alternatively, up to 10 cycles may be run. The HIC load was adjusted by adjusting the DEAE eluate with ammonium sulfate. The column was parallel and loaded with a conditioned, 0.2 μm filtered DEAE solution and then washed with 10 mM sodium phosphate / 600 mM ammonium sulfate containing propylene glycol pH 7.3. The material was eluted with reduced concentrations of ammonium sulfate and increased concentrations of propylene glycol at pH 7.3 and then stored at 2-8 ° C. until further processing.

  HIC eluate ultrafiltration and diafiltration (step 12)

The HIC eluate was concentrated and diafiltered into 10 mM sodium phosphate buffer at pH 6.8 to reduce the volume and prepare the material for the CHT column step. The cartridge was equilibrated with 10 mM sodium phosphate at pH 6.8. The HIC eluate was concentrated and then diafiltered against sodium phosphate buffer to achieve a pH of 6.8 ± 0.1 and a conductivity within 10% of the diafiltration buffer conductivity. If the pH and conductivity are determined to be within range, the system is drained and filtered into a sterile bag at 0.45 / 0.2 μm. The final volume of concentrated and diafiltered HIC eluate was stored overnight at ambient room temperature (18-26 ° C.). The retentate was tested bioburden, bacterial endotoxin, and for a particular protein by A _280.

  Ceramic hydroxyapatite (CHT) mixed mode chromatography (step 13)

  A CHT column packed with hydroxyapatite resin was used for this step. The column was packed to a default bed height. The column was equilibrated with sodium phosphate pH 6.8, loaded with concentrated and diafiltered HIC eluate, and washed with 4 column volumes (CV) of pH 6.8 sodium phosphate. Flow-through and wash materials were collected and held overnight at ambient temperature (18-26 ° C.) until further processing.

  SP-Sepharose chromatography (Step 14)

  A column packed with SP Sepharose resin was used for this step. The column was packed to a default bed height. The SP load was adjusted by adjusting the CHT flow-through fraction to pH 5.0-6.0 with citric acid. Following pH adjustment, the solution was loaded onto the column followed by a washing step. The flow-through was collected for further processing. The pH was adjusted to pH 6.0-6.5 with 0.1 M sodium hydroxide and the material was filtered through a 0.45 / 0.2 μm filter. The material was stored overnight at ambient temperature (18-26 ° C.) or up to 24 hours at 2-8 ° C. All chromatographic steps were performed in downflow mode.

  Virus inactivation by nanofiltration (step 15)

  Virus filtration was performed using an Asahi Planova 20N virus filter. A Sartopore 2 filter with a 0.45 / 0.2 μm or 0.1 μm membrane was used as a prefilter for nanofiltration. The virus filter is pre-equilibrated and the final formulation made with WFI is injected. The conditioned SP-flow-through was passed continuously through the filter train and collected in a sterile bioprocess bag. The filter train was washed away with formulation buffer to maximize product recovery. Filter integrity tests were performed before and after use according to the manufacturer's recommended procedures. Also, following the manufacturer's recommended procedures, post-use testing includes gold particle testing.

  Drug substance UFDF3 and filtration and storage (step 16)

  The virus filtrate was concentrated to the target final DS concentration (which can vary from 5 to 100 mg / ml) in preparation for bulk filtration and filling. A disposable or reusable cassette with a 3-30 KDa cut-off was used for this step. The product was concentrated in the first step to 5-25 mg / ml and diafiltered into 10 mM citrate, pH 6.4, 147 mM NaCl (≧ 7 DF volume). Alternatively, the product is concentrated in the first step to 5-25 mg / ml and a preservative to support repeated administration by the device, specifically 10 mM histidine citrate with 0.3% m-cresol Diafiltered into buffer and P-188 was added at 0.2% to reduce aggregates and particles not visible to the naked eye. The final product concentration was adjusted and filtered with a Millipak 100 filter. The filtered product solution was aliquoted and frozen at a temperature of -70 ± 5 ° C.

  MOD-4023 Pharmaceutical manufacturing

  The pharmaceutical (DP) formulation process begins with thawing of MOD-4023 DS. Drugs are achieved by dilution to the required concentration of drug substance (DS) using formulation buffer, sterile filtration, and filling into standard 2R vials or other direct packaging such as cartridges or pre-filled syringes. It was done. A description of a particular process is depicted in FIG.

