WO2012115638A1 - Compositions de pro-insuline glargine et procédés de production d'analogues de l'insuline glargine à partir de celles-ci - Google Patents

Compositions de pro-insuline glargine et procédés de production d'analogues de l'insuline glargine à partir de celles-ci Download PDF

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WO2012115638A1
WO2012115638A1 PCT/US2011/025922 US2011025922W WO2012115638A1 WO 2012115638 A1 WO2012115638 A1 WO 2012115638A1 US 2011025922 W US2011025922 W US 2011025922W WO 2012115638 A1 WO2012115638 A1 WO 2012115638A1
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proinsulin
sequence
glargine
arg
absent
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PCT/US2011/025922
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Ronald E. Zimmerman
David John STOKELL
Michael Patrick AKERS
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Elona Biotechnologies
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to compositions and preparations that comprise glargine proinsulin, in particular glargine proinsulin with modified C-peptide sequences.
  • the invention also relates to methods of manufacture for manufacturing glargine insulin analogs from modified proinsulin sequences.
  • Insulin is a hormone that regulates glucose metabolism in animals.
  • Insulin is a polypeptide hormone secreted by beta-cells of the pancreas. This hormone is made up of two polypeptide chains, an A-chain of 21 amino acids, and a B-chain of 30 amino acids. These two chains are linked to one another in the mature form of the hormone by two interchain disulphide bridges. The A-chain also features one intra-chain disulphide bridge.
  • Insulin analogs are altered forms of native insulin that are available to the body for performing the same action as native insulin.
  • a specific insulin analog known as glargine insulin has also been described in US Pat. Nos. 5,547,930, 5,618,913, and 5,834,422. This analog is used in the treatment of diabetes.
  • Glargine insulin is characterized as a slow release insulin analog that controls blood sugar when no food is being digested. Glargine insulin may form a hexamer when injected subcutaneously into the patient.
  • This insulin analog has been available commercially as LANTUS® (Sanofi Aventis).
  • LANTUS® is an insulin analog wherein the molecule includes a Gly(A 2 i)-Arg(B3 1 )-Arg(B32) amino acid sequence.
  • Proinsulin is a molecule comprised of a prepeptide of 24 amino acids, followed by the B-chain peptide, a C-peptide of 35 amino acids, and the A-chain peptide.
  • the C-peptide of this precursor insulin molecule (“proinsulin”) contains the two amino acids, lysine-arginine (KR) at its carboxy end (where it attaches to the A-chain), and the two amino acids, arginine-arginine (RR) at its amino end (where it attaches to the B-chain).
  • a third method utilizes yeast, especially Saccharomyces cerevisiae, to secrete the insulin precursor into the medium (Thim, et al. (1986), P.N.A.S., USA., 83: 6766-6770).
  • Chance et al. reported a process for preparing insulin by producing each of the A and B chains of insulin in the form of a fusion protein by culturing E. coli that carries a vector compromising a DNA encoding the fusion protein, cleaving the fusion protein with cyanogen bromide to obtain the A and the B chains, sulfonating the A and B chains to obtain sulfonated chains, reacting the sulfonated B chain with an excess amount of the sulfonated A chain; and then purifying the resultant products to obtain insulin.
  • Drawbacks associated with this process are that it requires two fermentation processes and the requirement of a reaction step for preparing the sulfonated A chain and the sulfonated B chain. This results in a low insulin yield.
  • proinsulin is produced in the form of a fusion protein by culturing E. coli which carries a vector comprising a nucleic acid sequence (DNA) encoding for the fusion protein, cutting the fusion protein with cyanogen bromide to obtain proinsulin, sulfonating the proinsulin and separating the sulfonated proinsulin, refolding the sulfonated proinsulin to form correct disulfide bonds, treating the refolded proinsulin with trypsin and carboxypeptidase B, and then purifying the resultant product to obtain insulin.
  • DNA nucleic acid sequence
  • the yield of the refolded proinsulin having correctly folded disulfide bonds is reported to sharply decrease as the concentration of the proinsulin increases. This is allegedly due to, at least among other reasons, misfolding of the protein, and some degree of polymerization being involved. Hence, the process entails the inconvenience of using laborious purification steps during the recovery of proinsulin.
  • Thim et al. reported a process for producing insulin in yeast, Saccharomyces cerevisiae. This process has the steps of producing a single chain insulin analog having a certain amino acid sequence by culturing Saccharomyces cerevisiae cells, and isolating insulin therefrom through the steps of: purification, enzyme reaction, acid hydrolysis and a second purification. This process, however, results in an unacceptably low yield of insulin.
  • Trypsin is a typical serine protease, and hydrolyses a protein or peptide at the carboxyl terminal of an arginine or lysine residue (Enzymes, pp. 261-262 (1979), ed. Dixon, M. & Webb, E. C. Longman Group Ltd., London).
  • This unwanted hydrolysis results in the unwanted Arg(Ao)-insulin byproduct, and typically constitutes about 10% of the reaction yield.
  • an additional purification step is required. The necessity of an additional purification step makes the process much more time consuming, and thus expensive, to use. Moreover, an additional loss of yield may be expected from the necessity of this additional purification step.
