JP7000153B2 - Methods for Producing Long-Acting CTP-Modified Growth Hormone Polypeptides - Google Patents

Description

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

Growth hormone (somatotropin) has been used clinically for over 50 years primarily to treat growth hormone deficiency in children with dwarfism. In 1985, recombinant DNA-derived human growth hormone (hGH) replaced hGH from the cadaveric pituitary gland, which was previously the only available source of material. hGH replacement therapy has become the standard of care for tens of thousands of patients and has proven safe and effective. The need to maintain hGH blood levels within the effective therapeutic range requires subcutaneous or intramuscular injection once daily or once every two days. Most of the commercially available hGH is produced in a bacterial system.

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

The methods disclosed herein include the production of long-acting hGH (MOD-4023; FIG. 1), which eliminates the need for numerous injections currently required in the treatment of GHD. This technique 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 the hCG with the long life required to maintain pregnancy (hCG). Initial T _1/2 about 10 hours, final T _1/2 about 37 hours). CTP has 28 amino acids and 4-6 O-linked sugar chains, all of which are bound to serine residues. The beta chain of luteinizing hormone (LH), a reproductive hormone that causes ovulation, is almost identical to hCG but does not contain CTP. As a result, LH has a significantly shorter half-life in blood (initial T _1/2 about 1 hour, final T _1/2 about 10 hours).

MOD-4023 is an hGH molecule fused to three copies of CTP, one fused at the N-terminus and two fused at the C-terminus (CTP-hGH-CTP-CTP set forth in SEQ ID NO: 7). (Fig. 1). MOD-4023 is a single-stranded protein with 275 amino acids and 12-18 O-linked carbohydrates. As demonstrated in animal models (weight gain in hypophysectomized rats), MOD-4023 is injected once-weekly to once-two-weekly, similar to once-daily injections of hGH. Has the potential to bring about clinical effects.

In one embodiment, a method for producing human chorionic gonadotropin carboxy-terminal peptide (CTP) -modified human growth hormone (hGH) polypeptide, wherein (a) a predetermined number of cells are encoded by the CTP-modified human growth hormone. A step of stably transfecting an expression vector containing a coding moiety, wherein the transfected cells express and secrete the CTP-modified hGH, and (b) overexpress the CTP-modified hGH. It contains a step of obtaining a cell clone, (c) a step of growing the clone in a solution to a predetermined scale, (d) a step of collecting the solution containing the clone, and (e) the clone. A step of filtering the solution to obtain a purified collection solution and (f) a step of purifying the purified collection solution to obtain a purified protein solution having a desired concentration of CTP-modified hGH. And, a method of producing human chorionic gonadotropin peptide (CTP) -modified human growth hormone (hGH) polypeptide and the amino acid sequence of the CTP-modified hGH is set forth in SEQ ID NO: 7 is disclosed herein. In a related embodiment, the produced CTP-modified hGH is highly glycosylated. In a related embodiment, the produced CTP-modified hGH is highly sialic acid-added. In another related embodiment, the glycosylated pattern of the produced CTP-modified hGH comprises glycosylation of at least 4 O-binding sites per CTP.

In one aspect, a human chorionic gonadotropin carboxy-terminal peptide (CTP) -modified human growth hormone (hGH) polypeptide produced by the methods disclosed herein is disclosed. In a related embodiment, 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 detailed description and examples show embodiments of manufacturing methods and consequent products, various changes and modifications are made within the principles and scope of the methods and products disclosed herein. It should be understood that these are provided for purposes of illustration only, as this detailed description may be obvious to those of skill in the art.

The subject matter considered to be the recombinant long-acting human chorionic gonadotropin carboxy-terminal peptide (CTP) -modified human growth hormone (hGH) polypeptide (CTP-modified hGH) and the method for producing it is specific to the conclusions herein. Pointed out and clearly asserted. However, CTP-modified hGH and the methods for its manufacture, as well as its objects, features and advantages, are best referred to in the following detailed description by reading in conjunction with the accompanying drawings for both configuration and operating methods. 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. The upstream process flow generation chart of a CTP modified polypeptide, eg MOD-4023, is shown. A flow generation chart of a CTP-modified polypeptide, eg MOD-4023, is shown. MOD-4023 A flow chart overview of the pharmaceutical (DP) manufacturing process is presented. A Coomassie-stained SDS-PAGE showing purification of the MOD-4023 glycosylated form by a first ion exchange chromatography (IEX) column is presented. Lane (1) Collected Total Protein, Step 8 of FIG. 3; Lane (2) Ultrafiltration / Dialysis Filtration, Step 1 (UFDF1) Concentration and Dialysis Filtered Total Protein of FIG. 4; Lane (3) IEX Flow Through , FIG. 4; lane (4) IEX cleaning material, FIG. 4; and lane (5) IEX elution, FIG. 4. Data showing the robust O-glycan content of MOD-4023 from different batches is presented. The O-glycan mol content per mole of MOD-4023 was determined for different batches of MOD-4023 API (DS) and Drug (DP). A batch-by-batch comparison between individual O-glycosylated peptides based on the response of mass spectrometry (MS) is presented. The note in the column is the amino acid sequence, where "O" is an abbreviation for O-linked glycans. The MS response of the first peptide was used for data normalization and was set to 100%. A reverse phase high performance liquid chromatography (RP-HPLC) profile of MOD-4023 is presented. Peak 4 corresponds to the main peak of MOD-4023. There are also four minor related morphologies (peaks 1, 2, 3, and 5). A size exclusion high performance liquid chromatography (SEC-HPLC) profile of MOD-4023 is presented. Peak 3 corresponds to the MOD-4023 monomer. Peaks 1 and 2 correspond to the MOD-4023 dimer and polymer, respectively. MOD-4023 reference standard SR-929SI analyzed by RP-HPLC at 220 nm. A comparison of 1 (upper profile) with MOD-4023 I15-PP (UFDF1 sample) (lower 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 of 1 (the profile above) with MOD-4023 C10-PP (Elution DEAE Sepharose) is presented. At this point in the production process, only glycosylation peaks were observed in the MOD-4023 sample. It shows activation of hGH receptor by MOD-4023 in stably transfected cells. Typical dose-response activation curve by MOD-4023. MOD-4023 activation of hGH receptors on Baf cells that stably express hGH receptors on their cell surface. Individual virus safety assessment studies using Abelson murine leukemia virus (A-MuLV), mouse microvirus (MVM), Leovirus type 3 (Reo-3), and pseudorabies virus (PrV). The clearance coefficients for the process steps and the overall process are presented. The viral load per theoretical dose was calculated at the maximum dose of 15 mg / dose. O-linked glycan structure and their abundance in 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 the removed sialic acid separated by ultra-high performance liquid chromatography (UPLC) and identified for commercial standards. 2 Shows the abundance of sialic acid forms in the two MOD-4023 product samples measured by HCl (DMB) labeling. Shown is an overlay of three total ion current chromatograms obtained from analysis of three MOD-4023 batches of tryptic digests by online liquid chromatograph / electrospray mass spectrometry (LC / ES-MS). Obtained from analysis of tryptic digestion of desialylated, reduced, and carboxymethylated MOD-4023 protein batches (shown in FIG. 16 and described in Example 2) by online LC / ES-MS. The evaluation of the signal is shown and the focus is on the signal modified with at least one HexNAc-Hex residue. The amino acid sequences 1-30 of SEQ ID NO: 7 of MOD-4023 are shown, with O-glycosylation on serine (S) residues at positions 10, 13, 15 and 21 (shown in red). Will be done. All of these serines (S) follow the proline (P) residue in the sequence. At least two of the S residues at positions 1 to 4 (1 to 4) are occupied by the O-glycosylation site (indicated by purple). The amino acid sequences 211-275 of SEQ ID NO: 7 of MOD-4023 are shown, and O-glycosylation is performed on the serine (S) residue shown in red. All of these serines (S) follow the proline (P) residue in the sequence. In addition, in the serine contiguous region, at least two of the S residues are occupied by the O-glycosylation site (indicated by purple). Deconvoluted mass spectra obtained during elution of UV-absorbing components at tR19.8 min obtained from analysis of MOD4023 lot 648-01-10-014A samples by online LC / ES-MS after desialization. Is shown. MOD-4023 (DS) purity assessed using reverse phase (RP) -HPLC. MOD-4023 (DS) purity as assessed using size exclusion chromatograph (SEC) -HPLC. Shows efficacy results. The results of impurity analysis for host cell protein (HCP), DNA, methotrexate (MTX), propylene glycol (PG), triton, insulin, DMSO, and bioburden are shown.

It will be appreciated that for the convenience and clarity of the illustration, the elements shown in the figure are not necessarily drawn to scale. For example, some dimensions of an element may be emphasized compared to other elements for clarity. In addition, reference numbers may be repeated between drawings to indicate corresponding or similar elements, where appropriate.

In the following detailed description, a 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 a method for producing the same. A number of specific details are given to provide a complete understanding of. In other cases, known methods, procedures, and components are not described in detail so as not to obscure the CTP-modified hGH polypeptide and the method for producing it.

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

Those skilled in the art will appreciate that the terms "CTP peptide", "carboxy-terminal peptide", and "CTP sequence" can be used interchangeably. In one embodiment, the carboxy-terminal peptide is a full-length CTP. In another embodiment, the carboxy-terminal peptide is a missing CTP.

Those skilled in the art will appreciate that the terms "signal sequence" and "signal peptide" can be used interchangeably. In addition, one of ordinary skill in the art will appreciate that the term "sequence" may include the coding portion of a polynucleotide sequence when it is associated with a polynucleotide.

Those skilled in the art will appreciate that the terms "polypeptide", "peptide", "peptide of interest", "polypeptide of interest", and "polypeptide sequence of interest" can be used interchangeably. There will be. 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 villous gonadotropin carboxy-terminal peptide (CTP) attached to the amino end of human growth hormone (hGH), and two human villous gonadotropin carboxy adhered 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 comprising the amino acid sequence set forth in SEQ ID NO: 7. In another embodiment, CTP comprises GH, a single CTP attached to the amino end of GH, two CTPs attached to the carboxy terminus of GH, and a signal peptide attached to the amino end of the amino end CTP. A modified hGH polypeptide is disclosed herein and the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4. Those of skill in the art will appreciate that mature secretory polypeptides are deficient in the signal peptide.

In one embodiment, it is a method for producing human chorionic gonadotropin carboxy-terminal peptide (CTP) -modified human growth hormone (hGH) polypeptide (CTP-modified hGH), wherein (a) a predetermined number of cells are modified with the CTP. A step of stably transfecting an expression vector containing a coding moiety encoding hGH, wherein the transfected cells express and secrete the CTP-modified hGH, and (b) the transfecting step. A step of obtaining a cell clone overexpressing CTP-modified hGH, a step of (c) growing the clone in a solution to a predetermined scale, and (d) a step of collecting the solution containing the clone, (c). e) The step of filtering the solution containing the clone to obtain purified collection solution and (f) Purification of the purified collection solution to have the desired concentration of CTP-modified hGH. A human chorionic gonadotropin carboxy-terminal peptide (CTP) -modified human growth hormone (hGH) polypeptide is produced by the step of obtaining a protein solution, and the amino acid sequence of the CTP-modified hGH polypeptide is set forth in SEQ ID NO: 7. The method is disclosed herein.

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, the method of making a CTP-modified hGH polypeptide with an increased glycosylated content disclosed herein is disclosed herein. In another embodiment, the CTP-modified hGH polypeptide disclosed herein, produced by the method disclosed herein, comprises an increased number of glycosylated O-linked glycosylation sites. Has. Those 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 of producing or producing and using them 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 the degradation of proteins or peptides derived from them. In another embodiment, CTP prolongs the circulating half-life of a protein or peptide derived from it. In some embodiments, CTP enhances the efficacy of the protein or peptide derived from it.

Those skilled in the art can use the terms "CTP peptide", "CTP", "human chorionic gonadotropin carboxy-terminal peptide", "carboxy-terminal peptide", and "CTP sequence" interchangeably herein. Will understand. In one embodiment, the carboxy-terminal peptide is a full-length CTP. In another embodiment, the carboxy-terminal peptide is a missing CTP.

In another embodiment, the poly comprises a GH, a single CTP attached to the amino terminus of the GH, two CTPs attached to the carboxy terminus of the GH, and a signal peptide attached to the amino terminus of the N-terminus CTP. The peptide is disclosed herein and the polypeptide has the amino acid sequence set forth in SEQ ID NO: 4. Those of skill in the art will appreciate that mature secretory polypeptides can be deficient in the 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 described herein. Disclosed, the polypeptide has the amino acid sequence set forth in SEQ ID NO: 7.

In another embodiment, the poly comprises a GH, a single CTP attached to the amino terminus of the GH, two CTPs attached to the carboxy terminus of the GH, and a signal peptide attached to the amino terminus of the amino terminus CTP. A method for producing a peptide is disclosed herein, and the polypeptide has the amino acid sequence set forth in SEQ ID NO: 4. In yet another embodiment, a method for producing a polypeptide consisting of GH, a single CTP bonded to the amino terminus of GH, and two CTPs bonded to the carboxy terminus of GH and containing no signal peptide is described herein. The polypeptide has the amino acid sequence set forth in SEQ ID NO: 7.

In another embodiment, the CTP-containing GH set forth in SEQ ID NO: 7 has enhanced in vivo biological activity as 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 a 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: DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILQ (SEQ ID NO: 1).

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

In some embodiments, the CTP-modified hGH polypeptide disclosed herein has enhanced protection against degradation of the polypeptide. In some embodiments, the CTP-modified hGH polypeptide disclosed herein has an extended half-life of the protein to which it adheres. In some embodiments, the CTP-modified hGH polypeptide disclosed herein has enhanced activity of hGH.

In another embodiment, the CTP-modified hGH polypeptide disclosed herein has an extended clearance time. In another embodiment, the CTP-modified hGH polypeptide disclosed herein has an enhanced C _max of hGH. In another embodiment, the CTP-modified hGH polypeptide disclosed herein has an enhanced _Tmax of hGH. In another embodiment, the CTP-modified hGH polypeptide disclosed herein has an enhanced T _1/2 of hGH. In some embodiments, the CTP-modified hGH polypeptide disclosed herein provides an extended half-life of hGH.

In another embodiment, the carboxy-terminal peptide (CTP) peptide disclosed herein comprises the amino acid sequence of amino acids 112-145 of human chorionic gonadotropin 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 set forth in SEQ ID NO: 2: SSSSKAPPPSLPSPSRLPGPSDTPILPQ. In another embodiment, the CTP sequence also begins at any position between positions 112 and 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 acid lengths 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 1-5 conserved 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 native CTP by one conserved amino acid substitution. In another embodiment, the CTP peptide is a variant of chorionic gonadotropin CTP that differs from native CTP by two conserved amino acid substitutions. In another embodiment, the CTP peptide is a variant of chorionic gonadotropin CTP that differs from native CTP by three conserved amino acid substitutions. In another embodiment, the CTP peptide is a variant of chorionic gonadotropin CTP that differs from native CTP by four conserved amino acid substitutions. In another embodiment, the CTP peptide is a variant of chorionic gonadotropin CTP that differs from native CTP by five conserved 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 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 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 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 peptide thereof.