  Characteristic evaluation of MOD-4023

  The MOD-4023 content in the high glycosylation collection was determined by a specific RP-HPLC method. Total protein in the collection was determined by Bradford analysis. The percentage of specific MOD-4023 protein in the collection produced by clone # 28 was greater than 70% compared to the total protein in the collection. This unique high level of specific protein is also present in the high and low glycosylated MOD-4023 bands compared to the intensity of additional host cell protein bands and USDF samples in the collection analyzed by SDS-PAGE Coomassie staining. It is also recognized by the strong strength (Fig. 6). In addition, the MOD-4023 manufacturing upstream process was developed to allow a high percentage of high glycosylated MOD-4023 protein compared to the low glycosylated form. The high glycosylation form is the target MOD-4023 form because it results in a longer extension of the GH half-life.

  O-glycan content

  Sugar profiles were performed by releasing MOD-4023 glycans, followed by labeling the glycans with 2-aminobenzamide (2AB), washed and analyzed by NP-HPLC. In brief:

  To calculate the number of moles of O-glycan per mole of MOD-4023 protein, an O-glycan content assay was performed. The terminal galactose unit of O-glycan was enzymatically cleaved from the protein with β-galactosidase. These free galactose units were separated on a CarboPac PA20 column and detected by amperometric electrochemical. Galactose (Gal) was quantified using an external standard with a galactose reference standard. The content of galactose can be directly related to the O-glycan structure, Gal-GalNAc. Sialic acid (SA) content is measured in the drug substance (DS) and should not differ from the SA content of the final drug product (DP). Both DS and DP can be used to indicate the level of CTP-modified hGH SA and O-glycan occupancy. Analysis of five different MOD-4023 drug substance and drug batches depicted in FIG. 7 shows robust batch-to-batch consistency. This unexpected robust glycosylation content is marked, indicating that the number of O-glycans per CTP in CTP-hGH-CTP-CTP has improved over that known in the art. The CTP-hGH-CTP-CTP produced herein has 4-6 O-glycans per CTP compared to levels known in the art where hCG has only 4 O-glycans. Had.

  Intact molecular weight analysis of MOD-4023 sample

  In order to obtain information on the number of O-linked glycosylation sites, molecular weight analysis of four different MOD-4023 DS batches was performed (see detailed results in Example 3). Intact MOD-4023 samples, as well as desialylated MOD-4023 samples using neuraminidase, and deglycosylated samples using glycosidases were analyzed by online LC / ES-MS. Data obtained from intact mass measurements of desialylated samples show that proteins modified at 12-18 glycosylation sites in GalNac-Gal, where the protein is modified at 15 glycosylation sites, are the most. Showed strong. This result, which shows a high percentage of serum occupancy, was unexpected compared to levels known in the art (4 compared to up to 6 in the CTP modified hGH produced herein). Only serum was glycosylated).

  O-linked glycosylation site occupation of MOD-4023 protein sample

  In order to obtain information on the number of O-linked glycosylation sites per MOD-4023 molecule, O-glycosylation site occupancy of four different MOD-4023 DS batches was performed with M-scan. The MOD-4023 sample was desialylated using neuraminidase followed by trypsin digestion of the reduced / carboxymethylated MOD-4023 sample. Finally, online LC / ES-MS was performed on the treated samples to interpret the MS data using the designated software. Evaluation of the data obtained from analysis of the trypsin digestion mixture led to a signal that allowed 100% of the protein sequence to be mapped. O-glycosylation occurs at both the N-terminal and C-terminal CTP regions. Occupied sites were identified as serine residues following proline, as well as two of the four serines in the contiguous region. A total of up to 18 serine residues can serve as adhesion sites for O-glycans. As presented in FIG. 8, no significant differences between batches were detected.

  The O-linked glycosylation site occupancy of the MOD-4023 protein sample MOD-4023 (SEQ ID NO: 7) contains one CTP at the N-terminus and two CTPs aligned at the C-terminus. O-linked glycosylation analysis showed that each CTP contained 4-6 O-linked glycans, all of which bound to serine (S) residues. Overall, there were 12-18 O-linked glycans per MOD-4023, the most abundant form containing 15 O-glycans per MOD-4023. (See the results and conclusions of Examples 2 and 3 below) No sugar chain was present on the GH sequence.