  • proinsulin constructs that do not have a conserved terminal dibasic amino acid sequence of the C-peptide region.
  • US Pat. No. 6,777,207 (Kjeldsen et al.) relates to a novel proinsulin peptide construct containing a shortened C-peptide that includes the two terminal amino acids, glycine-arginine or glycine-lysine at the carboxyl terminal end that connects to the A-chain of the peptide.
  • the B-chain of the proinsulin construct described therein has a length of 29 amino acids, in contrast to the native 30 amino acid length of the native B-chain in human insulin.
  • yeast provides a relatively low insulin yield, due to the intrinsically low expression levels of a yeast system as compared to E. coli.
  • the present invention provides processes for using a modified proinsulin sequence to produce glargine insulin analogs.
  • the modified proinsulin sequence has the formula
  • R ⁇ is a tag sequence containing one or more amino acids, preferably with a C- terminal Arg or Lys, or Ri is absent, with an Arg or Lys present prior to the start of the B chain;
  • (B1-B29) and (Ai-A 20 ) comprise amino acid sequences of native human insulin
  • B 30 is Gly, Ala, Ser, Thr, Val, Leu, lie, Asn, Gin, Cys, Met, Tyr, Phe, Pro, or Trp, preferably Thr;
  • R 2 , R 3 and R 5 are Arg
  • R4 is any amino acid other than Gly, Lys or Arg or is absent, preferably Ala;
  • X is a sequence that comprises one or more amino acids or is absent, provided that X does not comprise a C-terminal Gly, Lys, or Arg when R4 is absent;
  • a 2 i is Gly, Ala, Val, Leu, He, Pro, Phe, Trp, Met, Ser, Thr, Tyr, Asp, or Glu, preferably Gly;
  • R ⁇ 5 is a tag sequence containing one or more amino acids, preferably with a N- terminal Arg or Lys, or R 6 is absent;
  • One aspect of the present invention is related to a process for producing glargine insulin analogs comprising the steps of culturing E. coli cells under conditions suitable for expression of a modified proinsulin sequence as provided in Formula I; disrupting the cultured E. coli cells to provide a composition comprising inclusion bodies containing the modified proinsulin sequence; , solubilizing the composition of inclusion bodies; and recovering glargine insulin analogs from the solubilized composition.
  • Another aspect of the present invention is related to a process for producing glargine insulin analogs comprising the steps of: (a) providing a modified proinsulin sequence as provided for in Formula I; (b) folding the modified proinsulin sequence to provide a glargine proinsulin derivative peptide; (c) purifying the glargine proinsulin derivative using metal affinity chromatography; (d) enzymatically cleaving the glargine proinsulin derivative peptide to remove a connecting peptide and tag to provide an intermediate solution comprising glargine insulin analog; and (e) purifying the intermediate solution using chromatography columns to yield the glargine insulin analog.
  • FIG. 1 is a vector map of plasmid pTrcHis2A (Kan) with a glargine proinsulin gene insert.
  • FIG. 2 is a process flow scheme for the purification of glargine insulin analogs.
  • FIG. 3 is an analytical HPLC overlay of glargine product by the method (B) according to one aspect of the invention, compared with LANTUS® (A), manufactured by Sanofi Aventis. Detailed Description
  • the present invention generally relates to the preparation of insulin analogs, specifically glargine insulin analog, from modified proinsulin sequences.
  • Glargine insulin analog comprises a modified A-chain and B-chain having Gly(A 21 ), Arg(B 31 ), and Arg(B 32 ).
  • Modified proinsulin sequences refer to a single-chain polypeptide that may be converted into human insulin or insulin analogs and comprise a connecting peptide (C-peptide) having at least one non-dibasic terminal amino acid sequence.
  • non-dibasic terminal amino acid sequences may comprise (any amino acid except Lys or Arg-Arg ((any except R or K)R), and more preferably (any amino acid except Gly, Lys, or Arg-Arg ((any except G, R, or K)R).
  • the terminal amino acid sequence may comprise Ala-Arg.
  • the positioning of these particular terminal amino acids in the C-peptide provides for an improved method for producing recombinant glargine insulin analog, having fewer steps, improved yields of the recombinant glargine insulin analog and less contaminating byproducts.
  • the process for producing glargine insulin analogs of the invention presents many advantages, among them the advantage of reducing and/or eliminating the presence of unwanted and contaminating cleavage by-products characteristic of conventional manufacturing processes for producing recombinant human insulin in E. coli.
  • Previously undesirable by-products evident in yield mixtures using conventional methods of producing recombinant human insulin analogs included, by way of example, the production of an unwanted cleavage product, Arg(A 0 )-insulin analogs.
  • a highly efficient process for the production of recombinant human insulin analogs is presented that reduces and/or eliminates the presence of this and other unwanted and undesirable cleavage by-products, and that further presents the advantages of eliminating several time consuming, expensive, purification steps.
  • a process having fewer technician-assisted steps is thus devised, and illustrates the additional advantage of eliminating the degree of inconsistency and/or error associated with technician assisted steps in the manufacturing process.