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

In one embodiment, at least one of the chorionic gonadotropin CTP amino acid sequences is deficient. In another embodiment, both the chorionic gonadotropin CTP amino acid sequences are deficient. In another embodiment, two of the chorionic gonadotropin CTP amino acid sequences are deficient. 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 deficient. In one embodiment, the missing CTP comprises the first 10 amino acids of SEQ ID NO: 3: SSSSKAPPPSLP. 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 the 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, the entire chorionic gonadotropin CTP amino acid sequence is glycosylated. In one embodiment, the CTP sequence disclosed herein comprises at least one glycosylated site. In another embodiment, the CTP sequence disclosed herein comprises two glycosylation sites. In another embodiment, the CTP sequence disclosed herein comprises three glycosylation sites. In another embodiment, the CTP sequence disclosed herein comprises four glycosylated sites. In another embodiment, the CTP sequence disclosed herein comprises five glycosylated sites. In another embodiment, the CTP sequence disclosed herein comprises six glycosylated sites. In another embodiment, the CTP sequence disclosed herein comprises seven glycosylated sites. In another embodiment, the CTP sequence disclosed herein comprises eight glycosylated sites. In another embodiment, the glycosylation site is an O-glycosylation site. In another embodiment, glycosylation is at a serine residue. In another embodiment, 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 method described herein produces CTP-modified polypeptide human growth hormone (CTP-hGH), the CTP-modified hGH being highly glycosylated. In another embodiment, the method disclosed herein produces 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.

In another embodiment, the method disclosed herein produces CTP-modified hGH, to which at least one of the chorionic gonadotropin CTP amino acid sequences is glycosylated. In another embodiment, both the 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 method disclosed herein produces CTP-modified hGH, to which 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, the entire chorionic gonadotropin CTP amino acid sequence is glycosylated.

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

In one embodiment, the CTP-modified hGH of SEQ ID NO: 7 produced comprises two CTPs attached to the carboxy terminus of hGH and one CTP attached to the amino end of hGH, and the CTP-modified hGH is 12 and Includes glycosylation at between 18 glycosylation sites. In another embodiment, the CTP-modified hGH comprises glycosylation at the glycosylation site between 12 and 21. In another embodiment, the CTP-modified hGH comprises glycosylation at the glycosylation site between 12 and 24. 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 the glycosylation site between ~ 24. In another embodiment, the CTP-modified hGH has 12 sugar chain addition sites, 13 sugar chain addition sites, 14 sugar chain addition sites, 15 sugar chain addition sites, and 16 sugar chain addition sites. , 17 sugar chain addition sites, 18 sugar chain addition sites, 19 sugar chain addition sites, 20 sugar chain addition sites, 21 sugar chain addition sites, 21 sugar chain addition sites, 22 Includes sugar chain addition at 13 sugar chain addition sites, 23 sugar chain addition sites, or 24 sugar chain addition 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 the available serine (S) residues present within each CTP unit. In another embodiment, each CTP contains 4-6 O-linked glycans and the glycosylation is to the serine (S) residue present within each CTP unit. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 12-18 O-linked glycosylation. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 12-21 O-linked glycosylation. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 12-24 O-linked glycosylation. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 12 O-linked glycosylation. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 13 O-linked glycosylation. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 14 O-linked glycosylation. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 15 O-linked glycosylation. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 16 O-linked glycosylation. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 17 O-linked glycosylation. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 18 O-linked glycosylation. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 19 O-linked glycosylation. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 20 O-linked glycosylation. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 21 O-linked glycosylation. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 22 O-linked glycosylation. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 23 O-linked glycosylation. In another embodiment, the O-linked glycosylation at the serine (S) residue of each CTP unit comprises 24 O-linked glycosylation. In another embodiment, there is no O-linked sugar chain on the hGH sequence (SEQ ID NO: 8) of the CTP-modified hGH.

In another embodiment, the CTP-modified hGH polypeptide disclosed herein comprises at least 4 O-glycans per CTP. In another embodiment, the CTP-modified hGH polypeptide disclosed herein comprises at least 5 O-glycans per CTP. In another embodiment, the CTP-modified hGH polypeptide disclosed herein comprises at least 6 O-glycans per CTP. In another embodiment, the CTP-modified hGH polypeptide disclosed herein comprises at least 7 O-glycans per CTP. In another embodiment, the CTP-modified hGH polypeptide disclosed herein comprises at least 8 O-glycans per CTP. Those skilled in the art will appreciate that O-glycan occupancy can vary with each CTP within the CTP-modified hGH polypeptide.

In another embodiment, the CTP-hGH-CTP-CTP polypeptide disclosed herein comprises at least one CTP containing more than four O-glycan occupancy. In another embodiment, the CTP-hGH-CTP-CTP polypeptide disclosed herein comprises at least one CTP containing more than 5 O-glycan occupancy. In another embodiment, the CTP-hGH-CTP-CTP polypeptide disclosed herein comprises at least one CTP containing more than 6 O-glycan occupancy. In another embodiment, the CTP-hGH-CTP-CTP polypeptide disclosed herein comprises at least one CTP containing more than 7 O-glycan occupancy.

One of skill in the art will appreciate that the term "homologousness" may include deletions, insertions, or substitution mutations, including its amino acid substitutions and their bioactive polypeptide fragments. In one embodiment, the substitution mutation is one in which glutamine at position 65 of hGH is replaced with valine [Gellerfors et al. , J Palm Biomed Anal 1989, 7: 173-83].

In one embodiment, a "peptide" or "peptide fragment" comprises a compound in which a plurality of amino acids are bound by a peptide bond. 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 compound in this case is also collectively referred to as a peptide or peptide fragment.

Those skilled in the art will appreciate that the term "protected peptide fragment" can lead to undesired side reactions during the preparation of the peptide fragment or the condensation reaction of the peptide fragment. One or more reactive substituents selected from the group consisting of a guanidino group, an imidazolyl group, an indolyl group, a mercapto group, and a carboxyl group can comprise a fragment of the peptide protected by a protective 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 according to the teachings disclosed herein. In some embodiments, adhesion of the CTP sequence (s) to both the amino and carboxy ends of the hGH protein results in increased efficacy. In some embodiments, adhesion of the CTP sequence to both the amino and carboxy ends of the hGH protein results in extended 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.

Those skilled in the art have used the phrase "human growth hormone" (hGH) as a precursor containing a signal peptide that expresses hGH activity (ie, stimulation of growth), as described in Genbank Accession No. P01241 (SEQ ID NO: 5). You will understand that it can include body polypeptides. 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.

Those skilled in the art will appreciate that the "hGH" disclosed herein may include homologues. In another embodiment, the GH amino acid sequences of the methods and compositions disclosed herein have been determined herein using the National Center for Biotechnology Information (NCBI) BlastP software using default parameters. It is at least 50% homologous to the described hGH sequence. In another embodiment, the percentage of homology is 60%. In another embodiment, the percentage of homology is 70%. In another embodiment, the percentage of homology is 80%. In another embodiment, the percentage of homology is 90%. In another embodiment, the percentage of homology is 95%. In another embodiment, the percentage of homology is greater than 95%.

In one embodiment, following the 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 ~ 26, MATGSRTSLLLAFGLLCLPWLQEGSA (SEQ ID NO: 6) represents a signal peptide CTP modified hGH polypeptides, amino EsuesuesuesukeieiPipipiesueruPiesupiesuarueruPiJipiesuditiPiaieruPikyuefuPitiaiPieruesuarueruefudienueiemueruarueieichiaruerueichikyuerueiefuditiwaikyuiefuiieiwaiaiPikeiikyukeiwaiesuefuerukyuenuPikyutiesuerushiefuesuiesuaiPitiPiesuenuaruiitikyukyukeiesuenueruierueruaruaiesueruerueruaikyuesudaburyueruiPibuikyuefueruaruesubuiefueienuesuerubuiwaijieiesudiesuenubuiwaidieruerukeidieruiijiaikyutieruemujiarueruidijiesuPiarutijikyuaiefukeikyutiwaiesukeiefuditienuesueichienudidieieruerukeienuwaijierueruwaishiefuarukeidiemudikeibuiitiefueruaruaibuikyushiaruesubuiijiesushijiefuesuesuesuesukeieiPipipiesueruPiesupiesuarueruPiJipiesuditiPiaieruPikyuesuesuesuSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 7), the mature engineered lacking a signal peptide Represents a CTP-modified hGH polypeptide (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 for 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 represent 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 two C-termini to hGH.

In one embodiment, MOD-4023 (SEQ ID NO: 7) comprises two disulfide (SS) crosslinks, 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 a second disulfide bridge is with cysteine residue 210 and cysteine residue 217 of SEQ ID NO: 7. It is between.

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

In one embodiment, the manufacturing method disclosed herein produces an amino acid sequence of a mature CTP-modified hGH polypeptide lacking the signal peptide disclosed herein. In another embodiment, the manufacturing method disclosed herein produces 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 the CTP-modified hGH polypeptide set forth in SEQ ID NO: 7 that stimulates muscle growth.

In another embodiment, the method disclosed herein comprises the use of a nucleic acid sequence encoding the CTP-modified hGH polypeptide disclosed herein. In one embodiment, the method disclosed herein is the nucleic acid set forth in SEQ ID NO: 9, which encodes 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: ATGGCCACCGGCAGCAGGACCAGCCTGCTGCTGGCCTTCGGCCTGCTGTGCCTGCCATGGCTGCAGGAGGGCAGCGCCAGCTCTTCTTCTAAGGCTCCACCCCCATCTCTGCCCAGCCCCAGCAGACTGCCGGGCCCCAGCGACACACCCATTCTGCCCCAGTTCCCCACCATCCCCCTGAGCAGGCTGTTCGACAACGCCATGCTGAGGGCTCACAGGCTGCACCAGCTGGCCTTTGACACCTACCAGGAGTTCGAGGAAGCCTACATCCCCAAGGAGCAGAAGTACAGCTTCCTGCAGAACCCCCAGACCTCCCTGTGCTTCAGCGAGAGCATCCCCACCCCCAGCAACAGAGAGGAGACCCAGCAGAAGAGCAACCTGGAGCTGCTGAGGATCTCCCTGCTGCTGATCCAGAGCTGGCTGGAGCCCGTGCAGTTCCTGAGAAGCGTGTTCGCCAACAGCCTGGTGTACGGCGCCAGCGACAGCAACGTGTACGACCTGCTGAAGGACCTGGAGGAGGGCATCCAGACCCTGATGGGCCGGCTGGAGGACGGCAGCCCCAGGACCGGCCAGATCTTCAAGCAGACCTACAGCAAGTTCGACACCAACAGCCACAACGACGACGCCCTGCTGAAGAACTACGGGCTGCTGTACTGCTTCAGAAAGGACATGGACAAGGTGGAGACCTTCCTGAGGATCGTGCAGTGCAGAAGCGTGGAGGGCAGCTGCGGCTTCAGCTCCAGCAGCAAGGCCCCTCCCCCGAGCCTGCCCTCCCCAAGCAGGCTGCCTGGGCCCTCCGACACACCAATCCTGCCACAGAGCAGCTCCTCTAAGGCCCCTCCTCCATCCCTGCCATCCCCCTCCCGGCTGCCTGGCCCCTCTGACACCCCTATCCTGCCTCAG.

In one embodiment, the method comprises the use of a nucleic acid sequence comprising a coding moiety encoding the 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 of skill in the art will appreciate that the nucleic acid sequence may be part of an expression vector containing a coding moiety encoding the CTP-modified hGH disclosed herein.

In another embodiment, the method disclosed herein is a CTP for therapeutic use in a number of indications as provided in US Pat. No. 8,946,155, which is incorporated herein by reference. A nucleic acid sequence encoding a modified hGH peptide is provided. Further, such CTP-modified hGH polypeptides are fully described in US Patent Application Publication No. US-2014-0113860-A1, US Pat. No. 8,450,269, and US Pat. No. 8,304,386. All of these are incorporated herein by reference in their entirety.

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

In some embodiments, the polypeptides disclosed herein are biochemically synthesized 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 when it cannot be produced by recombinant techniques (ie, not encoded by a nucleic acid sequence). And therefore requires 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 198). .. In some embodiments, the synthetic polypeptide is purified by preparative high performance liquid chromatography [Creighton T. et al. (1983) Proteins, structures and molecular principles. WH Freeman and Co. N. Y. ], The composition can be identified via the amino acid sequence by methods known to those of skill in the art.

In some embodiments, recombinant protein techniques are used to produce the polypeptides disclosed herein. In some embodiments, recombinant protein techniques are used to produce relatively long polypeptides (eg, longer than 18-25 amino acids). In some embodiments, recombinant protein techniques are used to produce large quantities of the polypeptides disclosed herein. In some embodiments, the recombinant technique is described in Bitter et al. , (1987) Methods in Enzymol. 153: 516-544, Studio et al. (1990) Methods in Enzymol. 185: 60-89, Brisson et al. (1984) Nature 310: 51-514, Takamatsu et al. (1987) EMBO J. 6: 307-311, Coruzzi et al. (1984) EMBO J. 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 polynucleotides encoding the polypeptides disclosed herein. In some embodiments, the polynucleotide encoding the polypeptide disclosed herein is ligated to an expression vector comprising transcriptional regulation 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 the induced expression of the polypeptides disclosed herein.

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

Those skilled in the art have used the phrase "polynucleotide" in the form of isolated, RNA sequences, complementary polynucleotide sequences (cDNAs), genomic polynucleotide sequences, and / or complex polynucleotide sequences (eg, combinations described above). It will be appreciated that the provided single-stranded or double-stranded nucleic acid sequences may be included.

Those skilled in the art will appreciate that the phrase "complementary polynucleotide sequence" may include 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.

Those skilled in the art will appreciate that a "genome polynucleotide sequence" can include a sequence derived (isolated) from a chromosome, and thus it represents an adjacent portion of the chromosome.

Those skilled in the art will appreciate that the phrase "complex polynucleotide sequence" can include sequences that are at least partially complementary and at least partially genomic. In one embodiment, the complex sequence may comprise some exon sequences required to encode the polypeptides disclosed herein, as well as some intron sequences sandwiched between them. In one embodiment, the intron sequence may be of any source, including other genes, and will typically include a conserved splicing signal sequence. 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 the 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-terminal to the CTP sequence, which in turn is a sequence from the N-terminal to the polypeptide sequence of interest, eg, the sequence is (a) 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 of interest sequence and the polypeptide of interest itself, thereby blocking the wild-type sequence of interest.

In another embodiment, the polypeptide disclosed herein and the method of its production provide the mature CTP-modified GH set forth in SEQ ID NO: 7, lacking the signal peptide.

In some embodiments, the polynucleotides disclosed herein are prepared using PCR techniques, or any other method or procedure known to those of skill 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 of reducing the frequency of administration of GH to a subject is to the hGH, one villous gonadotropin carboxy-terminal peptide (CTP) adhered to the amino end of the GH, and the carboxy-terminal of the GH. The subject comprises administering to the subject a therapeutically effective amount of a polypeptide consisting of two adhered villous gonadotropin CTP, the polypeptide lacking a signal peptide, and the amino acid sequence of the polypeptide is SEQ ID NO: 7. And thereby reducing the number of doses of GH to a subject are disclosed herein.

In another embodiment, a method of improving the area under the curve (AUC) of GH to a subject is the hGH, one chorionic gonadotropin carboxy-terminal peptide (CTP) attached to the amino end of the hGH, and the said. It comprises administering to the subject a therapeutically effective amount of a polypeptide consisting of two villous 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, thereby disclosing methods herein to improve the area under the curve (AUC) of GH to the subject.

In one embodiment, a method of treating a subject in need of GH treatment, to the hGH, one chorionic gonadotropin carboxy-terminal peptide (CTP) attached to the amino end of the hGH, and the carboxy-terminal of the hGH. The subject comprises administering to the subject a therapeutically effective amount of a polypeptide consisting of two adhered villous gonadotropin CTP, the polypeptide lacking a signal peptide, and the amino acid sequence of the polypeptide is SEQ ID NO: 7. , And thereby treating the subject in need of GH in treatment is disclosed herein.