  Using the amino acid sequence of SEQ ID NO: 7 as a guide, the result of site occupancy analysis was that O-glycosylation was performed on serine residues at positions 10, 13, 15, 21 of the CTP unit on each CTP. This corresponds to positions 229, 232, 234, 240 on the second CTP sequence and positions 257, 260, 262, and 268 of the third CTP sequence. Two additions of O-glycosylation occupancy occurring on serine residues Ser1-Ser4 of N-terminal CTP (corresponding to positions 220-223 of the second CTP sequence and positions 248-251 of the third CTP sequence) The site of was identified. However, it was not possible to determine via mass spectrometry which two of the four serine residues were modified. Details of O-glycan occupancy and structure are provided in Example 2 below.

  O-glycan structure

  The major peak of O-glycan corresponds to core 1 added with monosialic acid (Neu5Acα2-3Galβ1-3GalNAc). Further, a small amount of neutral core 1 (Galβ1-3GalNAc), monosialic acid-added core 1 (Neu5Acα2-6 (Galβ1-3) GalNAc) and disialic acid-added core 1 (Neu5Acα2-3Galβ1-3 (Neu5Acα2-6) GalNAc) Admitted. The main sialic acid in the sample was Neu5Ac (NANA). The structure and sialic acid type of O-linked glycans are presented in FIGS. 15A and 15B, respectively.

  MOD-4023 purity

  RP-HPLC separates molecules by their polarity. A mobile phase gradient from a higher polarity solvent to a lower polarity solvent was used to elute molecules with strong polarity before molecules with lower polarity. RP-HPLC separates MOD-4023 drug substance into major and minor peaks observed at 21.0-25.0 minutes and represents product-related mutations (Figure 9). Related forms are separated from native proteins using UV detection at 220 nm. The relative peak area (area%) and main peak of the relevant form can be calculated by integrating the corresponding peak areas. The main peak of MOD-4023 drug substance and drug consists of a peak region of over 97%, which represents a highly purified product and an effective purification process (see FIG. 21).

  Size exclusion HPLC is a chromatographic technique that separates molecules by size. Within the selected preparative range, larger molecules elute before smaller molecules. The separation mechanism is non-absorbing and the molecules are eluted under isocratic conditions. SEC allows monomers to be separated from high molecular weight forms (eg dimers and polymers) of target molecules. The SEC method was developed to analyze the content of MOD-4023 dimers and polymers in drug substances and pharmaceuticals (FIG. 10). MOD-4023 monomer consists of more than 98% peak area, which represents a highly purified product and an effective purification process (see FIG. 22).

  RP-HPLC content method

  This method is used to determine the MOD-4023 content of MOD-4023 intermediate samples by reverse phase chromatography and to determine the percentage of non-glycosylated MOD-4023 in the intermediate samples. Reverse phase HPLC separates molecules by their polarity. While relatively non-polar molecules such as MOD-4023 are coupled to the column material while being loaded, polar molecules elute without interacting with the column.

  Linked molecules are eluted with the aid of a gradient from polar to less polar solvent. The most polar molecules eluted first, followed by the less polar molecules. Detection was performed via absorption at 214 nm.

  In the initial stage of purification (UFDF1 sample), two peaks were observed. Peak 1 is a high sugar chain addition type of MOD-4023, and peak 2 is a low sugar chain addition type (FIG. 11). The relative peak area (area%) of the two peaks can be calculated by integrating the corresponding peak areas. The relative peak area I is 75%. In the first step of purification, the column DEAE Sepharose separates between these peaks and the elution fraction contains only MOD-4023 glycosylated form (100%) (FIG. 12).

  MOD-4023 effect

Activation of human growth hormone by MOD-4023 was characterized using Baf cells that stably express the hGH receptor. Cells experienced starvation in GH-free medium, followed by a wash step, resuspended in assay buffer, and transferred to a 96-well plate (100 μl / well). MOD-4023 was added at increasing concentrations during a 30 minute cell incubation period at 37 ° C. and incubated overnight at 37 ° C. with 5% CO ₂ . The assay was terminated by adding 30 μl of CellTiter 96 Aqueous One Solution Reagent (MTS) to the cell wells. Three hours after MTS addition, the optical density (OD) of the culture wells was measured at 492 nm. The absorption at 492 nm is directly proportional to the number of viable cells in culture. MOD-4023 showed specific binding to Baf-hGH cells in a typical dose-dependent curve (FIGS. 13 and 23).