  • a second key advantage involves the citraconylation of Lys 29 of the B chain using citraconic anhydride.
  • the lysine in the following sequence of pro-insulin; pro-lys-thr-arg-arg (SEQ ID NO: 24), is cleaved by trypsin during the transformation reaction at a rate of approximately 5-8%, which creates the desthreonine insulin contaminant.
  • Citraconylation of the lysine prevents trypsin cleavage and in turn prevents desthreonine insulin.
  • the citraconylation also decreases trypsin cleavage at the arginine at position B 31 of the C-peptide.
  • Single arg(B 31 )- insulin represents approximately 20-30% during the trypsin digest.
  • the level of arg(B 31 )-insulin decreases to approximately 6-10% during the trypsin digest.
  • Decreased levels of desthreonine insulin and arg(B 31 )-insulin provides for a simpler purification, as both these impurities are difficult to remove due to their high similarity to final glargine material.
  • the modified glargine proinsulin sequence of the present invention has the formula
  • R] is a tag sequence containing one or more amino acids, preferably with a C- terminal Arg or Lys, or Rj is absent with an Arg or Lys present prior to the start of the B chain;
  • (B1-B29) and (Ai-A 2 o) comprise amino acid sequences of native human insulin
  • B 30 is Gly, Ala, Ser, Thr, Val, Leu, He, Asn, Gin, Cys, Met, Tyr, Phe, Pro, or Trp, preferably Thr;
  • R 2 , R 3 and R 5 are Arg
  • j is any amino acid other than Gly, Lys or Arg or is absent, preferably Ala;
  • X is a sequence comprising one or more amino acids or is absent, provided that X does not comprise a C-terminal Gly, Lys, or Arg when R4 is absent;
  • a 2 i is Gly, Ala, Val, Leu, He, Pro, Phe, Trp, Met, Ser, Thr, Tyr, Asp, or Glu, preferably Gly;
  • Rs is a tag sequence containing one or more amino acids, preferably with a N- terminal Arg or Lys, or R 6 is absent.
  • R ⁇ or Re in the modified glargine proinsulin of Formula I comprises a pre or post-peptide that may be a native pre-peptide or an N-terminal multiple His-tag sequence, or any other commercially available tag utilized for protein purification, e.g. DSBC, Sumo, Thioredein, T7, S tag, Flag Tag, HA tag, VS epitope, Pel B tag, Xpress epitope, GST, MBP, NusA, CBP, or GFP.
  • at least one of Rj or R5 is present in Formula I. It is preferred that the terminal amino acid of the pre or post-peptide that connects to the B-chain or A-chain comprise Arg or Lys.
  • Native pre-peptide has the sequence of MALWMRLLPLLALLALWGPDPAAA (SEQ ID NO: 2).
  • the N-terminal multiple His-tagged proinsulin construct comprises a 6-histidine (SEQ ID NO: 3) N-terminal tag and may have the sequence of MHHHHHHGGR (SEQ ID NO: 4).
  • the modified proinsulin sequence may replace the native 24 amino acid pre-peptide with the 6-histidine (SEQ ID NO: 3) N-terminal tag sequence.
  • Ri and/or Re may be a sequence of one or more amino acids, e.g., preferably from 1 to 30 and more preferably from 6 to 10.
  • Native insulin comprises an A-chain having the sequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 5) and a B-chain having the sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 6).
  • the A-chain and B-chain of Formula I is modified from native insulin and contains at least one amino acid mutation, substitution, deletion, insertion, and/or addition.
  • a 21 is modified and B 31 and B 32 of the B-chain are added.
  • the asparagine (A 21 ) of native insulin is substituted with glycine and the two arginine amino acids are added to (B30) of native insulin.
  • the two arginine amino acids may be amino acid residues from the C-peptide.
  • the A-chain and/or B-chain that is modified is a human insulin B-chain. In another embodiment, the A-chain and/or B-chain that is modified is porcine insulin B-chain.
  • connecting peptide or "C- peptide” is the connecting moiety "C" of the B-C-A polypeptide sequence of a single chain proinsulin molecule.
  • the N-terminus of the C-peptide connects to C-terminus of the modified B-chain, e.g., position 30 of the B-chain, and the C- terminus of the C-peptide connects to N-terminus of the A-chain, e.g., position 1 of the A-chain.
  • the C-peptide may have a sequence of the formula II:
  • R 2 , R 3 , R 4 , R 5 , and X have the same meaning as in Formula I.
  • X may be a sequence having up to 40 amino acids, preferably up to 35 amino acids or more preferably up to 30 amino acids.
  • X may be any amino acid sequence, in one embodiment, X is preferably not EAEALQVGQVELGGGPGAGSLQPLALEGSLQ (SEQ ID NO: 7).
  • the C-peptide sequences of the present invention may include:
  • RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQAR SEQ ID NO: 8
  • RREAEDLQVGQVGLGGGPGAGSLQPLALEGSLQAR SEQ ID NO: 9
  • Preferred modified glargine proinsulin sequences of the present invention may include:
  • the single chain glargine insulin analogs of the invention will include three (3) correctly positioned, disulphide bridges, as is characteristic of the native human insulin.