In another embodiment, a method of increasing insulin-like growth factor (IGF-1) levels of interest, hGH and one chorionic gonadotropin carboxy-terminal peptide (CTP) attached to the amino end of the hGH. It comprises administering to the subject a therapeutically effective amount of a polypeptide consisting of two villous gonadotropin CTPs attached to the carboxy terminus of the hGH, the polypeptide lacking a signal peptide and of the polypeptide. The amino acid sequence is set forth in SEQ ID NO: 7, thereby disclosing methods herein to increase insulin-like growth factor (IGF-1) levels of interest.

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

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

In another embodiment, the defined range is the therapeutic dose range achieved by administration of CTP-modified hGH disclosed herein. In another embodiment, the defined range is such that the Cmax and Ctroug of the sinusoideal effect of IGF-I are maintained after continuous administration of the CTP-modified hGH disclosed herein. In another embodiment, the defined range is the 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. be. In another embodiment, the defined range is equivalent to IGF-I levels in an individual considered normal. In another embodiment, the defined range is the normal range of IGF-I levels / values in a normal solid. In yet another embodiment, the defined range is within the normal range when the IGF-ISDS value is ± 2 SDS.

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

In another embodiment, the methods disclosed herein provide a nucleic acid sequence encoding the 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, this conjugate is administered less frequently than unmodified GH. In another embodiment, the CTP-modified GH described herein is administered once a week to once every two weeks. In another embodiment, the CTP-modified GH described herein is administered once every two weeks to once every three weeks. In another embodiment, the CTP-modified GH described herein is administered once daily to three times a week. In another embodiment, reduced dosing frequency results in improved patient compliance, leading to improved treatment outcomes as well as improved patient quality of life. In another embodiment, the GH CTP conjugate having the molecular weight and linker structure of the conjugate disclosed herein has improved efficacy as compared to a conventional GH conjugate bound to poly (ethylene glycol). It was found to have improved stability, increased AUC levels, and improved circulating half-life. In another embodiment, the GH having the molecular weight and linker structure of the conjugate disclosed herein has improved efficacy, compared to a conventional GH conjugate bound to poly (ethylene glycol). It was found to have stability, increased AUC levels, and improved circulating half-life. In another embodiment, the therapeutically effective amount of CTP-modified hGH is the amount of conjugate required for measurable and expected biological activity in vivo. In another embodiment, the GH utilized in accordance with the teachings disclosed herein exhibits increased efficacy. In some embodiments, adhesion of the CTP sequence to both the amino and carboxy ends of GH results in extended in vivo activity.

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

In one embodiment, the methods, polynucleotides, and polypeptides disclosed herein are used in veterinary medicine. In one embodiment, it is fed as a human companion (eg, dogs, cats, horses), has significant commercial value (eg, dairy cows, beef cattle, competition animals), and has significant scientific value (eg, endangered species). Treatment of domesticated mammals that are of value in captivity or other forms) is disclosed herein.

In one embodiment, the polypeptides or polynucleotides disclosed herein are animals (eg, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro pigs, chickens, camels, goats, horses, cows, 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, micro pigs, chickens, goats, cows, etc. Includes, but is not limited to, sheep, dogs, cats, or non-human primates.

In one embodiment, livestock are treated by the methods disclosed herein. In one embodiment, livestock include pigs, beef cattle, dairy cows, horses, goats, sheep, chickens, turkeys, cancers, ducks, and related species. In one embodiment, the laboratory animal is treated by the method disclosed herein. In one embodiment, laboratory animals include rats, mice, guinea pigs, rabbits, goats, monkeys, dogs, cats, and others. In one embodiment, the animals in the zoo are treated by the methods disclosed herein. In one embodiment, the zoo animals include all vertebrates kept in the zoo. In one embodiment, aquatic animals are treated by the methods disclosed herein. In one embodiment, aquatic animals include fish, eels, turtles, seals, penguins, sharks, whales, and related species. In one embodiment, the domestic animal is treated by the method disclosed herein. In one embodiment, the domestic animals include any pets such as cats and dogs, or animals reared by humans such as horses, cows, pigs, goats, rabbits, chickens, turkeys, cancers, ducks and the like. ..

Those skilled in the art will appreciate that the term "pig" includes pigs, piglets, meat pigs, heifers, castrated pigs, breeding pigs, and farmed pigs. Similarly, one of ordinary skill in the art will appreciate that the term "cow" includes calves, cows, dairy cows, heifers, steers, and bulls.

In some embodiments, CTP sequence modification is advantageous in that it allows the use of lower doses.

Manufacturing

In one embodiment, it is a method for producing a human chorionic villous gonadotropin carboxy-terminal peptide (CTP) -modified human growth hormone (hGH) polypeptide, wherein (a) a predetermined number of cells are encoded with the CTP modification (hGH). A step of stably transfecting an expression vector containing a coding moiety to be transfected, wherein the transfected cells express and secrete the CTP-modified hGH, and (b) the CTP-modified hGH. A step of obtaining a cell clone that overexpresses the above, (c) a step of growing the clone in a solution to a predetermined scale, (d) a step of collecting the solution containing the clone, and (e) the step. The step of filtering the solution containing the clone to obtain a purified collection solution and (f) purifying the purified collection solution to have a purified protein solution having the desired concentration of CTP-modified hGH. The amino acid sequence of the CTP-modified hGH produced, comprising the step of obtaining, is set forth in SEQ ID NO: 7, thereby producing a human chorionic gonadotropin carboxy-terminal peptide (CTP) -modified human growth hormone (hGH) polypeptide. The method is disclosed herein. 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. When the final highly expressed clone is selected, each production comprises thawing, growing, collecting, and purifying the working cell bank (WCB) (eg, steps 1-8 of FIG. 3 illustrating clone growth through collection; See steps 8-16 of FIG. 4 to illustrate purification). In an alternative embodiment, when the final high expression clone is selected, each production comprises thawing, growing, collecting, and purifying the master cell bank (MCB) (eg, illustrating clone growth through collection). Steps 1-8 of 3; see steps 8-16 of FIG. 4 illustrating purification).

In one embodiment, the polynucleotides disclosed herein are inserted into an expression vector (ie, a nucleic acid construct) to allow expression of the recombinant polypeptide. In one embodiment, the expression vector disclosed herein comprises an additional sequence that makes this vector suitable for replication and integration in prokaryotes. In one embodiment, the expression vector disclosed herein comprises an additional sequence that makes this vector suitable for replication and integration in eukaryotes. In one embodiment, the expression vector disclosed herein comprises a shuttle vector that makes this vector suitable for replication and integration in both prokaryotes and eukaryotes. In some embodiments, the cloning vector comprises a transcription and translation initiation sequence (eg, promoter, enhancer), as well as a transcription and translation terminator (eg, polyadenylation signal).

In one embodiment, the method of making a CTP-modified polypeptide, eg, CTP-modified hGH, comprises the use of an expression vector, wherein the expression vector comprises a promoter, a coding sequence for the CTP-modified polypeptide, and polyadenylation. Includes peptides and sequences. 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 for expressing the polypeptides disclosed herein. In some embodiments, these include yeast transformed with a recombinant yeast expression vector containing a microorganism, eg, a polypeptide coding sequence; recombinant virus expression vector (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, etc.). Includes, but is not limited to, plant cell lines infected with TMV) or transformed with recombinant plasmid vectors such as the Ti plasmid containing the polypeptide coding sequence.

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

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

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

In some embodiments, expression vectors containing regulatory elements derived from eukaryotic viruses such as retroviruses can be found in the vectors disclosed herein. The SV40 vector includes pSVT7 and pMT2. In some embodiments, the vector derived from bovine papillomavirus comprises pBV-1MTHA and the vector derived from Epstein-Barr virus comprises pHEBO and p2O5. Other representative examples include pMSG, pAV009 / A ⁺ , pMTO10 / A ⁺ , pMAMneo-5, baculovirus pDSVE, and SV-40 early promoter, SV-40 late promoter, metallotionine promoter, mouse breast cancer virus promoter, Included are the Raus sarcoma virus promoter, the polyhedrin promoter, or any other vector that allows expression of the protein under the direction of other promoters that have been shown to be effective for expression in eukaryotic cells.

In one embodiment, various methods can be used to introduce the expression vector encoding the CTP-modified hGH disclosed herein into cells. "Transfections" of eukaryotic host cells with polynucleotides or expression vectors that result in transgenic or transgenic cells are known in the art and are 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. It can be performed by any method described in [Biotechniques 4 (6): 504-512, 1986], including, for example, stable or transient transfection, and electroporation. Further, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive and negative selection methods. Transfection methods include liposome-mediated transfection, calcium phosphate co-precipitation, electroporation, polycation (DEAE-dextran, etc.) -mediated transfection, protoplast fusion, viral infection (including recombinant virus infection), and microinjection. However, but not limited to these. Preferably, the transfection is a stable transfection. Transfection methods that provide optimal transfection frequency and expression of the heterologous gene encoding the peptide of interest disclosed herein in a particular host cell line and type are recommended. A suitable method can be determined by a conventional method. For stable transfection, the construct is integrated into the genome or artificial chromosome / minichromosome of the host cell or is episomally positioned to remain stable in the host cell.

Unless otherwise indicated, the methods disclosed herein use conventional techniques of cell biology, molecular biology, cell culture, immunology, and similar ones in the art. These techniques are well disclosed in the latest literature. For example, Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd ^Ed . , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.C. Y. (1989), Ausubel et al. , Current Protocols in Molecular Biology (1987, updated); Browned. , Essential Molecular Biology, IRL Press (1991), Goddel ed. , Gene Expression Technology, Academic Press (1991), Bothwell et al. 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), Polard et al. , Eds. , Animal Cell Culture, Humana Press (1990), Freshney et al. , Eds. , Culture of Animal Cells, Alan R. et al. Liss (1987), Studzinski, ed. , Cell Growth and Optosis, A Practical Approach, IRL Press at Oxford University Press (1995), Melamed et al. , Eds. , Flow Cytometry and Sorting, Wiley-Liss (1990), Current Cytometry in Cytometry, John Wiley & Sons, Inc. (Updated), Wizard & Hauser, Genetic Engineering of Animals Cells, in: Biotechnology Vol. 2, Puller ed. , VCH, Weinheim 663-744, the series Methods of Enzymeology (Academic Press, Inc.), and Hallow 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 various methods, eg, by viral transformation. The heterologous gene of interest may be introduced into cells as linear DNA or as part of an expression vector. Many eukaryotic expression vectors are known that provide multiple cloning sites for the insertion and expression of one or more heterologous genes. Among the many commercial suppliers, Stratagene, La Jolla, California. , USA, Invitrogen, Carlsbad, California. , 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 by conventional methods. Suitable methods of transfection include, for example, liposome-mediated transfection, calcium phosphate co-precipitation, 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 / minichromosome of the host cell or is episomally contained in the host cell in a stable manner. Transfection methods that provide optimal transfection frequency and expression of one or more heterologous genes of interest in the host cell line are preferred. In one embodiment, the gene of interest encodes the CTP-modified GH disclosed herein.

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

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

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

Those of skill in the art will appreciate that the term "promoter" may include polynucleotide sequences that allow and control transcription of genes or sequences that are functionally bound to it. The promoter contains a recognition sequence for binding RNA polymerase and a start site for transcription (transcription start site). Suitable functional promoters must be selected to express the desired sequence in a particular cell type or host cell. Those of skill in the art will be aware of a variety of promoters from a variety of sources, including constitutive, inducible, and inhibitory promoters. These can be deposited in a databank such as GenBank and obtained as separate elements or as cloned elements within a polynucleotide sequence from a commercially available source or individual source. For inducible promoters, promoter activity can be reduced or increased in response to a 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, the binding of tTA to teto is inhibited. Examples of other inducible promoters are 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 the promoters particularly suitable for high expression in eukaryotes are, for example, hamster ubiquitin / S27a promoter (WO 97/15664), SV40 early promoter, adenovirus tumor late promoter, mouse metallothionein-1 promoter, Ussarcoma. There is a terminal repeat sequence region of the virus, and a human cytomegalovirus. Examples of other heterologous mammalian promoters are actin, immunoglobulins, or heat shock promoters (s).

For example, a promoter may functionally bind to an enhancer sequence to increase transcriptional activity. Several copies of one or more enhancers and / or enhancer sequences, such as CMV or SV40 enhancers, may be used for this purpose. For example, a promoter may functionally bind to an enhancer sequence to increase transcriptional activity. Several copies of one or more enhancers and / or enhancer sequences, such as CMV or SV40 enhancers, may be used for this purpose.

Those 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 functionally connects to this promoter. Let's do it. Unlike promoters, enhancer effects are position- and placement-independent, so they may be located in front of or behind the transcription unit, in an intron, and even within a coding region. 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. We have many enhancers from a variety of sources available as independent elements, or elements cloned within a polynucleotide sequence (eg, ATCC-deposited or of commercial or individual source). (And you will know the deposits in data banks such as GenBank, eg SV40 enhancers, CMV enhancers, polyoma enhancers, adenovirus enhancers). Many promoter sequences also contain enhancer sequences such as the frequently used 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, polyadenylation signal termination, and other expression control elements. Both inducible and structural regulatory sequences are known for various cell types. A "transcriptional regulatory element" generally comprises an upstream promoter of the expressed gene sequence, a transcription initiation and termination site, and a polyadenylation signal.

One of skill in the art will appreciate that the term "transcription initiation site" may include nucleic acids in constructs that correspond to the primary transcript, the first nucleic acid that integrates into the pre-mRNA. The transcription initiation site can overlap with the promoter sequence.

Those skilled in the art understand that the term "transcriptional termination site" is usually at the 3'end of the gene of interest or the gene section to be transcribed and may include nucleotide sequences that result in the termination of transcription by RNA polymerase. There will be.

Those skilled in the art have described the term "polyadenylation signal" as a sequence of approximately 100-200 adenine nucleotides at a specific site at the 3'end of eukaryotic mRNA, and at the cleaved 3'end (polyA). It will be appreciated that it may include a signal sequence that triggers the post-translational introduction of tile). The polyadenylation signal comprises a sequence AATAAA upstream of 10-30 nucleotides at the cleavage site, and a sequence located downstream. Various polyadenylation elements are known, such as tk polyA, late and early SV40 polyA, or BGH polyA (eg, described in US Pat. No. 5,122,458).

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

Those skilled in the art will appreciate that "polypeptides", "polypeptides", or synonyms thereof, are used for amino acid sequences or proteins and may include polymers of amino acids of any length. You will understand. The term also includes proteins that have been post-translated and 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 fusion with other proteins, while preserving its biological activity.

In order to produce one or more gene products of interest intracellularly, the cells may be grown in serum-free medium and in suspension culture medium under conditions that allow expression of the gene of interest. For example, if the gene of interest is under the control of a structural promoter, no special inducer needs to be added. 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 in a sufficient but non-toxic concentration. The cells can be proliferated 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 secretory.