  In addition, bioidentity studies were performed on hypophysectomized rats where MOD-4023 activity in all batches was greater than 2 US Pharmacopeia (USP) somatropin units.

  Virus clearance

  The ability of the manufacturing process to address and mitigate contamination of the final drug by endogenous and foreign viruses has been the subject of preliminary research. GLP-enabled studies follow a applicable guidance for clinical trial drugs, and scaled-down segments of the manufacturing process to quantify the ability to inactivate or clear the virus population to which these steps were added. This has been done using three model viruses added. The amount of virus expressed as a log10 adjusted titer, the log10 clearance coefficient is determined by simply subtracting the output value from the input value. As a log10 value, the clearance factor is additive to derive the overall clearance factor for all evaluation steps. The clearance coefficients for the individual steps are calculated in FIG. Virus safety evaluation studies were performed using Ebelson murine leukemia virus (A-MuLV), mouse microvirus (MVM), reovirus type 3 (Reo-3), and pseudorabies virus (PrV). The theoretical viral load per dose was calculated with a maximum dose of 15 mg / dose. A-MuLV is considered to be a model virus that represents the possibility of the presence of CHO retroviruses, and measures taken to inactivate and remove contaminating A-MulV virus are at least 10 and 22.74. A clearance factor was achieved, indicating that the overall MOD-4023 process has excellent virus removal capabilities.

  impurities

  Results of analysis of the extent of impurities present in purified MOD-4023 protein are presented in FIG. Host cell proteins (HCP) below 100 ng / mg (ppm) were discovered. The presence of DNA was 10 pg / mg (ppb) or less. Residual methotrexate (MTX) levels were less than 50 ng / mL. The residual propylene glycol (PG) was 60 μg / ml or less. Triton below 2.5 μg / mL was found. Also 115 pg / mL insulin was present in the final purified MOD-4023 protein. Finally, less than 250 μg / mL DMSO was present in the purified MOD-4023 protein product. The results of the bioburden analysis are also presented in FIG. 24 and indicate that it is present in the purified pharmaceutical product at 10 cfu / 10 mL or less.

  Example 2 Occupied glycosylation site of MOD-4023 protein

  the purpose

  The purpose of this study was to provide information on glycosylation site occupancy of MOD-4023 protein (a glycoprotein with approximately 12 O-linked glycosylation sites).

  Method

  Desialylation of MOD-4023 using neuraminidase

  A Mod-4023 sample with a concentration of approximately 21 mg / ml was used. Approximately 200 ug of sample was buffer exchanged into water and lyophilized. The dried sample was suspended in neuraminidase and incubated at 37 ° C.

  Trypsin digestion of reduced / carboxymethylated MOD-4023 protein

  Following desialation, approximately 200 μg of MOD-4023 was buffer exchanged into Tris / guanidine hydrochloride buffer, reduced with dithiothreitol, and carboxymethylated using iodoacetic acid. Samples were then buffer exchanged into ammonium bicarbonate and trypsin digested at 37 ° C.

  Online liquid chromatography / electrospray mass spectrometry LC / ES-MS

Online liquid chromatography / electrospray mass spectrometry (LC / ES-MS) using a Jupiter Phenomenex column (C18, 5μ, 250 × 2.1 mm; room temperature; 214 nm wavelength; 0.2 ml / min flow rate) Was performed on an aliquot of the digested sample obtained. Solvent A was 0.05 mL formic acid in 1 L H ₂ O. Solvent B was 900 mL acetonitrile in addition to 0.05 mL formic acid in 100 mL H ₂ O. For the intact and reduced product, the slope presented in Table 1 below was used.

  The use of nitrogen dry gas enhanced ionization and increased the source temperature. A slightly increased cone voltage was ignited to promote source fragmentation. The mass range scan was m / z 200-m / z 2000.

  Data interpretation

  Interpretation of mass spectrometry data is based on General Protein / Mass Analysis for Windows (combined with the protein sequence of CTP modified hGH (SEQ ID NO: 7), where the sequence expected for O-glycosylation is present in the CTP unit). Assisted in the use of GPMAW) software (Lighthouse data).

  result

  Although the entire sequence of SEQ ID NO: 7 was analyzed, evaluation of the data led to a complete mapping of the sequence of the MOD-4023 protein between amino acids 37 and 224 of SEQ ID NO: 7.