  • the folded modified proinsulin, or proinsulin derivative peptide may include three (3) correctly positioned, disulphide bridges.
  • the C-peptide of the proinsulin glargine derivative peptide is removed to produce the glargine insulin analog.
  • Glargine insulin analogs of the invention have a sequence (SEQ ID NOS 22-23, respectively, in order of appearance) of formula III, where the disulphide bridges are represented as -S-S-: Formula III
  • the present invention provides modified proinsulin sequences having the modified C- peptide and methods for using these in a process to provide high yields of mature recombinant glargine insulin analog.
  • the positioning of these particular terminal amino acids in the C-peptide may provide for an improved method for producing recombinant glargine insulin analog, having fewer steps, improved yields of the recombinant glargine insulin analog and less contaminating byproducts.
  • insulin precursor or "proinsulin” are described as a single-chain polypeptide in which, by one or more subsequent chemical and/or enzymatic processes, may be converted into human insulin or insulin analog.
  • a proinsulin analog or modified proinsulin is defined as a proinsulin molecule having one or more mutations, substitutions, deletions, and or additions, of the A, B and/or C chains relative to the native human proinsulin nucleic acid sequence.
  • the proinsulin analogs are preferably such wherein one or more of the naturally occurring nucleic acids have been substituted with another nucleic acid within a triplet encoding for a particular amino acid.
  • proinsulin analog is understood to refer to glargine proinsulin analog, unless otherwise specified.
  • insulin analog includes insulin molecules having one or more mutations, substitutions, deletions, additions, or modifications to one or more amino acids of a native insulin sequence.
  • the native insulin sequence is porcine insulin, while in another embodiment, the native insulin sequence is human.
  • insulin analog is understood to refer to glargine insulin analog, unless otherwise specified.
  • the term "about” is intended to be inclusive of and to encompass both an exact amount as well as an approximate amount or range of values or levels of the item, ingredient, element, activity, or other feature or characteristic to which it references. Generally, and in some embodiments, the term “about” is intended to reference a range of values relatively close to the specific numerical value specifically identified. For example, “about 3 grams to about 5 grams” is intended to encompass a measure of in or around a value of 3 grams, concentration values between 3 grams and 5 grams, concentration values in and around 5 grams, as well as concentration values that are exactly 3 grams and exactly 5 grams.
  • a high protein concentration of the proinsulin or insulin analog product is defined as a protein yield concentration of at least about 3 grams/liter, or between about 3 grams to about 5 grams per liter.
  • the expression yield to be expected may be defined as a protein/peptide yield that is sufficient to detect via polyacrylamide gel electrophoreses (PAGE).
  • the invention provides a process for producing highly purified glargine insulin analog that is more efficient than current techniques.
  • the invention in a general and overall sense relates to an improved process for preparing a heterologous recombinant protein in a prokaryotic host cell. This process is characterized in that it employs a recombinant protein that provides a useful and efficiently processed modified proinsulin sequence analog as described herein.
  • heterologous protein is intended to mean that the protein in the prokaryotic host cell is not native, i.e., it occurs as a protein in peculiar or foreign (i.e., not native to) the host prokaryotic cell.
  • recombinant is intended to mean produced or modified by molecular- biological methods.
  • a recombinant protein is made using genetic engineering techniques and is not found in nature.
  • heterologous recombinant protein is defined as any protein known to the skilled person in the molecular biological arts, such as, for example, insulin, insulin analog, proinsulin, proinsulin analog, C-peptide, and proteins containing these together with any other protein or peptide fragment.
  • Prokaryotic host cells may be any host cells known to the skilled artisan in the molecular biological arts, and by way of example, Escherichia coli. Such types of cells available from public collections and useful in the practice of the present invention include, by way of example, the Deutsche Sammlung von Mikrooganismen and Zellkulturen GmbH, raunschweig, Germany, e.g., E. coli Strain K12 JM107 (DSM 3950).
  • the following reference table, Table 1 provides the triplet codons corresponding to each of the various amino acids that are used in the description of the present invention.
  • the amino acid that may be used in any particularly defined position as part of any of the peptide, protein, or constructs otherwise defined herein by reference to a particular nucleotide triplet base pair may be encoded by a number of different nucleotide triplets that function to encode the same amino acid.
  • the amino acid of the sequence defined herein is alanine (Ala, or A)
  • the triplet codon of nucleic acids that may encode for this amino acid are: GCT, GCC, GCA, or GCG.
  • the following table illustrates this definition of variables at and substitutions as can be applied to all of the naturally occurring amino acids sequences of the disclosure.
  • the glargine insulin analog prepared by the present invention may be formulated as liquid glargine insulin analog or crystalline glargine insulin analog.
  • a preparation of recombinant liquid glargine insulin analog is in a substantially liquid form and that has not been through a crystallization process. Eliminating these steps has no negative impact on the purity of the liquid glargine insulin analog produced, but has the added advantage of reducing the amount of inactive insulin multimers in the liquid glargine insulin analog of the invention.
  • Glargine insulin analog reconstituted from lyophilized and crystallized insulin may be contaminated with inactive insulin multimers and is less preferred.