In one embodiment, the step of producing the CTP-modified hGH disclosed herein stabilizes an expression vector comprising a coding portion encoding the CTP-modified hGH disclosed herein in a predetermined number of cells. Includes transfection. In another embodiment, the nucleotide sequence of the coding moiety is set forth in SEQ ID NO: 9. In another embodiment, the step of producing CTP-modified hGH comprises transfecting cells with an expression vector comprising a coding moiety encoding 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 (FIG. 2). In another embodiment, the transfected cells express the CTP-modified hGH disclosed herein. In another embodiment, the CTP-modified hGH produced and expressed comprises two villous gonadotropin carboxy-terminal peptides attached to the carboxy terminus of the hGH and one villous gonadotropin carboxy-terminal peptide attached to the amino terminus of the hGH. 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 is two villous gonadotropin carboxy-terminal peptides attached to the carboxy-terminal of the hGH and one villous property attached to the amino-terminal of the hGH. The amino acid sequence of mature CTP-modified hGH consisting of a gonadotropin carboxy-terminal peptide and expressed and secreted is set forth in SEQ ID NO: 7. In other embodiments, the expression of CTP-modified hGH is at high expression levels. In another embodiment, the CTP-modified hGH is highly glycosylated. In another embodiment, the CTP-modified hGH is highly sialic acid-added. As described in detail above, CTP-modified hGH can have different glycosylation content and patterns. The CTP-modified hGH produced by the method disclosed herein may contain any of the glycosylated patterns and contents disclosed above. In general, the production methods presented herein provide CTP-modified hGH with a high glycosylated content and a high percentage of glycosylated 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 clone of step (b) is propagated in the medium 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 products disclosed herein are obtained from cell culture media as secretory gene products. One of skill 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 secretory signal, the gene product can also be isolated from cytolysis. 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 lysates. The desired gene product is then subjected to, for example, immunoaffinity and fractionation on an ion exchange column, ethanol precipitation, sephadex, hydroxyapatite, silica, or reverse phase HPLC or chromatography on a cation exchange resin, such as DEAE. , Mixed soluble proteins, polypeptides, and nucleic acids (see examples herein). Methods known in the art that result in purification of heterologous proteins expressed by recombinant host cells are known to those of skill in the art and are known to those of skill in the art, such as Harris et al., Protein Purification: A Practical Approach, Pickwood and. It is described by 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 chromatographic methodology based on the needs and knowledge of the art. For example, one of ordinary skill in the art may choose an alternative cation exchange column instead of the DEAE column as long as the purpose of separation by cation exchange is maintained. In the art for preparative, immunoaffinity, protein precipitates, nucleic acid precipitates, ethanol precipitates, reverse phase HPLC, or chromatography on cefadex, hydroxyapatite, silica, anion exchange or cation exchange resins. Similar known alternatives will be understood by those skilled in the art.

Another embodiment is a method of preparing one or more products in mammalian cells under serum-free conditions, wherein (i) the mammalian cells are the CTP-modified hGH polypeptides disclosed herein. (Iii) The mammalian cells are proliferated under serum-free conditions that allow replication of the mammalian cells, and (iii) in each case, these mammalian cells. At least one of (s) is deposited in cell culture vessels under serum-free conditions, and (iv) suitably deposited mammalian cells are replicated under serum-free conditions. , (V) The replicated cells are cultured under serum-free conditions where 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 the method characterized by that is 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 the gene of interest has been introduced. Therefore, the method of preparing the recombinant gene product disclosed herein is that the mammalian cells are transfected with at least the nucleic acid encoding the gene of interest prior to step (i) of the above process. Can be characterized by. Stable transfection of the corresponding mammalian cells is preferred.

Examples of serum-free, protein-free, or known composition media include, for example, commercially available media such as Ham F12 (Sigma, Disenhofen, DE), RPMI 1640 (Sigma), Dalbeco Modified Eagle's Medium (DMEM; Sigma). ), Minimal essential medium (MEM; Sigma), Iskov modified Darvecco medium (IMDM; Sigma), CDCHO (Invitagen, Carlsbad, Calif., USA), CHOS-SFMII (Invitagen), Serum-free CHO medium (Sigma), CD- Examples include PowerCHO2 medium (Lonza) and protein-free CHO medium (Sigma). Each of these media, if desired, hormones and / or other growth factors (eg, insulin, transferase, epidermal growth factor, insulin-like growth factor), salts (eg, sodium chloride, calcium, magnesium, etc.) Phosphate), buffers (eg HEPES), nucleosides (eg adenosine, thymidin), glutamine, glucose or equivalent nutrients, antibiotics and / or trace elements, or commercially available Power Feed A (eg,) It can be supplemented with a variety of compounds, such as feeds such as Lonza) or Cell Boost 6 (HyCLone). If the replicable cells are recombinant cells expressing one or more selectable markers, then one or more suitable selective agents, such as antibiotics, may also be added to the medium.

In addition to containing the elements required for transcription and translation of the inserted coding sequence (encoding the polypeptide), the expression constructs disclosed herein are the stability, production, purification, of the expressed polypeptide. It may also include sequences engineered to optimize yield or activity.

In some embodiments, the transformed cells are cultured under effective conditions that allow for 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, the effective medium may include any medium in which cells are cultured to produce the recombinant polypeptides disclosed herein. In some embodiments, the medium typically comprises an absorbable carbon, nitrogen, and phosphate source, as well as an aqueous solution having suitable salts, minerals, metals, and other nutrients such as vitamins. In some embodiments, the cells disclosed herein may be cultured in conventional fermentation bioreactors, shaking flasks, test tubes, microtiter dishes, and petri dishes. In some embodiments, the culture is carried out at a temperature, pH, and oxygen content appropriate for the recombinant cells. In some embodiments, the culture conditions are based on the expertise of one of ordinary skill 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 condition comprises a pH that starts at one temperature and shifts to another temperature during production. 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. It shifts 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, recovery of the recombinant polypeptide is provided after a predetermined time in culture.

Those of skill in the art will appreciate that the phrase "recovering a recombinant polypeptide" may include collecting the entire medium containing the polypeptide and may 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. Purification is performed using a variety of standard protein purification techniques such as, but not limited to, A chromatography, hydroxyapatite chromatography, isoelectric point electrophoresis and differential solubilization.

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

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

In one embodiment, the polypeptides disclosed herein are withdrawn in "substantially pure" form.

One of ordinary skill in the art will appreciate that the phrase "substantially pure" may include a purity that allows for effective use of the protein in the applications described herein. Such forms include highly glycosylated and highly sialic acid-added forms 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, recombinant DNA technology is used to generate CTP-modified GH.

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

In one embodiment, the method of producing CTP-modified hGH disclosed herein comprises obtaining a clone from the WCB that optimally expresses and secretes the CTP-modified hGH and proliferating the clone. In another embodiment, the method of producing CTP-modified hGH comprises the step of obtaining a clone optimally expressing the CTP-modified hGH from the MCB and propagating the clone. In another embodiment, the method of manufacture comprises a clone that optimally expresses and secretes CTP-modified hGH. In another embodiment, cell clones are grown in solution to the level of the producing bioreactor through a series of subculture steps. 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 includes a stainless steel bioreactor, a locking motion bioreactor such as GE's Wave system, a perfusion bioreactor, or any other bioreactor system known in the art. In one embodiment, removal of cells from the bioreactor is accomplished by the use of a disposable filtration system. If large-scale production is carried out, continuous centrifugation can be used prior to the use of the filtration system.

In one embodiment, cell clones are further grown or grown by sequentially culturing the cells in a progressively larger bioreactor 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 repetitive batch mode. In another embodiment, the bioreactor is operated in 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 viable cell density of the bioreactor used in the manufacturing method disclosed herein is approximately 0.2 × 10 ⁶ to 1.4 × 10 ⁶ cells / ml. .. In another embodiment, the peak viable cell density of the bioreactor used in the manufacturing method disclosed herein is from about 0.05 × ^{106 to 100 × 10 6 .} In another embodiment, the peak viable cell density of the bioreactor is from about 0.05 × ¹⁰⁶ to 0.5 × ¹⁰⁶ . In another embodiment, the peak viable cell density of the bioreactor is about 0.5 × 10 ^6-5 × 10 ⁶ . In another embodiment, the peak viable cell density of the bioreactor is about 5.0 × ¹⁰⁶ to 50 × ¹⁰⁶ . In another embodiment, the peak viable cell density of the bioreactor is about ^50x106 to ^100x106 . Each possibility represents a separate embodiment disclosed herein.

Supply schemes for bioreactor utilization can vary, for example, depending on whether they are repeatedly supplied once a day from a particular day or fixed for several days, plus the percentage of added supply is a few percent. It can vary from up to 50% or more. 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 of producing CTP-modified hGH comprises purifying the purified 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 contains at least 50% CTP-modified hGH. In another embodiment, the purified protein solution contains at least 60% CTP-modified hGH. In another 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 protein solution contains at least 90% CTP-modified hGH.

In one embodiment, the purified collection is kept at 2-25 ° C. for up to 24 hours. In another embodiment, the purified 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 particles, and / or mycoplasma, or these. Tested for any combination.

In one embodiment, purification of the purified collection in step (f) is (g) a step of concentrating, dialysis filtering, and purifying the purified collection solution, wherein the concentration, dialysis filtration, and Purification is accomplished by a hollow thread cassette or a tangential flow cassette in which the purified collection solution is sequentially passed through an anion exchange column and a hydrophobic interaction column, and the purification is performed following step (h). The steps to obtain the collection, (i) the inactivating step present in the purified collection by incubating in a solution toxic to the virus, and (j) the purified collection solution. The step of concentrating, dialysis filtering, and purifying the purified collection solution obtained from (h), wherein the concentration, dialysis filtration, and purification is the hydroxyapatite mixed mode column and the purified collection solution. Following the step of sequentially passing through a cation exchange column, and (j) step (i), the purified collection solution is obtained, and the purified collection solution is physically obtained from the virus by nanofiltration. Purification containing the highly purified form of the CTP-modified polypeptide, with the removal step followed by (k) step (j) to obtain the purified collection solution and maximally purifying. It is accomplished by sequentially performing steps including, and: In another embodiment, the method disclosed herein comprises a centrifugation step prior to final filtration. Those skilled in the art will appreciate that both centrifugation and / or filtration are methods of purifying the solution.

In one embodiment, extrafiltration and dialysis filtration to concentrate and filter the purified collection can be performed using hollow fiber cartridges or equivalent TTF-based UFDF steps. The nominal molecular weight cutoff size of the cartridge is 10,000 kDa. In another embodiment, the membrane cartridge may comprise a PES / PS / RC membrane with a cutoff of 3 kDa to 30 kDa.

In another embodiment, the anion exchange column of step (g) is a DEAE-Sepharose Fast Flow column. In another embodiment, the DEAE column purifies the 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 include other anion exchange columns known in the art, such as Capto DEAE anion exchange columns or other resins such as 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 from about 1-10. In one embodiment, one to three 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 cleaning and elution. In one embodiment, the elution buffer comprises ammonium sulphate containing propylene glycol. In one embodiment, the elution buffer comprises ammonium sulphate 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 from about 1 to 10. In one embodiment, one to three 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, elution is carried out at about 3 CV. In another embodiment the elution is carried out at about 4 CV. In another embodiment the elution is carried out at about 5 CV. In another embodiment the elution is carried out at about 6 CV. In another embodiment the elution is carried out at about 7 CV. In another embodiment the elution is carried out at about 8 CV. In another embodiment the elution is carried out at about 9 CV. In another embodiment the elution is carried out at about 10 CV.

In one embodiment, the virus that may be present in the purified collection due to the mixture is inactivated in the purified 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 a 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 a 5-10% Triton-X100 solution. In another embodiment, virus inactivation in Triton-X100 solution lasts 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 Triton-X100 solution lasts about 1-2 hours. In another embodiment, virus inactivation in Triton-X100 solution lasts about 2-3 hours. In another embodiment, virus inactivation in Triton-X100 solution lasts about 3-4 hours. In another embodiment, virus inactivation in Triton-X100 solution lasts about 4-6 hours. In another embodiment, virus inactivation in Triton-X100 solution lasts about 6-8 hours. In another embodiment, virus inactivation in Triton-X100 solution lasts about 8-10 hours. In another embodiment, virus inactivation in Triton-X100 solution lasts about 10-12 hours. In another embodiment, virus inactivation in Triton-X100 solution lasts about 12-24 hours.

Other concentrates or other solutions available in the art and toxic to these viruses, including but not limited to sodium cholic acid and Tween80, can be used in the methods disclosed herein. That 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 inactivating the virus comprises lowering the pH. Those skilled in the art will appreciate that the inactivation of the virus may include changing the conditions of the solution as is known in the art.

In one embodiment, the virus is physically removed by using nanofiltration. Those skilled in the art will appreciate that any filter for virus removal known in the art may 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 a method that requires confirmation of virus clearance from purified collections 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 the purposes herein.

In one embodiment, the methods disclosed herein achieve a recovery of at least 20% for 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. Achieve 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.9% recovery.

In one embodiment, following the purification of the highly glycosylated CTP-modified hGH disclosed herein, the methods disclosed herein further comprise characterizing the CTP-modified polypeptide. .. In another embodiment, the purity of CTP-modified hGH is determined. In another embodiment, the glycosylated 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, the cell clones utilized in the methods disclosed herein are stored in a frozen cell bank. In another embodiment, the 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, cellbanks are intended for the production of clinical grade materials. In another embodiment, CellBank complies with regulatory practices for human use. In another embodiment, the cell bank is any other type of cell bank known in the art.

"Good Manufacturing Practice" is another embodiment defined by Code of Federal Regulations (21 CFR 210-211). In another embodiment, "Good Manufacturing Practice" is defined by other standards for the production or human consumption of clinical grade materials, 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 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 appreciated that the term "medium" may include liquids or gels or powders suitable for the growth or culture of cells containing the CTP-modified hGH disclosed herein. Such media are optionally referred to as "proliferation media" or "culture media" and may include, but are not limited to, nutrient media, fortified media, minimal media, confirmation media, transport media, or selective media. In a further embodiment, the selective medium may be suitable for selecting a particular group of cells during the production 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" is associated with 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 that it may include glycosylated levels of (see, eg, those measured by reverse phase HPLC in FIG. 11). In another embodiment, the collection containing highly glycosylated CTP-modified hGH has a glycosylated level of at least 50% of the CTP-modified hGH polypeptide in the purified solution. In another embodiment, the collection containing highly glycosylated CTP-modified hGH has a glycosylated level of at least 60% of the CTP-modified hGH polypeptide in the purified solution. In another embodiment, the collection containing highly glycosylated CTP-modified hGH has a glycosylated level of at least 70% of the CTP-modified hGH polypeptide. In another embodiment, the highly glycosylated CTP-modified hGH has a glycosylated level of at least 80% of the CTP-modified hGH polypeptide in the purified solution. In another embodiment, the collection comprises about 81-90% glycosylated levels of total CTP-modified hGH polypeptide in purified solution. In another embodiment, the highly glycosylated CTP-modified hGH has a glycosylated level of at least 90% of the CTP-modified hGH polypeptide in the purified solution. In another embodiment, the collection comprises about 91-95% glycosylated levels of total CTP-modified hGH polypeptide in purified solution. In another embodiment, the collection comprises a glycosylation level of about 95.1-99% of the total CTP-modified hGH polypeptide in the purified solution. In another embodiment, the collection comprises 100% glycosylated levels of total CTP-modified hGH polypeptide in purified solution.

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

Those skilled in the art will appreciate that the term "drug substance" (DS) may include or be equivalent to the 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. Those skilled in the art will also appreciate that the term "pharmaceutical" (DP) may include a drug that is aseptically formulated into a final container, eg, a vial, and is finally formulated. In one embodiment, the CTP-modified hGH polypeptide drug substance (DP) set forth in SEQ ID NO: 7 comprises the finally formulated CTP-modified hGH.