  LC-ES-MS of desialylated, reduced, and carboxymethylated MOD-4023 trypsin digestion mixture

  Online LC / ES-MS was performed on trypsin digestion of desialylated, reduced, and carboxymethylated MOD-4023. For a batch-to-batch comparison, FIG. 16 shows an overlay of three total ion current (TIC) chromatograms. The data acquired from the three batches were highly similar for both detected peptides and intensity.

  FIG. 17 presents an evaluation of the signal obtained from on-line LC / ES-MS analysis of trypsin digestion of desialylated, reduced, and carboxymethylated MOD-4023 protein batch, showing at least one HexNAc-Hex residue. Focus on the group modified signal. The results presented in FIG. 17 show O-linked glycosylation site occupancy at the N-terminal region as shown in FIG. FIG. 18 presents amino acid sequences 1-30 of SEQ ID NO: 7 of MOD-4023, and O-glycosylation is performed at serine (S) at positions 10, 13, 15, and 21 (shown in red). ) Done on residues. All of these serines (S) follow a proline (P) residue in the sequence. At least two of the S residues at positions 1-4 (1-4) are occupied by O-glycosylation sites (shown in purple).

  An additional signal of the complete CTP unit was observed consistent with the expected mass of amino acids 1-36 of SEQ ID NO: 7 with up to 6 O-glycosylation sites. The generation of such a large tryptic peptide containing a missing cleavage site is consistent with the presence of O-glycosylation, which indicates that glycan residues adjacent to arginine or lysine residues are sterically and electrostatically impaired. As a result, trypsin cleavage is disturbed.

  The data presented in FIG. 17 also shows comparability with previous data (not shown), ie, data for the C-terminal CTP-CTP region of MOD-4023. As a result, it can be said that O-glycosylation is carried out on the serine residue following the proline residue (shown in red) in FIGS. In addition, in regions where serine residues are contiguous, at least two of the four serines are occupied by O-glycosylation.

  From the results obtained, the following conclusions were drawn regarding the N-terminal CTP unit. O-glycosylation was performed on serine residues at positions 10, 13, 15, and 21 (amino acids 10, 13, 15, and 21 of SEQ ID NO: 7). All of these serines followed a proline residue in the sequence. Of the four serine residues (amino acids 1 to 4 of SEQ ID NO: 7) at positions 1 to 4 at the N-terminal of the protein, at least two are occupied by O-glycosylation sites. In the C-terminal CTP-CTP unit: amino acids 229, 232, 234, 240, 257, 260, 262, and 268 of SEQ ID NO: 7. In addition, in the region where serine is continuous, at least two of the four serines were occupied by O-glycosylation sites (amino acids 1-4 of N-terminal CTP unit SEQ ID NO: 7, as well as C-terminal CTP-CTP). Amino acids 220-223 and 248-251 of the unit SEQ ID NO: 7).

  It is understood that peptides with a large number of O-glycosylation sites may have escaped detection by mass spectrometry as a result of poor ionization. For this reason, identical samples were injected with higher amounts and altered ionization conditions favored on high mass trypsin peptides.

  Conclusion

  In the course of this study, evidence was collected in support of the following statement. Evaluation of the data obtained from analysis of the trypsin digestion mixture led to a signal that allowed 100% of the protein sequence to be mapped. O-glycosylation occurs at both the N-terminal and C-terminal CTP and CTP-CTP regions, respectively. Occupied sites were identified as serine residues following the proline residue, as well as two of the four serines in the region where serine is contiguous. In this way, a total of up to 18 serine residues can serve as O-glycan attachment sites. Thus, surprisingly and unexpectedly, using the methods of manufacture disclosed herein, O-glycan occupancy is CTP of each MOD-4023 (CTP-hGH-CTP-CTP polypeptide). There were up to 6 O-glycans per unit. Molecular weight analysis of MOD-4023 protein after desialation showed that the protein was modified with 12-18 glycosylation sites of the HexNAc-Hex structure (see Example 3).

  Example 3 Intact Molecular Weight Analysis of MOD-4023 Sample

  the purpose

  The purpose of this study was to provide accurate intact molecular weight information for three batches of MOD-4023 protein (a glycoprotein with approximately 12 O-linked glycosylation sites).