  • the methods of producing glargine insulin analog described herein generally include the following steps: fermentation/expression, Inclusion body isolation, of solubilization glargine proinsulin analog; refolding processing and transformation of glargine proinsulin analog to glargine insulin analog; and purification of glargine insulin analog.
  • FIG. 2 illustrates a flow chart of preferred process steps in producing glargine insulin analog according to embodiments of the present invention.
  • the recombinant expression system is a working cell bank (WCB) containing glargine proinsulin analog expressing vectors.
  • the cells of the WCB may be vertebrate or invertebrate cells, such as prokaryote or eukaryote cells, and most preferably the cells may be mammalian, bacterial, insect, or yeast cells.
  • the cell is a bacterial cell and in a further embodiment, the bacteria is E, coli.
  • the cell is a yeast cell and in a further embodiment, the yeast cell is S. cerevisiae or S. pombe,
  • E. coli cells may be cultured and disrupted to provide a composition comprising inclusion bodies.
  • the inclusion bodies contain the modified proinsulin sequence.
  • the glargine proinsulin analogs expressed by cells of the WCB according to the method of the invention may be secreted from the cells and include a secretory sequence.
  • glargine proinsulin analogs expressed by cells of the WCB are not secreted from the cells, and thus do not include a secretory sequence.
  • the step of solubilizing the composition of inclusion bodies may involve adjusting the pH to achieve complete solubilization of the modified glargine proinsulin sequences.
  • the inclusion bodies may be solubilized by adjusting the pH to at least 10.5, preferably from 10.5 to 12.5, preferably from 11.8-12.
  • the pH may be adjusted by adding an alkali hydroxide such as NaOH or KOH to the composition of inclusion bodies.
  • the step of solubilization may use one or more reducing agents and/or chaotropic agent.
  • Suitable reducing agents may include those selected from the group consisting of 2-mercaptoethanol, L- cysteine hydrochloride monohydrate, dithiothreitol, dithierythritol, and mixtures thereof.
  • Suitable chaotropic agents include those selected from the group consisting of urea, thiourea, lithium perchlorate or guanidine hydrochloride, and mixtures thereof.
  • the solubilized inclusion bodies may be mixed in a refolding buffer, such as glycine or sodium carbonate, at a pH of 7-12, preferably from 10-11, preferably from 10.5-11, to refold the modified proinsulin sequences to a proinsulin derivative peptide, e.g., glargine proinsulin derivative peptide.
  • a refolding buffer such as glycine or sodium carbonate
  • the solution with refolded material should be pH adjusted to 7-9, preferably 7.8-8.2, with or without the addition of an alkaline salt, preferably sodium chloride to a final concentration of lOOmM to 1M final concentration, preferably 500mM to 1M, preferably 700mM, and may be filtered and loaded onto a column, such as an immobilized metal-ion affinity chromatography (IMAC) column.
  • an alkaline salt preferably sodium chloride to a final concentration of lOOmM to 1M final concentration, preferably 500mM to 1M, preferably 700mM
  • IMAC immobilized metal-ion affinity chromatography
  • Commercially available resins suitable for embodiments of the present invention include Nickle Sepharose 6 Fast Flow (GE Healthcare), Nickle NTA Agarose (GE Healthcare), Chelating Sepharose Fast flow(GE Healthcare), IMAC Fast Flow (GE Healthcare).
  • one or more of the amino acids may be protected to prevent side reactions and impurities during the cleavage step.
  • the addition of a protecting group to glargine insulin analog may be added prior to addition of trypsin.
  • protecting groups may be used to protect the lysine residue of the B-chain.
  • a preferred protecting group is citriconic anhydride. In native human proinsulin, citriconic anhydride is preferably used to block Lys(B 29 ) in the proinsulin pro-lys-thr-arg-arg (SEQ ID NO: 24) amino acid sequence, and thus reducing the formation of desthreonine insulin impurity.
  • citriconic anhydride may also be used to block Lys(B 29 ) in the pro-lys-thr-arg-arg (SEQ ID NO: 24) amino acid sequence.
  • the citriconic anhydride protecting group may reduce the formation of impurities such as desthreonine insulin and arg(B 31 )-insulin.
  • an excess molar ratio of citriconic anhydride to glargine proinsulin analog may be used.
  • about 10 fold molar excess or more of citriconic anhydride to glargine proinsulin analog may be suitable, and more preferably, about 20 fold molar excess or more.
  • There is no upper limit on the excess molar ratio and the molar ratio may be as high as about 200 fold or about 300 fold.
  • glargine proinsulin analog is subject to buffer exchange and concentration by tangential flow filtration or diafiltration.
  • Proinsulin derivative peptide, with the blocking groups may be enzymatically cleaved, preferred by subjecting the proinsulin derivative peptide to trypsin digestion.
  • embodiments of the present invention may use commercially available rat, bovine, porcine or human trypsins or other isoenzymes or derivatives or variants thereof, it is also possible to use the following enzymes: trypsin from Fusarium oxysporum and from Streptomyces (S. griseus, S. exfoliatus, S. erythraeus, S. fradiae and S.
  • tryptase occurs at pH from about 7 to 10, and more preferably from 8.8 to 9.2.