The highly glycosylated CTP-modified hGH polypeptide is a long-acting hGH (eg, MOD-4023) that aids in reduced dosing frequency, eg, once a week, in patients with growth hormone deficiency. ) May have beneficial properties in the usage. 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 extended circulation time for 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 adhered CTP is at least 4-6. In another embodiment, the number of O-glycans per CTP-modified hGH polypeptide with one adhered CTP is at least 6-8. In another embodiment, the number of O-glycans per CTP-modified hGH polypeptide with one adhered CTP is at least 4-8. In another embodiment, the number of O-glycans per CTP-modified hGH polypeptide with two bonded CTPs is at least 8-12. In another embodiment, the number of O-glycans per CTP-modified hGH polypeptide with two adhered CTPs is at least 12-16. In another embodiment, the number of O-glycans per CTP-modified hGH polypeptide with two bonded CTPs is at least 8-16. In another embodiment, the number of O-glycans per CTP-modified hGH polypeptide with three adhered CTPs is at least 12-18. In another embodiment, the number of O-glycans per CTP-modified hGH polypeptide with three adhered CTPs is at least 18-24. In another embodiment, the number of O-glycans per CTP-modified hGH polypeptide with three adhered CTPs is at least 12-24. In another embodiment, the number of O-glycans per CTP-modified hGH polypeptide with 4 adhered CTPs is at least 16-24. In another embodiment, the number of O-glycans per CTP-modified hGH polypeptide with four adhered CTPs is at least 24-32. In another embodiment, the number of O-glycans per CTP-modified hGH polypeptide with 4 adhered CTPs is at least 16-32. In another embodiment, the number of O-glycans per CTP-modified hGH polypeptide with 5 adhered CTPs is at least 20-30. In another embodiment, the number of O-glycans per CTP-modified hGH polypeptide with 5 adhered CTPs is at least 30-40. In another embodiment, the number of O-glycans per CTP-modified hGH polypeptide with 5 adhered CTPs is at least 20-40.

In another embodiment, the number of O-glycans in each CTP-hGH-CTP-CTP (also known as "MOD-4023") is at least 12-18. 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 sialic acid-added. When the term "highly sialic acid-added" is associated with CTP-modified hGH, it may include about 60-80% sialic acid addition levels of the CTP-modified hGH polypeptide in the purified solution. Those skilled in the art will understand. In another embodiment, it may comprise about 60-70% sialic acid addition levels of the total CTP-modified hGH polypeptide in the purified solution. In another embodiment, it may comprise about 70-80% sialic acid addition levels of the total CTP-modified hGH polypeptide in the purified solution. In another embodiment, about 80-90% sialic acid addition levels of the total CTP-modified hGH polypeptide in the purified solution. In another embodiment, about 90-95% sialic acid addition levels of the total CTP-modified hGH polypeptide in the purified solution. In another embodiment, about 95.1-99% sialic acid addition levels of the total CTP-modified hGH polypeptide in the purified solution. In another embodiment, 100% sialic acid addition levels of the total CTP-modified hGH polypeptide in the purified solution. In another embodiment, the O-glycan bond in CTP-modified hGH comprises monosialic acid-added core 1.

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

In one embodiment, CTP-modified hGH is expressed at a level of at least 600 mg / L. In another embodiment, 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 to 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.

It will be appreciated by those skilled in the art 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 is the amount of corresponding mRNA or cDNA present in the cell, or the desired sequence encoded as in the examples of the invention. Can be determined based on either the amount of the polypeptide / protein of interest in. For example, mRNA transcribed from selected sequences can be obtained by Northern blot hybridization, ribonuclease RNA protection, in-situ hybridization to intracellular RNA, or by PCR (Sambrook et al., 1989, Ausubel et al.,. It can be quantified by (see 1987 hybridized). The protein encoded by the selected sequence is subjected to FACS analysis (Sambrook et) following immunostaining of the protein by assaying for the biological activity of the protein by various methods, eg, ELISA, Western blotting, radiation sedimentation. It can be quantified by (see al., 1989, Ausube et al., 1987 updated) or by a heterologous time-resolved fluorescence assay (HTRF) assay. In one embodiment, quantification of CTP-modified hGH comprises the use of reverse phase high performance liquid chromatography (RP-HPLC). In another embodiment, the RP-HPLC comprises a C-18 column. In another embodiment, the RP-HPLC comprises a C-8 column. In another embodiment, the method disclosed herein uses RP-HPLC to quantify CTP-modified hGH in the collection (see steps 3-8 of the examples). In another embodiment, the method disclosed herein uses RP-HPLC to quantify CTP-modified hGH in the first step of purification (see Steps 9-12 of Examples). ).

In another embodiment, the cellbank or frozen stock disclosed herein exhibits a survival rate of> 90% upon thawing. In another embodiment, storage extends for an indeterminate amount of time. Each possibility represents a separate embodiment disclosed herein.

In another embodiment, storage lasts for 2 weeks. In another embodiment, storage lasts for 3 weeks. In another embodiment, storage lasts for one month. In another embodiment, storage lasts for 2 months. In another embodiment, storage lasts for 3 months. In another embodiment, storage lasts for 5 months. In another embodiment, storage lasts up to 6 months. In another embodiment, storage lasts for 9 months. In another embodiment, storage lasts for one year.

In another embodiment, the cell bank or frozen stock disclosed herein allows the growth of cell culture medium in the specified medium disclosed herein and the culture medium in a solution containing glycerol. It is cryopreserved by methods including freezing and storing cell clones below -20 ° C. In another embodiment, the temperature is about −70 degrees Celsius. In another embodiment ^, the temperature is about ^-70-80 degrees Celsius. In another embodiment, any defined medium disclosed herein may be used in this manner. Each defined medium represents a separate embodiment disclosed herein.

In another embodiment of the methods and compositions disclosed herein, the culture is planted from Cellbank. In another embodiment, the culture is planted from frozen stock. In another embodiment, the culture is planted from seed culture. In another embodiment, the culture medium is planted from the cell colony. In another embodiment, the culture medium is planted in the mid-logarithmic growth phase. In another embodiment, the culture medium is planted approximately in the mid-logarithmic 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 comprises 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 colligative additive known in the art or an additive having antifreeze properties. Each possibility represents a separate embodiment disclosed herein.

In one embodiment, the frozen solution for the composition used in this method and disclosed herein comprises a culture supernatant and DMSO. In one embodiment, the frozen solution for the composition used in this 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 the growth of the cell culture medium. 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 the growth of the cell culture medium. 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 the saturated state. In another embodiment the concentration is 15% of the saturated state. In another embodiment the concentration is 16% of the saturated state. In another embodiment the concentration is 18% of the saturated state. In another embodiment the concentration is 22% saturated. In another embodiment the concentration is 25% of the saturated state. In another embodiment the concentration is 30% of the saturated state. In another embodiment the concentration is 35% of the saturated state. In another embodiment the concentration is 40% of the saturated state. In another embodiment the concentration is 45% of the saturated state. In another embodiment the concentration is 50% of the saturated state. In another embodiment the concentration is 55% of the saturated state. In another embodiment the concentration is 60% of the saturated state. In another embodiment the concentration is 65% of the saturated state. In another embodiment the concentration is 70% of the saturated state. In another embodiment the concentration is 75% of the saturated state. In another embodiment the concentration is 80% of the saturated state. In another embodiment the concentration is 85% of the saturated state. In another embodiment the concentration is 90% of the saturated state. In another embodiment the concentration is 95% of the saturated state. In another embodiment the concentration is 100% saturated. In another embodiment the concentration is close to 100% of the saturated state.

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

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

In another embodiment, the freezing step is performed when the culture medium has a density of 1 × 10 ⁶ live cells (VC) / ml. In another embodiment, the biomass is 1.5 × ¹⁰⁶ VC / ml. In another embodiment, the biomass is 1.5 × ¹⁰⁶ 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 × ¹⁰⁶ VC / ml. In another embodiment, the biomass is 20 × ¹⁰⁷ VC / ml. In another embodiment, the biomass is 25 × ¹⁰⁶ VC / ml. In another embodiment, the biomass is 30 × ¹⁰⁷ VC / ml. In another embodiment, the biomass is 33 × ¹⁰⁶ VC / ml. In another embodiment, the biomass is 40 × ¹⁰⁶ VC / ml. In another embodiment, the biomass is 50 × ¹⁰⁶ VC / ml. In another embodiment, the biomass is greater than 50 × ¹⁰⁶ VC / ml.

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

The terms "cell culture" and "tissue culture" are used interchangeably for cells in vitro, in suspension culture medium 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 that it means 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 required nutrients and is osmotic to the particular cells maintained while controlling the temperature and gas phase within suitable limits. Can be adapted to. Cell culture techniques are known in the art. For example, Morgan et al. 1993 Animal Cell Culture, BIOS Scientific Publics, Oxford, UK, and Adams, R. et al. L. P. See 1990 Cell Culture for Biochemists, Second Edition, Elsevier.

It will be appreciated by those skilled in the art that the term "passage" may include the effects of subculture of cell populations. A "passage culture" can include a cell culture established by planting a sterile medium with a sample from a previous culture, which is a fresh sterile medium in one embodiment.

It will also be appreciated by those skilled in the art that the term "cell line" may include 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 in a regular sense over several months to maintain the cell line. Subculture 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 particular functional markers such as keratin, hormones and growth factor receptors.

In some embodiments, the culture may be carried out in a serum-free defined medium containing added growth factors. In another embodiment, the medium contains serum with or without added growth factors. Such changes can be empirically determined by one of ordinary skill in the art to optimize cell proliferation.

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

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

In another embodiment of the methods and compositions disclosed herein, the storage temperature of the culture is ^between -20 ^and -80 degrees Celsius (° C.). In another embodiment the temperature is significantly lower than ^-20 ° C. In another embodiment the temperature is no higher than ^-70 ° C. In another embodiment ^, the temperature is −70 ° C. In another embodiment the temperature is about ^-70 ° C. In another embodiment the temperature is ⁻²⁰ ° C. In another embodiment the temperature is about ⁻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 −30 ^to −70 ° C. In another embodiment ^, the temperature is −40 ^to −70 ° C. In another embodiment ^, the temperature is -50 ^to -70 ° C. In another embodiment, the temperature is ^between -60 ^and -70 ° C. In another embodiment ^, the temperature is -30 ^to -80 ° C. In another embodiment ^, the temperature is -40 ^to -80 ° C. In another embodiment ^, the temperature is -50 ^to -80 ° C. In another embodiment, the temperature is ^between -60 ^and -80 ° C. In another embodiment ^, the temperature is −70 ^to −80 ° C. In another embodiment the temperature is below ^-70 ° C. In another embodiment the temperature is below ^-80 ° 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 they are maintained at a temperature below -130 ° C. The vial is transferred to a liquid nitrogen freezer for this purpose.

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

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

In another embodiment of the methods and compositions disclosed herein, cells exhibit proliferation after long-term cryopreservation or freeze storage followed by thawing. In another embodiment, cells show proliferation within about 15-22 hours after planting cells from cell bank or seed culture in fresh medium. In another embodiment, cells show proliferation within about 12-20 hours after planting cells from cell bank or seed culture in fresh medium. In one embodiment, the cell line must be maintained in exponential growth (via regular subculture) to ensure viability, genetic stability, and phenotypic stability.

Those skilled in the art will appreciate that the term "long term" of cryopreservation or cryopreservation can include 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 two 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> 90% after thawing following cryopreservation. In another embodiment, the survival rate on thawing following the 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 vaccinated cell bank, frozen stock, or batch disclosed herein is grown in a defined cell culture medium. Such media are known in the art and include Dalveco Modified Eagle's Medium (DMEM) (ATCC® No. 30-2002), Iskov Modified Dalveco Medium (IMDM) (ATCC® No. 30-2005), Hybrid-Care medium (ATCC® No. 46-X), McCoy 5A and RPMI-1640 (ATCC® No. 30-2007), ham nutritional mixture (ATCC®). Includes, but is not limited to, CCL-61 ™), PowerCHO ™ known composition serum-free CHO medium (Lonza Cat. No. 12-771Q), or any other medium known in the art. .. In another embodiment, these media may be supplemented with antibiotics or animal serum, as determined empirically by those of skill in the art.

In one embodiment, bioreactors and methods that allow the cultivation of mammalian cells in large quantities are disclosed herein. Yet yet, in another embodiment, the bioreactor and method allow the culture of mammalian cells under optimal conditions even when grown in large quantities, thus a process independent of the size of the bioreactor. Enables performance and production quality. The duration of incubation within the bioreactor can vary by simply changing the scale and bioreactor system, for example, the duration can be between 8-9 days, or between 15-16 days. In another embodiment, the incubation period 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 of the possibilities listed above is an embodiment disclosed herein.

In another embodiment, while maintaining a well-mixed cell suspension in the bioreactor and mixing the nutrient feed, heterogeneous with respect to process parameters such as pH, dissolved oxygen partial pressure (DOT), and temperature. Large-scale bioreactors that allow the cultivation of mammalian cells in the environment are disclosed herein. In another embodiment, the bioreactor is a disposable bioreactor.

The manufacturing methods disclosed herein are the technical underlying methods 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 500 L. In another embodiment, the volume 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, cells are subcultured in progressively larger volumes of bioreactor (see Examples herein).

Composition

In some embodiments, growth and weight are associated with CTP-modified human growth hormone (hGH) polypeptides disclosed herein and produced using the methods disclosed herein. Symptoms such as growth deficiency disorder, AIDS exhaustion, aging, immune dysfunction in HIV infected subjects, degradative diseases, postoperative recovery, congestive myocardium, liver transplantation, liver regeneration after liver resection, chronic renal failure, kidney Chronic inflammatory or malnutrition such as osteodystrophy, osteoporosis, aplasia / hypochondral dysplasia, skeletal dysplasia, Crohn's disease, short bowel syndrome, juvenile rheumatoid arthritis, cystic fibrosis, Subjects with male infertility, X-chain hypophosphorus disease, Down's syndrome, spinal rupture, Noonan's syndrome, obesity, muscle weakness, and fibromyalgia can be treated. In one embodiment, the polypeptides disclosed herein may be provided to the individual itself. In one embodiment, the polypeptide disclosed herein can be provided to an individual as part of a pharmaceutical composition in which it is mixed with a pharmaceutically acceptable carrier.

Those skilled in the art will appreciate the formulation of one or more active ingredients described herein in which the term "pharmaceutical composition" is combined with other chemical components such as physiologically suitable carriers and excipients. You will understand that it can be included. The purpose of the pharmaceutical composition is to facilitate the administration of the compound to the organism.

The modified peptides disclosed herein are mixed with carriers, diluents and the like known per se, which can be administered orally or parenterally to mammals (eg, bovine, horse, pig, sheep, human). It can be formulated into a suitable pharmaceutical preparation such as capsules and injections.

Those skilled in the art will appreciate that the term "active ingredient" may include a polypeptide sequence of interest that can explain the biological effect.

In some embodiments, each of the compositions disclosed herein will comprise at least two CTP sequences bound to the protein of interest in any form. In one embodiment, the combined formulation is disclosed herein. Those skilled in the art will appreciate that the term "combination formulation" is used by the combination partners defined above independently or by the use of different fixed combinations in prominent amounts of the combination partners, ie, simultaneously (simultaneously). It will be appreciated that a "parts kit" can be included in the sense that it can be administered concurrently, separately or sequentially. In some embodiments, the parts of the parts kit may then be administered, for example, simultaneously or at different times for any part of the parts kit, and at equal or different time intervals, staggered over time. In some embodiments, the ratio of the total amount of the combination partner can be administered to the combination formulation. In one embodiment, a single patient may have different needs, eg, due to a particular disease, the severity of the disease, age, gender, or body weight. Can vary to meet the needs of the population, which can be readily made by one of ordinary skill in the art.