  Method

  Molecular weight analysis of acquired conditions and samples after treatment

Analyzed samples were 21 mg / ml and 41 mg / ml concentrations. Online LC / ES-MS was performed using the following conditions:
HPLC: GE AKTAmicro Liquid Chromatography System This includes a P-905 pump, UV-900 3-wavelength absorbance, conductivity detector, and A-905 autosampler, fraction collector Frac-950 MS: Micromass / Waters Q-TOF Micro quadrupole time-of-flight mass spectrometer Wavelength: 280nm
Column: Phenomenex Jupiter 3p C18 300A 150 × 2.0 mm
(SGS M-Scan GmbH column # 74)
Column temperature 40 ° C
Flow rate: 0.2 mL / min Solvent A: 1.0 mL FA in 1 L H20
Solvent B: in 100 mL H20, 1.0 mL FA plus 800 mL acetonitrile and 100 mL tetrahydrofuran

  The gradient described below was used for the analysis of the desialylated product. Note that the retention time during data evaluation is adjusted at the beginning of the slope (tRo = 10 minutes).

  The use of nitrogen dry gas enhanced ionization and increased the source temperature. The scanned mass range was m / z 200 to m / z 2000. The instrument was calibrated using Glu-fibrinopeptide fragment ions in MS / MS mode.

  Desialylation using neuraminidase

  Approximately 100 pg of sample was buffer exchanged into water and lyophilized. The dried sample was suspended in neuraminidase and incubated at 37 ° C. Approximately 10 pg was analyzed as described above.

  Data interpretation

  Interpretation of mass spectrometry data was aided by the use of GPMAW software (Lighthouse data) in conjunction with the MOD-4023 protein sequence (SEQ ID NO: 7).

  result

  Intact molecular weight analysis of acquired samples

  FIG. 20 shows the decon obtained during elution of the UV absorbing component at tR 19.8 minutes obtained from analysis of MOD4023 lot 648-01-10-014A sample by online LC / ES-MS after desialation. A voluteed mass spectrum is shown.

  Table 3 presents a summary of the results obtained from the mass spectrometric detection obtained during on-line LC / ES-MS analysis of MOD4023 lot 648-01-10-014A after desialation.

  Conclusion

  In order to obtain information on the number of O-linked glycosylation sites, three batches of MOD4023 were subjected to molecular weight analysis on the desialylated sample. Consistently in all three batches, data obtained from intact mass measurements of desialylated samples are consistent with the average chemical mass of MOD4023 protein with 12-18 glycosylation sites of the HexNAc-Hex structure. Signal (within the experimental error of the instrument). For all three batches, the protein modified with 15 glycosylation sites was the strongest.

Each corresponding mass spectrometric signal was characterized by the presence of a satellite signal at approximately +16.5 Da. This mass shift may have been the result of the addition of oxygen or NH ₃ residues. The latter can be derived from NH ₄ ⁺ protonation rather than H + protonation causing net addition of NH ₃ (+17 Da). Since the oxidation could alternatively result in a shift in elution time (as observed by the presence of a UV signal at 18.5 minutes, consistent with the mass of the main peak + 1 oxygen), the data NH ₄ ⁺ result of protonation). However, this could not be finally clarified by the intact mass measurement alone.

  Findings derived from this study show that a previous analysis of the MOD 4023 protein found that a maximum of 18 O-glycosylation residues were found and the protein modified at 15 glycosylation sites was the strongest (data is (Not shown).

  While particular features disclosed herein have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those skilled in the art. Accordingly, it is to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit disclosed herein.

Claims (25)