  • the trypsin digest is quenched by adding glacial acetic acid. While it is contemplated that other additives may be employed, acetic acid appears to be most preferred and stable for this purpose.
  • Trypsin is an enzyme that has specific cleavage activity at the c-terminal side of arginine residues, and to a lesser extent, lysine residues, of the C-peptide. In the transformation reaction, it is required that the terminal arginine or lysine residues of the C-peptide be removed. In native human proinsulin, when trypsin cleaves at the lysine in position 64, it will be unable to remove the arginine at position 65, due to the fact that it requires at least one residue on both sides of a cleavage site. What results is the production of an unwanted by-product, arg(A 0 )-insulin.
  • This by-product constitutes a small loss in yield and generates an undesired contaminant.
  • the arg(Ao)- insulin byproduct is preferentially not formed. When formed, is less than 10%, and more preferably is less than 0.3% of total byproducts from the trypsin transformation reaction may be arg(A 0 ). This is because the trypsin no longer acts to cleave at this particular site of the proinsulin derivative peptide.
  • the glargine insulin analog is subjected to deblocking after digestion with trypsin.
  • Citriconic anhydride deblocking occurs by permitting the glargine insulin to be warmed to a temperature of 15°C to 25°C, more preferably 18°C to 20°C, and the pH is adjusted to 2.5 to 3.5, more preferably 2.8 to 3.0.
  • the glargine insulin after deblocking the glargine insulin is purified in a chromatography column, such as an ion exchange column. Following the ion exchange chromatography, the glargine insulin may be further purified using reverse phase chromatography.
  • the intermediate solution may be purified in a chromatography column by eluting the glargine insulin analog using a buffer comprising an alcohol or organic solvent, n-propanol or acetonitrile.
  • the buffer may also further comprise an alkali metal salt, preferable sodium sulfate.
  • the buffer may also further comprise an organic acid, preferable phosphoric acid.
  • insulin having two additional arginine residues at the carboxyl terminal end of the B chain, along with glycine substituted for asparagines at the carboyl terminal end of the A chain allows glargine insulin to form a precipitate (hexamer) when injected subcutaneously.
  • this glargine insulin analogue upon administration of this glargine insulin analogue to a patient, it can maintain a peakless level for up to 24 hours.
  • this analogue is particularly suitable for moderate control of serum glucose levels that more closely resemble typical basal insulin secretion. For example, if administered prior to sleep, insulin glargine can reduce the risk of nocturnal hypoglycaemia.
  • the insulin glargine analogue is provided to a patient in combination with a rapid acting insulin to provide optimal glycemic control.
  • the glargine insulin analog is provided to a patient in combination with human insulin or another insulin analog to provide optimal glycemic control.
  • the preparations comprise a pharmaceutically acceptable preparation comprising recombinant glargine insulin analog and being essentially free of modified proinsulin sequences.
  • Step 1 Construction of a purified glargine proinsulin gene segment for insertion into the vector.
  • the initial gene construct was synthesized in a basic cloning vector.
  • the gene construct included the N-terminal histidine tag, MHHHHHHGGR (SEQ ID NO: 4), modified B- chain, and modified C-peptide with the alanine codon in place of the native lysine and having the amino acid sequence
  • MHHHHHHGGRFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGG PGAGSLQPLALEGSLQARGIVEQCCTSICSLYQLENYCG (SEQ ID NO: 16).
  • the gene was flanked by Ndel and EcoRl restriction sites, for subsequent subcloning into the desired expression vector.
  • the codons selected were optimized for expression in E.coli.
  • the following sequence represents the pTrcHis2a(Kan) vector with glargine proinsulin insert (FIG. 1).
  • the IPTG inducible promoter region which regulates the transcription rate is shown by the dotted underline, while the glargine proinsulin insert, adjacent the promoter region, is shown by the solid underlined.
  • the sequence shown in bold and italics is the Kanamycin gene, which provides the antibiotic selection marker for the vector.
  • Step 2 Generation of the pTrcHis2A(Kan) vector containing glargine proinsulin.
  • Commercially available pTrcHis2A(Kan) vector was modified to include a Kanamycin resistance gene in the middle of the Ampicillin resistance gene to negate the Ampicillin resistance prior to insertion of the proinsulin sequence into the vector.
  • Ampicillin resistance heightens the potential for allergic reactions to preparations made using vector constructs that include the Ampicillin resistance gene. Therefore it is preferable to eliminate the Ampicillin resistance in the constructs that are prepared and used.
  • the pTrcHis2A(Kan) vector was modified at the start codon in the multiple cloning site by replacing the Ncol restriction site with an Ndel site to simplify subsequent subcloning work.
  • the proinsulin gene was isolated from the DNA 2.0 plasmid using Ndel to cleave at the N-terminal side of the gene and EcoRl to cleave at the C-terminal side of the gene.
  • the Digested DNA was run over a 2% agarose gel to separate the plasmid DNA from the glargine proinsulin gene.
  • a QIAquickTM (Qiagen) gel purification kit was then used to purify the gene construct.
  • Step 3 Transformation.