Those skilled in the art will appreciate that the terms "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" that can be used interchangeably do not cause significant irritation to the organism and that the bioactivity of the administered compound and It will be appreciated that carriers or diluents that do not suppress properties may be included. The adjuvant is included in these phrases. In one embodiment, one of the components contained in a 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)).

Those skilled in the art will appreciate that the term "excipient" may include an inert substance added to a 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 glycol.

The technique of drug formulation and administration is described in "Remington's Pharmaceutical Sciences" Mac Publishing Co., Ltd. , Easton, PA, latest edition, which is incorporated herein by reference.

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

In one embodiment, the formulation is administered locally rather than in a systemic manner, eg, via direct injection of the formulation into a particular 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 in a 0.1-5 ml solution at a dose of 1-50 micrograms. In another embodiment, the CTP-modified hGH polypeptide is administered in a 0.1-5 ml solution at a dose of 1-25 micrograms. In another embodiment, the CTP-modified hGH polypeptide is administered in a 0.1-5 ml solution at a dose of 50-90 micrograms. In another embodiment, the CTP-modified hGH polypeptide is administered in a 0.1-5 ml solution at a dose of 10-50 micrograms.

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

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

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

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

In another embodiment, the dose of CTP-modified hGH polypeptide given to a subject is a reference subject from the same subject population (eg, children, the elderly, men, women, GH deficiency, specific nationalities, etc.). 50% of the standard dose given. In another embodiment, the dose is 30% of the dose given to a subject from a particular population of subjects. In another embodiment, the dose is 45% of the dose given to a subject from a particular population of subjects. In another embodiment, the dose is 100% of the dose given to a subject from a particular population of subjects.

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

In another embodiment the dose is 1-5 mg / dose. In another embodiment the dose is 2 mg / dose. In another embodiment the dose is 4 mg / dose. In another embodiment the dose is 1.2 mg / dose. In another embodiment the dose 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 into 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 in the range of 0.0001 mg to 0.6 mg. In another embodiment, the CTP-modified hGH polypeptide is administered to the subject at a dose in the range of 0.001 mg to 0.005 mg. In another embodiment, the CTP-modified hGH polypeptide is administered to the subject at a dose in the range of 0.005 mg to 0.01 mg. In another embodiment, the CTP-modified hGH polypeptide is administered to the subject at a dose in the range of 0.01 mg to 0.3 mg. In another embodiment, the CTP-modified hGH polypeptide is administered to the subject at a dose in the range of 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 in the range of 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, the CTP-modified GH is administered to the subject at a dose in the range of 0.2 mg to 2 mg. In another embodiment, the CTP-modified hGH polypeptide is administered to the subject at a dose in the range of 2 mg to 6 mg. In another embodiment, the CTP-modified hGH polypeptide is administered to the subject at a dose in the range of 4 mg to 10 mg. In another embodiment, the CTP-modified hGH polypeptide is administered to the subject in doses ranging from 5 mg and 15 mg.

In another embodiment, the CTP-modified hGH polypeptide disclosed herein is 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 treatment, providing the subject in need thereof with the CTP-modified hGH polypeptide disclosed herein. And thereby improve compliance in the use of GH treatment.

In another embodiment, protein agents having a molecular weight of less than 50,000 daltons, such as the CTP-modified hGH polypeptide 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 polypeptide disclosed herein prolongs the half-life of a protein agent having a molecular weight of less than 50,000 daltons, such as GH.

In another embodiment, the immunogenicity of CTP-modified hGH is equal to that of isolated GH. In another embodiment, the immunogenicity of CTP-modified hGH is comparable to that of isolated GH. In another embodiment, modifying GH with a CTP peptide as described herein reduces the immunogenicity of GH. In another embodiment, CTP-modified hGH is as active as isolated GH. In another embodiment, the CTP-modified hGH polypeptide is more active than the 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 compliance of a subject suffering from a chronic disease requiring 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 reduction in frequency of administration of GH is achieved by CTP modification that makes the CTP modified GH more stable. In another embodiment, a reduction in the frequency of administration of GH is achieved as a result of increased GH T1 / 2. In another embodiment, a reduction in the frequency of administration of GH is achieved as a result of an increase in GH glialance 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 the subject once daily. 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 three 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 dose is in the range of 50-500 mg / day. In another embodiment the dose is in the range of 50-150 mg / day. In another embodiment the dose is in the range of 100-200 mg / day. In another embodiment the dose is in the range of 150-250 mg / day. In another embodiment the dose is in the range of 200-300 mg / day. In another embodiment the dose is in the range of 250-400 mg / day. In another embodiment the dose is in the range of 300-500 mg / day. In another embodiment the dose is in the range of 350-500 mg / day.

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

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

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

In one embodiment, oral administration comprises a unit dosage form comprising tablets, capsules, sweetened tablets, chewable tablets, suspensions, emulsions and the like. Such unit dosage forms include safe and effective amounts of the desired compound (s), each of which in one embodiment is 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 inert diluents such as conventional pharmaceutically compatible adjuvants 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; abundant agents such as magnesium stearate, stearate, and talc. In one embodiment, lubricants such as silicon dioxide can be used to improve the flow properties of the powder mixture. In one embodiment, colorants such as FD & C dyes may be added for appearance. Sweets and flavors 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 ancillary considerations such as taste, cost, and shelf life that are not important for the purposes disclosed herein and may be readily accomplished by one of ordinary skill in the art. ..

In one embodiment, the oral dosage form comprises a predetermined release profile. In one embodiment, the oral dosage form disclosed herein comprises a sustained release tablet, capsule, sweetened tablet, or chew. In one embodiment, the oral dosage form disclosed herein comprises a sustained release tablet, capsule, sweetened tablet, or chew. In one embodiment, the oral dosage form disclosed herein comprises an immediate release tablet, capsule, sweetened tablet, or chew. In one embodiment, the oral dosage form is formulated according to the desired release profile of the pharmaceutically active ingredient, as known to those of skill in the art.

In some embodiments, the oral composition comprises a liquid solution, an emulsion, a suspension, 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 is 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, the composition for use in the methods disclosed herein comprises a solution or emulsion, which in some embodiments is disclosed in safe and effective amounts herein. An aqueous solution or emulsion comprising the compound to be formulated and optionally other compounds intended for topical intranasal administration. In some embodiments, the composition comprises a subject compound of about 0.01% to about 10.0% w / v, more preferably about 0.1% to about 2.0, which is the compound. Used for systemic delivery by intranasal route.

In another embodiment, the pharmaceutical composition is administered by intravenous, intraarterial, or intramuscular injection of the liquid formulation. In some embodiments, the liquid formulation comprises a solution, dispersion, emulsion, oil, etc. In one embodiment, the pharmaceutical composition is administered intravenously and thus is formulated into a form suitable for intravenous administration. In another embodiment, the pharmaceutical composition is administered intra-arterial and thus formulated into a form suitable for intra-arterial administration. In another embodiment, the pharmaceutical composition is administered intramuscularly and thus is formulated into a form suitable for intramuscular administration.

In another embodiment, the pharmaceutical composition is administered topically to the surface of the body and thus is formulated into a form suitable for topical administration. Suitable topical formulations include gels, ointments, creams, lotions, infusions 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 pharmaceutical carriers. Combined with additional suitable therapeutic agents (s).

In one embodiment, the pharmaceutical compositions disclosed herein are subjected to, for example, conventional mixing, dissolution, granulation, sugar preparation, wet grinding, emulsification, encapsulation, by processes known in the art. Manufactured by encapsulation or lyophilization process.

In one embodiment, the pharmaceutical composition for use in accordance with the disclosure herein is one or more physiology that comprises an excipient and an adjunct that facilitates the processing of the pharmaceutically usable active ingredient into a pharmaceutical product. It is formulated in the conventional manner using a carrier that is acceptable to the patient. In one embodiment, the formulation depends on the route of administration chosen.

In one embodiment, the injectables disclosed herein are formulated in an aqueous solution. In one embodiment, the injectables disclosed herein are formulated in a physiologically compatible buffer, such as Hanks, Ringer's, or sodium chloride buffer. In some embodiments, for transmucosal administration, a penetrant suitable for the permeated barrier is 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, the formulation for injection is presented in unit dosage form, eg, in an ampoule, or in a repeat-dosing container, with a preservative added optionally. 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 agent, a stabilizer, and / or a dispersant.

The composition also, in some embodiments, is a preservative such as benzalconium 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, mannitol; antioxidants such as ascorbic acid, acetylcysteine, sodium disulfate; viscosity regulators such as polymers containing cellulose and derivatives thereof; and polyvinyl alcohol, and if required Accordingly, it contains acids and bases for adjusting the pH of these aqueous compositions. The composition also comprises, in some embodiments, local anesthesia or other active substance. The composition can be used as a spray, spray, drip or the like.

In some embodiments, the pharmaceutical composition for parenteral administration comprises an aqueous solution of the activated formulation in water-soluble form. In addition, in some embodiments, the active ingredient suspension is prepared as a suitable oily or aqueous injectable suspension. 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. The water-soluble injectable suspension, in some embodiments, contains a substance that increases the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. In another embodiment, the suspension also contains a suitable stabilizer or agent that increases the solubility of the active ingredient to allow the preparation of high concentration 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, Lipez-Berestin and Fiddler (eds.), Liss, New York, pp. 353-365 (1989), Lipez-Berestin, ibid, pp. 317-327; in general. ).

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 form of administration. In one embodiment, a pump is used (Langer, supra, Lefton, 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 located in close proximity to the therapeutic target, i.e. in the brain, thereby requiring only a small amount of systemic administration (eg, Goodson, in Medical Applications of Polypropylene). See Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems are described in the overview by Ranger (Science 249: 1527-1533 (1990)).

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

In one embodiment, the formulations disclosed herein are formulated into a composition for the rectum, eg, a suppository or a retention enema, using a conventional suppository base such as cocoa butter or other glycerides. ..

In some embodiments, a pharmaceutical composition suitable for use in the context disclosed herein comprises a composition in which the active ingredient is contained in an amount effective to achieve the intended purpose. include. In some embodiments, a therapeutically effective amount means the amount of active ingredient that is effective in preventing, alleviating, or ameliorating the symptoms of the disease or prolonging the survival of the subject being treated.

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

The composition is also a preservative such as benzalconium chloride and thimerosal; a chelating agent such as sodium edetate and others; a buffering agent such as phosphates, citrates, acetates; sodium chloride, potassium chloride, glycerin. , Isotonic agents such as mannitol; antioxidants such as ascorbic acid, acetylcysteine, sodium disulfate; viscosity modifiers such as polymers containing cellulose and derivatives thereof; and polyvinyl alcohols, and optionally these aqueous compositions. Contains acids and bases to regulate the pH of. The composition also comprises local anesthesia or other active material. The composition can be used as a spray, spray, drip or the like.

Some examples of substances that can act as pharmaceutically acceptable carriers or components thereof are sugars such as lactose, glucose, sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose. , And methyl cellulose; powdered tragant; malt; gelatin; starch; 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; polyol. Emulsifiers such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween ™ brand emulsifiers; wetting agents such as sodium lauryl sulfate; coloring agents; flavoring agents; tablets, stabilizers; antioxidants. Agents; preservatives; pyrogen-free water, isotonic saline; as well as starch buffers. The choice of pharmaceutically acceptable carrier to be used in conjunction with the compound is essentially 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. Be adjusted.

In addition, the composition includes excipients (eg acacia, corn starch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, povidone), disintegrants (eg corn starch, potato starch, alginic acid, silicon dioxide, croscarmé). Sodium loin, crospovidone, guar gum, sodium starch glycolate,), buffers of various pH and ionic strengths (eg Tris-HCI, acetates, phosphates), additives such as to prevent absorption on the surface. Albumin or gelatin, detergents (eg Tween 20, Tween 80, Polymeric F68, bile acid salt), protease inhibitors, surfactants (eg sodium lauryl sulfate), permeation enhancers, solubilizers (eg glycerol, preethylene glycerol) antioxidants. Agents (eg ascorbic acid, sodium metabisulfate, butyl hydroxyanisole), stabilizers (eg hydroxypropyl cellulose, hydroxypropylmethyl cellulose), thickeners (eg carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweeteners (eg guar gum). Aspartame, citric acid), preservatives (eg thimerosal, benzyl alcohol, parabens), lubricants (eg steaic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow aids (eg colloidal silicon dioxide), plastics Agents (eg diethyl phthalate, triethyl citrate), emulsifiers (eg carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (eg poroxamer or poroxamine), coatings or film-forming agents (eg ethyl cellulose, acrylate, polymethacrylate) And / or further include an 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), tragant, and sodium alginate, and typical wetting agents include. Examples include lecithin and polyethylene oxide sorbitan (eg, polysorbate 80). Typical preservatives include methylparaben and sodium benzoate. In another embodiment, the liquid composition for oral use also contains one or more components such as sweeteners, flavors, and colorants disclosed above.

The composition is also in or on a granular formulation of a polymer compound such as polylactic acid, porglycolic acid, hydrogel, or into liposomes, microemulsions, micelles, monolayer or multilayer vesicles, blood cell shadows, or spheroplasts. Including the incorporation of active materials. Such compositions will affect physiological conditions, solubility, stability, rate of in vivo release, and rate of in vivo clearance.

In another embodiment, the composition is a granular composition coated with a polymer (eg, poloxamer or poloxamin), and a tissue-specific receptor, ligand, or antibody to an antigen or tissue-specific receptor. Contains compounds bound to the 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, polyvinylpyrrolidone, or polyproline. In another embodiment, the modified compound exhibits a substantially longer half-life in the blood after intravenous injection than that of 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 polymeric 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 initially predicted by an in vitro assay. In one embodiment, the dose can be formulated in an animal model 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 in vitro, cell culture, or standard pharmaceutical procedures in laboratory animals. In one embodiment, data obtained from these in vitro and cell culture assays as well as animal studies can be used to formulate a range of doses for use in humans. In one embodiment, the dosage will vary 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 consideration of the patient's physical condition. [For example, Finger, et al. , (1975) "The Pharmacological Basics of Therapeutics", Ch. 1 p. Please refer to 1.].

In one embodiment, administration can be a single or multiple doses, depending on the severity and responsiveness of the condition being treated, which can range from days to weeks or result in recovery of this condition. It involves treatment over a period of time until the reduction is achieved.

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

In one embodiment, the composition comprising the formulations disclosed herein formulated in a compatible pharmaceutical carrier is also prepared, placed in a suitable container and labeled for the treatment of indications. Will be 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 can be a nonionic surfactant (ie, surfactants), various sugars, organic polyols, and / or human serum albumin, and the like. A lyophilized (ie, freeze-dried) formulation in combination with a composite organic excipient and stabilizer. In another embodiment, the pharmaceutical composition comprises a described CTP-modified lyophilized GH in sterile water for injection. In another embodiment, the pharmaceutical composition comprises the lyophilized CTP-modified hGH described herein in sterile PBS for injection. In another embodiment, the pharmaceutical composition comprises the lyophilized CTP-modified hGH described herein in sterile 0.9% NaCl for injection.

In another embodiment, the pharmaceutical composition comprises a CTP-modified GH described herein and a composite carrier such as human serum albumin, polyols, sugars, and anionic surfactants. See, for example, WO89 / 10756 (containing Hara et al.-polyol and p-hydroxybenzoate). In another embodiment, the pharmaceutical composition comprises the CTP-modified hGH disclosed herein, lactobionic acid, and an acetic acid / glycine buffer. In another embodiment, the pharmaceutical composition is an isotonic agent with a CTP-modified lyophilized GH described herein, glycine or human serum albumin (HSA), a buffer (eg, acetic acid). (For example, NaCl) and. In another embodiment, the pharmaceutical composition comprises the CTP-modified lyophilized GH described herein, a phosphate buffer, glycine, and HSA.