A method for producing a human chorionic gonadotropin carboxy terminal peptide (CTP) modified human growth hormone (hGH) polypeptide comprising:
(A) stably transfecting a predetermined number of cells with an expression vector comprising a coding portion encoding the CTP-modified hGH,
i. The transfected cells expressing and secreting the CTP modified hGH;
(B) obtaining a cell clone that overexpresses the CTP-modified hGH;
(C) growing the clone in solution to a predetermined scale;
(D) collecting the solution containing the clones;
(E) filtering the solution containing the clones to obtain a purified collection solution;
(F) purifying the clarified collection solution to obtain a purified protein solution having a desired concentration of the CTP-modified hGH;
A CTP-modified hGH is thereby produced, and the amino acid sequence of the produced CTP-modified hGH is set forth in SEQ ID NO: 7.   The method of claim 1, wherein the purity of the CTP-modified hGH protein is at least 90%.   The method according to claim 2, wherein the produced CTP-modified hGH is highly glycosylated.   The method according to claim 3, wherein the glycosylation pattern of the produced CTP-modified hGH includes glycosylation of at least 4 O-linked glycosylation sites for each CTP.   The method according to claim 2, wherein the produced CTP-modified hGH is highly sialylated.   The method of claim 1, wherein step (c) comprises propagating a clone obtained from a working cell bank (WCB) that optimally expresses and secretes the CTP modified hGH.   The method of claim 1, wherein step (c) comprises propagating a clone obtained from a master cell bank (MCB) that optimally expresses and secretes the CTP-modified hGH.   The method of claim 1, wherein the manufacturing method is an animal free process.   2. The method of claim 1, wherein in step (c), the clone expresses and secretes CTP modified hGH at a level of at least 600 mg / L.   2. The method of claim 1, wherein in step (c), the clone is grown in solution to a production bioreactor level through a series of subculture steps.   The method of claim 10, wherein the bioreactor comprises a disposable bioreactor or a stainless steel bioreactor.   11. The method of claim 10, wherein the bioreactor is operated as a fed-batch mode bioreactor.   2. The method of claim 1, wherein in step (f), 60% of the purified CTP modified hGH in the clarified collection comprises a high glycosylation form of the CTP modified hGH. The purification (step f) of the purified collection
(G) concentrating, diafiltrating, and purifying the purified collection solution,
i. The concentration, diafiltration, and purification are accomplished by sequentially passing the clarified collection solution through an anion exchange column and a hydrophobic interaction column;
(H) obtaining the clarified collection obtained following step (g) and inactivating the virus present in the clarified collection by incubating in a solution toxic to the virus; And steps to
(I) obtaining the purified collection solution from step (h) and concentrating, diafiltration and purifying the purified collection solution,
i. Sequentially passing the purified collection solution through a hydroxyapatite mixed-mode column and a cation exchange column after the concentration, diafiltration, and purification;
(J) obtaining the purified collection solution following step (i) and physically removing the purified collection solution from the virus by nanofiltration;
(K) Obtaining the clarified collection solution following step (j) and concentrating the clarified collection solution until a clarified collection containing the maximally purified CTP modified hGH is reached. And diafiltering in a sequential manner.   15. The method of claim 14, wherein the anion exchange column is a DEAE-Sepharose column.   15. The method of claim 14, wherein the hydrophobic column is a Phenyl HIC column.   15. The method of claim 14, wherein the solution that is toxic to the virus is a 1% Triton-X100 solution.   The method according to claim 14, wherein the cation exchange column is an SP-Sepharose column.   15. The method of claim 14, wherein the viral clearance exhibits a virus reduction rate (LRF) of about 22.   The method of claim 1, wherein the method achieves a recovery of at least 20% of highly glycosylated CTP-modified hGH. Human chorionic gonadotropin carboxy terminus (CTP) modified human growth hormone (hGH),
(A) stably transfecting a predetermined number of cells with an expression vector comprising a coding portion encoding the CTP-modified hGH,
i. The transfected cells expressing and secreting the CTP modified hGH;
(B) obtaining a cell clone that overexpresses the CTP-modified hGH;
(C) growing the clone in solution to a predetermined scale;
(D) collecting the solution containing the clones;
(E) filtering the solution containing the clones to obtain a purified collection solution;
(F) purifying the clarified collection solution to obtain a purified protein solution having a desired concentration of CTP-modified hGH, and
Human chorionic gonadotropin carboxy terminus (CTP) modified human growth hormone (hGH), wherein the produced CTP modified hGH comprises the amino acid sequence set forth in SEQ ID NO: 7.   The CTP-modified hGH according to claim 21, wherein the produced CTP-modified hGH is highly glycosylated.   23. The CTP-modified hGH of claim 22, wherein the glycosylation pattern of the CTP-modified hGH comprises at least 4 O-linked glycans per CTP.   23. The CTP modified hGH of claim 22, wherein said glycosylation comprises at least 4-6 O-glycans per CTP unit.   A composition comprising the CTP-modified hGH of claim 21 and a pharmaceutically acceptable carrier.