  • One microliter of the ligation reaction was used to transform competent E. coli cells BL21 with the pTrcHis2A(Kan) plasmid containing the proinsulin gene.
  • the transformed E. coli BL21 cells were plated on LB-Kan agar plates and incubated overnight at 37°C. Several clones were selected and sequenced. Clones with the correct sequence were then screened for expression.
  • the resulting clone is referred to as the glargine proinsulin pTrcHis2A(Kan) vector.
  • Step 4 Preparation of the working cell bank (WCB).
  • WCB working cell bank
  • sterile growth medium was inoculated with the recombinant BL21 E. coli containing the glargine proinsulin Met-His-tagged/pTrcHis2A(Kan) vector and incubated to allow cell growth.
  • the cells were harvested in an IS05 (class 100) environment under a biosafety cabinet and then sterile filtered. Sterile medium and glycerol were added to the cells. 1 mL aliquots of the cells were then dispensed into sterile ampoules and stored at -80°C. Aseptic techniques were utilized to generate the WCB.
  • Step 1 Culturing of E. coli transformed with glargine modified proinsulin sequence from the WCB of Example 1.
  • yeastolate purchased from VWR, Prod. # 90004-426 or - 488)
  • select phytone sodium chloride
  • purified water purified water
  • sterile Kanamycin solution sterile Kanamycin solution
  • Step 2 Disruption— Cells containing inclusion bodies expressing glargine modified proinsulin sequence are lysed in a basic Tris/salt buffer, using a Niro Soavi homogenizer (1100- 1200 bar).
  • Step 3 Inclusion Body Washing— Contaminant protein removal is accomplished via two sequential washes with a Tris/Triton X-100 buffer, followed by two sequential washes with a Tris/Tween-20 buffer, and finally a single wash with a Tris/NaCl buffer.
  • Step 4 - Solubilization Inclusion bodies enriched with the modified glargine proinsulin peptide are solubilized in 4-8M urea, preferably 6-8M urea, containing reducing agents (2-mercaptoethanol, L-cysteine hydrochloride monohydrate). Complete solubilization is achieved by adjusting the pH to 10.5-12, preferably 11.8-12 with NaOH.
  • Step 5 Dilution refolding— The solubilized glargine insulin analog protein is then diluted into refolding buffer (20 mM Glycine, pH 10-11 at 6-10°C.) to a final concentration of 1.5 mg/ml and permitted to refold for 24 to 72 hours, preferentially about 48 hours, at 6-10°C. Higher protein concentration may be used in the refold if desired, however, overall refold efficiency will decrease.
  • Sodium Chloride and Phosphate are then added to final concentrations of 700 mM and 25 mM respectively, followed by pH adjustment to 7.0 to 9.0, preferably 7.9-8.0 with 6M HC1.
  • Step 6 IMAC Chromatography— The dilute proinsulin derivative is loaded onto an IMAC column to a maximum capacity of ⁇ 26.5 mg main peak protein per ml of resin. A 75mM imidizole buffer is used to isocratically strip the majority of impurities from the column. Tagged glargine proinsulin is then eluted isocratically using ⁇ 300 mM imidizole.
  • Step 7 Citriconic anhydride(CA) Blocking— To the AC pool, add citriconic anhydride at a molar ratio of 20:1 (CA to Glargine tagged proinsulin), while stirring at 4-10°C. Allow the sample to stir for not less than 3 hour at 4-10°C.
  • Step 8 - Buffer exchange To the blocked material, add EDTA to a final concentration of 20 mM. Exchange the buffer using a membrane with a suitable molecular weight cutoff (e.g. 3000 Da). The final buffer should be at least 97% exchanged to a 20 mM Tris-Cl, pH 7.0-10.0, preferably 8.8 to 9.2 at 8-10°C. A protein concentration of approximately 5 mg/ml is desirable.
  • Step 9 Trypsin Enzymatic Transformation/Proteolysis—
  • the buffer exchanged sample is digested with a 1000:1 mass ratio of main peak protein to trypsin, in the presence of 5mM CaCl.
  • the ratio of trypsin may be increased or decreased depending on the desired length of time for the reaction.
  • the digest is then quenched by the addition of acetic acid to > 700 mM.
  • Step 10 Citriconic anhydride Deblocking- The trypsin digest solution is then warmed to 18 to 20°C and the pH is adjusted to 2.8 to 3.0. The digest is stored at room temperature for not less than 10 hours to permit release of the citriconic anhydride.
  • Step 11 Ion Exchange Chromatography— The digested material is loaded onto a cation exchange column and eluted with a NaCl gradient, in the presence of 20% n-propanol or acetonitrile at pH 2-5, preferably 4.0. RP-HPLC is used to pool the appropriate fractions containing the Glargine insulin peak of interest at the desired purity level.
  • Step 12 Reverse Phase Chromatography— The S-column pool containing the Glargine insulin is loaded onto an RPC30 or CI 8 reverse phase column and eluted using an n- propanol or acetonitrile gradient in the presence of 200mM sodium sulfate and 0.136% phosphoric acid. Fractions are immediately diluted 1:4 with water if n-propanol is used for elution; or 1 :2 with water if acetonitrile is used for elution, or no dilution if acetonitrile is used for elution.