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

In another embodiment, the pharmaceutical composition comprising CTP-modified GH disclosed herein is a liquid composition comprising a stabilizer which is an amino acid between about 0.3% by weight and 5% by weight. Is.

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

In another embodiment, the pharmaceutical composition comprising the CTP-modified GH disclosed herein is a liquid that allows long-term storage in the liquid state, facilitating storage and transport prior to administration. Provide formulation.

In another embodiment, the pharmaceutical composition comprising CTP-modified GH disclosed herein comprises a solid lipid as a matrix material. In another embodiment, the injectable pharmaceutical composition comprising the 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 Speecher (Speeser and al., Charm. Res. 8 (1991) 47-54), followed by lipid nanopellets for oral administration (Speeser). 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, the CTP-modified GH-containing pharmaceutical composition disclosed herein is in the form of liposomes (JE Diedrichs and al., Pharma./nd. 56 (1994). ) 267-275).

In another embodiment, the CTP-modified GH-containing pharmaceutical composition disclosed herein comprises polymer fine particles. In another embodiment, the injectable pharmaceutical composition comprising the CTP-modified GH disclosed herein comprises polymeric microparticles. In another embodiment, the pharmaceutical composition comprising CTP-modified GH disclosed herein comprises nanoparticles. In another embodiment, the pharmaceutical composition comprising CTP-modified GH disclosed herein comprises liposomes. In another embodiment, the pharmaceutical composition comprising CTP-modified GH disclosed herein comprises a fat emulsion. In another embodiment, the pharmaceutical composition comprising CTP-modified GH disclosed herein comprises microspheres. In another embodiment, the pharmaceutical composition comprising CTP-modified GH disclosed herein comprises lipid nanoparticles. In another embodiment, the CTP-modified GH-containing pharmaceutical composition disclosed herein comprises lipid nanoparticles comprising amphipathic lipids. In another embodiment, the CTP-modified GH-containing pharmaceutical composition 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 packs or dispenser devices such as FDA-approved kits containing one or more unit dosage forms containing the active ingredient. In one embodiment, the pack comprises a plastic foil such as, for example, a metal or blister pack. In one embodiment, the pack or dispenser device is attached with dosing instructions. In one embodiment, the pack or dispenser is contained in a notice relating to the container in the form prescribed by a government agency that regulates the manufacture, use, or sale of the drug, which notice is for composition or human or animal administration. Represents a form of institutional approval. Such notice is, in one embodiment, a label approved by the US Food and Drug Administration for prescription drugs or infusions into 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 it can be done. In another embodiment, means (eg, administration and selection of complementary agents) are taken to minimize the side effects associated with the combination therapy.

In another embodiment, a kit comprising the compositions, cells, plasmids, etc. disclosed herein, an applicator, and teaching materials describing the use of the methods disclosed herein is disclosed herein. Will be done. Model kits are described below, but the contents of other useful kits will be apparent to those of skill in the art in the light of the present disclosure. Each of these kits represents a separate embodiment disclosed herein.

Additional objectives, advantages, and novel features disclosed herein will be apparent to those of skill in the art by verification of the following examples that are not intended to be limiting. In addition, each of the various embodiments and embodiments disclosed herein, described above, and claimed in the following claims finds experimental support in the following examples.

Example 1 Generation of hGH construct

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: first the gene was introduced into the cell, then the cell nucleus, and finally the chromosomal DNA. Was done. After gene transfer, cells were cultured in medium containing selectable markers such as dihydrofolate reductase (DHFR). Briefly:

The mouse dihydrofolate reductase (dhfr) gene derived from pSV2-dhfr was subcloned into the neomycin site of the pCI neoplasmid. To create a modified expression vector, pCI-dhfr (Fig. 2). Therefore, the MOD-4023 expression vector contains a cytomegalovirus (CMV) IE (earliest) enhancer / promoter and a 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). Therefore, the MOD-4023 expression or set consists of the CMV promoter, the CTP-hGH-CTP-CTP gene sequence, and the SV40 polyadenylation sequence.

Clonal selection-Limited dilution selection of single cells by plating on 96-well plates may be applied to single cell cultures and repeated several times to obtain 100% clone purity. The most productive clones based on growth curve, clone characterization, specific productivity and protein profile were propagated in 1 L shaking flask prior to planting in the bioreactor. Briefly:

MOD-4023 protein-producing cell lines were produced by recombinant DNA technology. Animal element-free medium was used through induction of master and working cell banks (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 the dilution steps during cell culture. The most productive clones were amplified with increasing concentrations of methotrexate (MTX). Based on clone population doubling level (PDL), MOD-4023 productivity (daily cell-by-cell picogram, PCD), and maximum cell density obtained in selected media, the most productive clones are simply selected. Separately used for the preparation of R & D banks, followed by the production of qualified master cell banks (MCBs) and working cell banks (WCBs).

Upstream process

The upstream process of MOD-4023 consists of two types of medium formulations, 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 was used for cell culture propagation (steps 1-4 in FIG. 3) prior to seeding in a 200 liter bioreactor, and medium 2 was N-2 step (2 prior to the final product). Two breeding. N refers to the final product), used in 200 liters of bioreactor (steps 5-6 in FIG. 3) and 1000 liters of production bioreactor (step 7 in FIG. 3).

Manufacturing process: MOD-4023

Clone # 28 was produced on a scale of 1000 liters (L) in serum-free culture medium. Culturing of CHO cell lines was carried out in several steps from a single vial of Working Cell Bank (WCB) to the culture volume of the final product. Production cell culture supernatants were tested for bioburden, bacterial endotoxin, productivity, and foreign viruses. This process was performed using 50 liter and 200 liter seeding bioreactors, as well as 1000 L bioreactors for growth. All product contact surfaces are disposable, while non-disposable product contact devices are product-only. Parts of these devices were cleaned and disinfected between batches. For growth, cultures were grown in 50 L and 200 L seeded bioreactors prior to planting in 1000 L bioreactors. Final proliferation and fed-batch bioreactor production were performed in 1000 L disposable bioreactor. Cell removal was accomplished using a disposable filtration system (Millipore deep filter).

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

50 liter and 200 liter seeding bioreactors were used as seeding bioreactors. Prior to planting, a 200 L bioreactor was filled with approximately 50 L of 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 the specified range, the inoculum is started to be transported. No additional feed was applied to the process during cell proliferation in the 50 L and 200 L seeded bioreactors. The expected culture time in the seeded bioreactor was 3-4 days before the cells were transferred to the 1000 L production bioreactor. Samples for in-process control (IPC) were taken daily.

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

Cultures are incubated in a 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 (fat-free Power Feed A) is added. The feed capacity is equal to 33% of the final bioreactor capacity. On day 6, DMSO is added to the bioreactor. A glucose feed was added to the culture to maintain the desired concentration, and a bolus of 1 M sodium bicarbonate was added to maintain the desired culture concentration (HCO ₃ ). The collection was carried out using the default criteria. Cell cultures were sampled daily for cell number, viability, and metabolic analysis for the first 4 days. From day 5 onwards, cells were sampled twice daily for cell number, viability, and metabolic analysis, and from day 9 onwards also for specific productivity by reverse phase HPLC. The productivity of MOD-4023 is at least 500 gr / L and the glycosylated form consists of at least 70% of the total hGH-CTP protein in the collection.

Although the examples presented herein show production using a fed-batch mode, one of ordinary skill in the art would be able to develop a perfusion mode using a generally similar purification scheme. Alternatively, one of ordinary skill in the art would be able to develop a perfusion mode in which 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. Depth filtration and 0.2 μm filtration were performed to purify the collection. Following purification, 0.45 / 0.2 μm filtration was performed. The deep filter was rinsed with water and residual moisture was blown out of the system with air. The filtration process was carried out at a pump speed of ≤15 L / min and a maximum predetermined pressure. The filter was later washed with Tris-HCl buffer and blown with pressurized air to increase product recovery.

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

Purification process

The purification scheme is shown in FIG.

Ultrafiltration and dialysis filtration 1-UFDF1 (step 8)

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

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

The material was filtered through a sterile Sartopore 2 XLG 10 "filter following a Millipore 1.55 m2 filter into a sterile mixing bag (deep filtration step). Then Tris / 10% Triton solution was added to the final filtration volume and Triton. The concentration was set to 1% (w / w). After incubation, the product solution was filtered through a 0.2 μm filter unit prior to 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 columns were packed to the default bed height. Due to the interference caused by Triton in the assay, certain proteins in the load were determined prior to the addition of Triton. DEAE columns were equilibrated and loaded in a virus-inactivated pool and then washed. A second wash is performed, the material is eluted with 20 mM Tris-HCl / 150 mM NaCl at pH 8.2, then at ambient temperature (18-26 ° C.) for the next day's treatment, or for a longer period of 2 Stored at -8 ° C. All chromatographic steps were performed in downward flow mode. Alternatively, the steps may be performed in ascending current mode. The eluate was tested for specific proteins by RP-HPLC (target: major 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 columns were packed to the default bed height. HIC chromatography was performed in 1-5 cycles depending on the amount of product. Alternatively, it may be run up to 10 cycles. The HIC load was prepared by adjusting the DEAE eluate with ammonium sulfate. The column was paralleled and loaded with a conditioned, 0.2 μm filtered DEAE solution and then washed with 10 mM sodium phosphate / 600 mM ammonium sulphate containing propylene glycol at pH 7.3. The material was eluted with reduced concentrations of ammonium sulphate and increased concentrations of pH 7.3 propylene glycol and then stored at 2-8 ° C. until further treatment.

HIC eluate ultrafiltration and dialysis filtration (step 12)

The HIC eluate was concentrated and dialysis filtered into 10 mM sodium phosphate buffer at pH 6.8 to reduce volume and prepare materials 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 dialysis-filtered against sodium phosphate buffer to achieve a pH of 6.8 ± 0.1 and a conductivity of less than 10% of the conductivity of the dialysis-filter buffer. Once the pH and conductivity are determined to be in range, they are drained from the system and filtered to a sterile bag at 0.45 / 0.2 μm. The final volume of concentrated, dialysis-filtered HIC eluate was stored overnight at ambient room temperature (18-26 ° C.). Concentrated water was tested by A280 for _bioburden , bacterial endotoxin, and specific proteins.

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

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

SP-Sepharose Chromatography (Step 14)

A column packed with SP Sepharose resin was used for this step. The columns were packed to the default bed height. The SP load was prepared 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 wash step. Flow-throughs were 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 at 2-8 ° C. for up to 24 hours. All chromatographic steps were performed in downward flow mode.

Virus inactivation by nanofiltration (step 15)

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

UFDF3 and filtration and storage of API (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 cutoff of 3-30 kDa was used for this step. The product was concentrated to 5-25 mg / ml in the first step and dialysis filtered through 10 mM citrate with pH 6.4 and 147 mM NaCl (≧ 7 DF volume). Alternatively, the product is concentrated to 5-25 mg / ml in the first step and a preservative to support repeated doses by the device, specifically 10 mM histidine citrate containing 0.3% m-cresol. The buffer was dialysis filtered and P-188 was added at 0.2% to reduce aggregates and particles invisible to the naked eye. The final product concentration was adjusted and filtered with a Millipak 100 filter. The filtered product solution was fractionated and frozen at a temperature of −70 ± 5 ° C.

MOD-4023 Pharmaceutical manufacturing

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

Characteristic evaluation of MOD-4023

The MOD-4023 content in the glycosylated 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 proteins 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 also has a high and low sugar chain addition MOD-4023 band compared to the strength of the additional host cell protein band and USDF sample in the collection analyzed by SDS-PAGE Coomasie staining. It is also recognized by strong strength (Fig. 6). In addition, the MOD-4023 production upstream process was developed to allow a higher percentage of high glycosylated MOD-4023 protein compared to the low glycated form. The hyperglycosylated 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. Briefly:

An O-glycan content assay was performed to calculate the number of moles of O-glycans per mole of MOD-4023 protein. 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 methods. Galactose (Gal) was quantified using an external standard in the galactose reference standard. The content of galactose may be directly related to the O-glycan structure, Gal-GalNAc. The sialic acid (SA) content is measured in the drug substance (DS) and should not differ from the SA content of the final drug (DP). Both DS and DP can be used to indicate the level of SA and O-glycan occupancy in CTP-modified hGH. Analysis of the five different MOD-4023 APIs and drug batches depicted in FIG. 7 shows robust batch-by-batch consistency. This unexpectedly robust sugar chain addition content is significant, indicating that the number of O-glycans per CTP in CTP-hGH-CTP-CTP has improved above what is known in the art. The CTP-hGH-CTP-CTP produced herein is 4-6 O-glycans per CTP compared to levels known in the art in which hCG has only 4 O-glycans. Had.

Intact molecular weight analysis of MOD-4023 sample

Molecular weight analysis of four different MOD-4023 DS batches was performed for the purpose of obtaining information on the number of O-linked glycosylation sites (see detailed results in Example 3). Intact MOD-4023 samples, as well as desialylated MOD-4023 samples using neuraminidase, and glycosylated samples using glycosidases were analyzed by online LC / ES-MS. The data obtained from the intact mass measurement of the desialylated sample shows that the protein is most modified with 15 glycosylated sites, and the protein modified with 12 to 18 glycosylated sites of GalNac-Gal is the most. Showed to be strong. This result, showing a high percentage of serum occupancy, was unpredictable compared to levels known in the art (4 compared to up to 6 in the CTP-modified hGH produced herein). Only serum was glycosylated).

Occupation of O-linked glycosylation site in MOD-4023 protein sample

For the purpose of obtaining information on the number of O-linked glycosylation sites for each MOD-4023 molecule, occupancy of O-glycosylation sites in four different MOD-4023 DS batches was performed with M-scan. The MOD-4023 sample was desialylated using neuraminidase, followed by tryptic digestion of the reduced / carboxymethylated MOD-4023 sample. Finally, online LC / ES-MS was performed on the treated sample and the MS data was interpreted using the specified software. Evaluation of the data obtained from the analysis of the tryptic digestion mixture led to a signal that allowed 100% of the protein sequences to be mapped. O-glycosylation is performed 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 contiguous regions of serine. A total of up to 18 serine residues can serve as adhesion sites for O-glycans. No significant differences between batches were detected, as presented in FIG.

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 sugar chains per MOD-4023, with the most abundant form containing 15 O-glycans per MOD-4023. (See the results and conclusions of Examples 2 and 3 below) No sugar chains were present on the GH sequence.

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

O-glycan structure

The major peak of the O-glycan corresponds to the monosialic acid-added core 1 (Neu5Acα2-3Galβ1-3GalNAc). In addition, 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 major 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 polar solvent to a lower polar solvent was used to elute the molecules with higher polarity before the molecules with lower polarity. RP-HPLC separates the MOD-4023 drug substance into major and minor peaks observed between 21.0 and 25.0 minutes, representing product-related mutations (FIG. 9). The relevant form is separated from the intrinsically disordered protein using UV detection at 220 nm. The relative peak region (region%) and main peak of the relevant form can be calculated by integrating the corresponding peak regions. The main peak of MOD-4023 APIs and pharmaceuticals consists of a peak region of> 97%, which represents a highly purified product and an effective purification process (see Figure 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-absorbent and the molecule is eluted under constant composition conditions. The SEC allows the monomer to separate from the high molecular weight form of the target molecule (eg, dimer and polymer). The SEC method was developed to analyze the content of MOD-4023 dimers and polymers in drug substances and pharmaceuticals (Fig. 10). The MOD-4023 monomer consists of a peak region greater than 98%, which represents a highly purified product and an effective purification process (see Figure 22).

RP-HPLC content method

This method is used to determine the MOD-4023 content of the MOD-4023 intermediate sample by reverse phase chromatography and to determine the percentage of non-glycosylated MOD-4023 in the intermediate sample. Reversed phase HPLC separates molecules by their polarity. Relatively non-polar molecules such as MOD-4023 bind to the column material while loaded, and polar molecules elute without interacting with the column.

The linked molecules are eluted with the help of a gradient from polar to lower polar solvent. The most polar molecule elutes first, followed by the lower polar molecule. Detection was performed via absorption at 214 nm.

Two peaks were observed in the early stages of purification (UFDF1 sample). 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 region (region%) of the two peaks can be calculated by integrating the corresponding peak regions. The relative peak region I is 75%. In the first step of purification, the column DEAE Sepharose is separated between these peaks and the elution fraction contains only the MOD-4023 glycosylated form (100%) (FIG. 12).

MOD-4023 efficacy

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 96-well plates (100 μl / well). During the 30 minute cell incubation period at 37 ° C, MOD-4023 was added at increasing concentrations 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 the addition of MTS, the optical density (OD) of the culture wells was measured at 492 nm. 492 nm absorption 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 tests were performed on hypophysectomized rats in which MOD-4023 activity in all batches was the United States Pharmacopeia (USP) somatropin unit per mg of 2 or greater.

Virus clearance

The ability of the manufacturing process to address and mitigate the endogenous and foreign viral contamination of the final drug was the subject of preliminary research. GLP-compliant studies follow applicable guidance for clinical trial drugs into scaled-down segments of the manufacturing process to quantify the ability of these steps to inactivate or clear viral populations added. It has been done using the three model viruses added. Log10 A quantity of virus expressed as a regulated titration, the log10 clearance factor is determined by simply subtracting the output value from the input value. As a log10 number, the clearance factor is additive to derive the overall clearance factor for all evaluation steps. The clearance factor for each step is calculated in FIG. A virus safety assessment study was performed using Abelson murine leukemia virus (A-MuLV), mouse microvirus (MVM), leovirus type 3 (Reo-3), and pseudorabies virus (PrV). The viral load per theoretical dose was calculated at the maximum dose of 15 mg / dose. A-MuLV is considered to be a model virus for the possible presence of CHO retroviruses, and the measures taken to inactivate and eliminate contaminated A-MulV virus are at least 10, 22.74 antilogarithms. A clearance factor was achieved, indicating that the overall MOD-4023 process has excellent virus removal capacity.

impurities

The results of an analysis of the range of impurities present in the purified MOD-4023 protein are presented in FIG. 24. A host cell protein (HCP) of 100 ng / mg (ppm) or less was discovered. The presence of DNA was less than 10 pg / mg (ppb). Residual methotrexate (MTX) levels were less than 50 ng / mL. The residual propylene glycol (PG) was 60 μg / ml or less. Triton of 2.5 μg / mL or less 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, showing that they are present in purified pharmaceuticals at 10 cfu / 10 mL or less.

Example 2 Occupancy of glycosylated site of MOD-4023 protein

Purpose

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

Method

Desialization 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 in water and lyophilized. The dried sample was suspended in neuraminidase and incubated at 37 ° C.

Tryptic digestion of reduced / carboxymethylated MOD-4023 protein

Following desialation, approximately 200 μg of MOD-4023 was buffer exchanged in Tris / guanidine hydrochloride buffer, reduced with dithiothreitol and carboxymethylated with iodoacetic acid. The sample was then buffer exchanged in ammonium bicarbonate and trypsinized 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; wavelength at 214 nm; flow rate of 0.2 ml / min). It was carried out on a certain amount of the digested sample obtained in the above. Solvent A was 0.05 mL of formic acid in 1 L of _H2O . Solvent B was 900 mL of acetonitrile in addition to 0.05 mL of formic acid in 100 ml of _H2O . For intact and reduced products, the gradients presented in Table 1 below were used.

The use of dry nitrogen 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

The interpretation of the mass spectrometric data is that the sequence expected to be O-sugar chain addition is present in the CTP unit, along with the protein sequence of CTP-modified hGH (SEQ ID NO: 7), General Protein / Mass Analysis for Windows ( 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 a desialylated, reduced, and carboxymethylated tryptic digested mixture of MOD-4023

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

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

Further signaling of the complete CTP unit was consistent with the expected mass of amino acids 1-36 of SEQ ID NO: 7 with up to 6 O-glycosylation sites. The development of large tryptic peptides containing such defective cleavage sites is consistent with the presence of O-glycosylation, which is that glycan residues close to arginine or lysine residues are sterically and electrostatically damaged. As a result, it interferes with trypsin cleavage.

The data presented in FIG. 17 also show comparison 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 the O-glycosylation is performed on the serine residue following the proline residue (indicated in red) in FIGS. 18 and 19. In addition, in regions where serine residues are contiguous, at least two of the four serins are occupied by O-glycosylation.

From the obtained results, 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 the proline residue in the sequence. Of the four serine residues at positions 1 to 4 of the N-terminal of the protein (amino acids 1 to 4 of SEQ ID NO: 7), at least two are occupied by the O-glycosylation site. 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 serine contiguous region, at least two of the four serines were occupied by the O-glycosylation site (amino acids 1-4 of SEQ ID NO: 7 of the N-terminal CTP unit, as well as the C-terminal CTP-CTP). Amino acids 220-223 and 248-251 of SEQ ID NO: 7 of the unit).

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, the same sample was injected in higher quantities and with modified ionization conditions preferred on high mass tryptic peptides.

Conclusion

In the course of this study, evidence was gathered in support of the following statements. Evaluation of the data obtained from the analysis of the tryptic digestion mixture led to a signal that allowed 100% of the protein sequences to be mapped. O-glycosylation is performed 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 the serin is contiguous. In this way, a total of up to 18 serine residues can serve as adhesion sites for O-glycans. Thus, surprisingly and unexpectedly, by using the manufacturing methods disclosed herein, O-glycan occupancy is CTP for each MOD-4023 (CTP-hGH-CTP-CTP polypeptide). There were up to 6 O-glycans per unit. Molecular weight analysis of the 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 Samples

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 the obtained condition and the sample after treatment

The samples analyzed had concentrations of 21 mg / ml and 41 mg / ml. 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 absorptiometer, transmission detector, and A-905 autosampler, fraction collector Frac-950 MS: Spectromass / Waters Q-TOF. Micro quadrupole flight time type mass spectroscope Wavelength: 280 nm
Column: Phenomenex Jupiter 3p C18 300A 150 × 2.0mm
(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 of H20
Solvent B: In 100 mL of H20, in addition to 1.0 mL of FA, 800 mL of acetonitrile and 100 mL of tetrahydrofuran

The gradients described below were used for the analysis of desialation products. Note that the retention time during data evaluation is adjusted at the beginning of the gradient (tRo = 10 minutes).

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

Desialization using neuraminidase

Approximately 100 pg of sample was buffer exchanged in water and lyophilized. Dry samples were suspended in neuraminidase and incubated at 37 ° C. Approximately 10 pg of it was analyzed as described above.

Data interpretation

Interpretation of mass spectrometric data was aided in 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 the obtained sample

FIG. 20 shows the decon obtained during elution of UV-absorbing components at tR19.8 minutes obtained from the analysis of MOD4023 lot 648-01-10-014A samples by online LC / ES-MS after desialization. The volumen mass spectrum is shown.

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

Conclusion

Molecular weight analysis of three batches of MOD4023 was performed on the desialylated sample for the purpose of obtaining information on the number of O-linked glycosylation sites. Consistently in all three batches, the data obtained from the intact mass measurement of the desialized sample is consistent with the average chemical mass of the MOD4023 protein with 12-18 sugar chain addition sites of the HexNAc-Hex structure. (Within the experimental error of the instrument). For all three batches, the protein modified with 15 glycosylated sites was the strongest.

Each of the corresponding mass spectrometric signals was characterized by the presence of satellite signals at approximately + 16.5 Da. This mass shift may have been the result of the addition of oxygen or _NH3 residues. The latter may be derived from NH ₄ ⁺ protonation rather than H + protonation that causes net addition of NH ₃ (+17 Da). Oxidation can also 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 +1 oxygen in the main peak), so the data are based on the latter explanation (satellite). It is the result of NH ₄ ⁺ protonation). However, this could not be finally clarified by the intact mass measurement alone.

The findings derived from this study were a previous analysis of the MOD4023 protein that up to 18 O-glycosylated residues were found and the protein modified at the 15 glycosylated sites was the strongest. Not shown).

Although the particular features disclosed herein have been exemplified and described herein, many modifications, substitutions, modifications, and equivalents will now be apparent to those of skill in the art. Accordingly, it should be understood that the appended claims are intended to cover all such amendments and changes that fall within the scope of the true intent disclosed herein.

Claims (18)

Human chorionic gonadotropin carboxy-terminal peptide (CTP) -modified human growth hormone (hGH) polypeptide.
It consists of two CTPs bonded to the carboxy terminus of hGH and one CTP bonded to the amino terminus of hGH.
The amino acid sequence of the polypeptide is set forth in SEQ ID NO: 7, and the CTP-modified hGH polypeptide comprises a human CTP-modified hGH poly containing glycosylation at 13 to 24 O-linked glycosylation sites. peptide. The human CTP-modified hGH polypeptide according to claim 1, wherein the glycosylation occurs on a CTP unit and there is no glycosylation of the hGH. The human CTP-modified hGH polypeptide according to claim 1 or 2, wherein each CTP contains 4, 5 or 6 O-linked glycans. The human CTP-modified hGH polypeptide according to any one of claims 1 to 3, wherein the glycosylation of the CTP unit comprises at least 15 O-linked glycans. The human CTP-modified hGH polypeptide comprises O-linked glycosylation of residues 10, 13, 15, 21, 229, 232, 234, 240, 257, 260, 262, and 268 of SEQ ID NO: 7. Item 6. The human CTP-modified hGH polypeptide according to any one of Items 1 to 4. The glycosylation pattern of the human CTP-modified hGH polypeptide is O-linked glycosylation of at least two residues selected from amino acid residues 1-4 of SEQ ID NO: 7, amino acid residues of SEQ ID NO: 7. O-linked glycosylation of at least 2 residues selected from 220-223, O-linked glycosylation of at least 2 residues selected from amino acid residues 248-251 of SEQ ID NO: 7 or The human CTP-modified hGH polypeptide according to claim 5, further comprising a combination thereof. The human CTP-modified hGH polypeptide according to any one of claims 1 to 6, wherein the O-glycan structure comprises a sialic acid-added core structure. Glycosylation at each of the O-linked glycosylation sites resulted in monosialic acid-added core 1 (Neu5Acα2-3Galβ1-3GalNAc), neutral core 1 (Galβ1-3GalNAc), and monosialic acid-added core 1 (Neu5Acα2-6). (Galβ1-3) GalNAc), or glycan core structure selected from the group containing disialic acid-added core 1 (Neu5Acα2-3Galβ1-3 (Neu5Acα2-6) GalNAc), according to any one of claims 1 to 7. The human CTP-modified hGH polypeptide according to the above. A composition comprising the CTP-modified hGH polypeptide according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier. The composition of claim 9 , wherein the most abundant form of the glycosylated CTP-modified hGH polypeptide present in the composition comprises 15 O-glycans. The composition according to claim 9 or 10 , wherein at least about 60% of the CTP-modified hGH polypeptide present in the composition is sialic acid-added. 13 . Composition. The composition according to any one of claims 9 to 12 , wherein the main O-glycan in the composition is core 1 (Neu5Acα2-3Galβ1-3GalNAc) to which monosialic acid is added. The composition according to any one of claims 11 to 13 , wherein the main sialic acid in the composition is Neu5Ac. The following characteristics,
i. Host cell protein of 100 ng / mg (ppm) or less,
ii. DNA of 10 pg / mg or less,
iii. Methotrexate less than 50 ng / mL,
iv. Polyethylene glycol of 60 μg / ml or less,
v. Triton, 2.5 μg / mL or less,
vi. Insulin of 115 pg / mL or less,
vii. DMSO <250 μg / mL, or viii. In bioburden analysis, 10 cfu / 10 mL or less,
The composition according to any one of claims 9 to 14 , comprising one or more of the above. The human chorionic gonadotropin carboxy-terminal peptide (CTP) -modified human growth hormone (hGH) polypeptide according to any one of claims 1 to 8, or the composition according to any one of claims 9 to 15 . There,
(A) A step of stably transfecting a predetermined number of cells with an expression vector containing a coding moiety encoding the CTP-modified hGH, wherein the transfected cells express and express the CTP-modified hGH. Secreting, stepping,
(B) A step of obtaining a cell clone that overexpresses the CTP-modified hGH, and
(C) A step of growing the clone in solution to a predetermined scale,
(D) A step of collecting the solution containing the clone, and
(E) A step of filtering the solution containing the clone to obtain a purified collection solution.
(F) Produced by a method comprising purifying the purified collection solution to obtain a purified protein solution having a desired concentration of CTP-modified hGH.
The polypeptide consists of two CTPs bonded to the carboxy terminus of hGH and one CTP bonded to the amino terminus of hGH.
A human chorionic gonadotropin carboxy-terminal peptide (CTP) -modified human growth hormone (hGH) polypeptide or composition, wherein the produced CTP-modified hGH comprises the amino acid sequence set forth in SEQ ID NO: 7. The human chorionic gonadotropin carboxy-terminal peptide (CTP) -modified human growth hormone (hGH) polypeptide according to any one of claims 1 to 8, or the composition according to any one of claims 9 to 15 . It ’s a manufacturing method,
(A) A step of stably transfecting a predetermined number of cells with an expression vector containing a coding moiety encoding the CTP-modified hGH, wherein the transfected cells express and express the CTP-modified hGH. Secreting, stepping,
(B) A step of obtaining a cell clone that overexpresses the CTP-modified hGH, and
(C) A step of growing the clone in solution to a predetermined scale,
(D) A step of collecting the solution containing the clone, and
(E) A step of filtering the solution containing the clone to obtain a purified collection solution.
(F) The step comprises purifying the purified collection solution to obtain a purified protein solution having a desired concentration of CTP-modified hGH.
The method for producing CTP-modified hGH thereby, wherein the amino acid sequence of the produced CTP-modified hGH is set forth in SEQ ID NO: 7. The purification (step f) of the purified collection is:
(G) A step of concentrating and dialysis filtering the purified collected solution, and
(H) The purified collection obtained following step (g) is obtained and incubated in a solution toxic to the virus to inactivate the virus present in the purified collection. Steps and
(I) A step of obtaining the purified collected solution from step (h) and purifying the purified collected solution.
i. The purification is accomplished by sequentially passing the purified collection solution through an anion exchange column and a hydrophobic interaction column, followed by a concentration step and a dialysis filtration step.
ii. After the purification, the purified collection solution is sequentially passed through a hydroxyapatite mixed mode column and a cation exchange column.
(J) Following step (ii), the purified collection solution is obtained, and the purified collection solution is physically removed from the virus by nanofiltration.
(K) Following step (j), the purified collection solution is obtained and the purified collection solution is concentrated and dialyzed until a maximally purified purified collection containing the CTP-modified hGH is reached. 17. The method of claim 17 , comprising sequentially performing the steps of filtering.

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