  • Step 13 - Buffer Exchange Exchange the sample into WFI (water for injection) using a membrane with a suitable molecular weight cutoff (e.g. 3000 Da).
  • the pH of the solution should be monitored and maintained at 2-5, preferably 3.5 to 4.0.
  • the final sample is concentrated to 5-8 mg/ml, with an adjusted pH of 2-5, preferably 3.8 to 4.2, chilled to 6-10°C.
  • This material represents the liquid API form of the presently disclosed preparations of Glargine Insulin Analog.
  • the API should be stored in the dark at 6-10°C.
  • the glargine Insulin Analog purified by Example 3 is formulated by diluting the API material with cold WFI to a final concentration of 4.54725 mg/ml.
  • a concentrated formulation buffer stock containing 85 mg/ml glycerol, 13.5 mg/ml meta cresol, 0.150 mg/ml zinc chloride and polysorbate(20) 0.1 mg/ml is added to the API material in a 1/5 ratio of formulation buffer stock to API.
  • the solution is mixed, followed by sterile filtration into appropriate vials in 10 ml aliquots.
  • Example 1 The cloning procedure outlined in Example 1 is utilized to create the initial vector.
  • Purified His Tagged Glargine proinsulin pTrcHis2A(Kan) vector is transformed into competent BL21 E. coli cells and plated on sterile LB-Kan plates. From the plates, an isolated colony is used to inoculate sterile LB-Kan media (-lOOmls). The cells are grown at 37°C to mid log phase ( ⁇ 4-5hours) OD 6 oo nm of -1.5-2.0. Culture media containing cells is then aliquoted into sterile cryovials, combined with glycerol at a 20% final concentration. The vials are then stored at - 80°C.
  • FIG. 3 depicts an analytical HPLC overlay of LANTUS® (A) and the glargine analog (B).
  • the glargine analog demonstrates increased purity with respect to related substances and multimeric species over LANTUS®.
  • the glarine analog shows noticeably lower levels of contaminants in both the related substance region and the multimeric region.
  • Most notably, the number of multimeric species is much lower in the glargine analog.
  • Overall purity for the LANTUS® material (A) in the current profile was 98.8%, while the glargine product produced by the herein described method (B) was 99.6%.

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Abstract

La présente invention concerne des constructions de pro-insuline glargine qui présentent une séquence d'acide nucléique et/ou d'acide aminé de C-peptide modifiée pour la production d'analogues de l'insuline glargine. L'invention concerne également des procédés hautement efficaces pour la préparation d'analogues de l'insuline glargine et des préparations améliorées contenant des analogues de l'insuline glargine préparés selon le procédé de la présente invention.
PCT/US2011/025922 2011-02-23 2011-02-23 Compositions de pro-insuline glargine et procédés de production d'analogues de l'insuline glargine à partir de celles-ci WO2012115638A1 (fr)

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CN103694339A (zh) * 2013-12-04 2014-04-02 珠海联邦制药股份有限公司 一种甘精胰岛素前体的复性方法
CN106117345A (zh) * 2015-05-05 2016-11-16 广东东阳光药业有限公司 一种制备甘精胰岛素结晶的方法
WO2020069011A1 (fr) * 2018-09-25 2020-04-02 Absci, Llc Procédés de purification de protéines
CN112789290A (zh) * 2018-08-08 2021-05-11 株式会社大熊制药 使用梭菌蛋白酶制备长效胰岛素类似物缀合物的活性形式的方法
EP4206220A4 (fr) * 2020-09-11 2024-03-06 Amphastar Nanjing Pharmaceuticals, Inc. Nouvelle pro-insuline glargine et procédé de préparation d'insuline glargine à partir de celle-ci

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103694339A (zh) * 2013-12-04 2014-04-02 珠海联邦制药股份有限公司 一种甘精胰岛素前体的复性方法
CN106117345A (zh) * 2015-05-05 2016-11-16 广东东阳光药业有限公司 一种制备甘精胰岛素结晶的方法
CN106117345B (zh) * 2015-05-05 2020-11-24 宜昌东阳光长江药业股份有限公司 一种制备甘精胰岛素结晶的方法
CN112789290A (zh) * 2018-08-08 2021-05-11 株式会社大熊制药 使用梭菌蛋白酶制备长效胰岛素类似物缀合物的活性形式的方法
CN112789290B (zh) * 2018-08-08 2024-05-17 株式会社大熊制药 使用梭菌蛋白酶制备长效胰岛素类似物缀合物的活性形式的方法
WO2020069011A1 (fr) * 2018-09-25 2020-04-02 Absci, Llc Procédés de purification de protéines
CN113286810A (zh) * 2018-09-25 2021-08-20 Absci有限责任公司 蛋白质纯化方法
US11584785B2 (en) 2018-09-25 2023-02-21 Absci, Llc C-peptides and proinsulin polypeptides comprising the same
EP4206220A4 (fr) * 2020-09-11 2024-03-06 Amphastar Nanjing Pharmaceuticals, Inc. Nouvelle pro-insuline glargine et procédé de préparation d'insuline glargine à partir de celle-ci

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