CA2908295C - Method for purifying teriparatide - Google Patents

Abstract

The present invention relates to a novel method for purifying teriparatide, a therapeutically active polypeptide fragment of full-length human parathyroid hormone. The method is based on ion exchange chromatography and is effective in separating the therapeutically active polypeptide from undesirable variants, such as truncated polypeptides. The method of the invention can be used at a preparative scale which allows it to be implemented into the production process for teriparatide. Accordingly, the invention also provides a method for the production of teriparatide which includes a step in which teriparatide is purified by the novel ion exchange chromatography method of the invention.

Description

CA 2,908,295 Blakes Ref.: 12770/00001

2 The present invention relates to a novel method for purifying teriparatide, a therapeutically active

3 polypeptide fragment of full-length human parathyroid hormone. The method is based on ion

4 exchange chromatography and is effective in separating the therapeutically active polypeptide from undesirable variants, such as truncated polypeptides. The method of the invention can be 6 used at a preparative scale which allows it to be implemented into the production process for 7 teriparatide. Accordingly, the invention also provides a method for the production of teriparatide 8 which includes a step in which teriparatide is purified by the novel ion exchange chromatography 9 method of the invention.
BACKGROUND OF THE INVENTION
11 Parathyroid hormone (PTH) is a polypeptide hormone which is naturally produced in the 12 parathyroid of mammals. The human polypeptide consists of 84 amino acids and is involved in 13 the regulation of the calcium concentration in blood plasma. If the calcium level drops below a 14 threshold level, PTH is secreted by cells of the parathyroid glands into the blood and induces a release of calcium from bone tissue. At the same time, PTH supports calcium absorption from the 16 small intestine and enhances calcium reabsorption from the primary urine, thereby suppressing 17 calcium loss via the kidneys. Due to its calcium releasing effects, an excessive amount of PTH in 18 the blood as commonly observed in primary and secondary hyperparathyroidism has been found 19 to be associated with reduced bone density and bone atrophy (osteoporosis). Considering the physiological effects of PTH, it appears odd that the hormone nevertheless proved useful in the 21 therapy of osteoporosis.
22 However, animal studies in rats for the first time revealed that a short-term exposure to PTH
23 supports bone formation due to transient activation of osteoclasts, whereas a sustained exposure 24 ultimately results in bone atrophy. Subsequent clinical studies in humans using the pharmaceutically active fragment PTH1-34 of human PTH confirmed that the fragment can be 26 used for treating osteoporosis. PTH1-34 is a polypeptide having a molecular mass of 4.7 kDa that 27 consists of the first 34 amino acids of the human PTH hormone. It was approved for osteoporosis 28 therapy in 2002 under the product name "teriparatide". Teriparatide is sold by Lilly Pharma under 29 the trade name Forteo (in the US) and Forsteo (in Europe).
CPST Doc: 375089.2 1 Date recue / Date received 2021-11-09 CA 2,908,295 Blakes Ref.: 12770/00001 1 At present, PTH1-34 for therapeutic use is produced by heterologous expression in bacterial host 2 cells and subsequent purification of the pharmacological active polypeptide. However, it has been 3 .. found that all known fragments and variants of PTH1-34 occur during the production process 4 demonstrate a reduced potency compared to PTH1-34. In addition a negative effect on overall purity is observed. For example, a number of fragments derived from PTH1-34 were identified in 6 .. the final batch obtained after heterologous expression, including amongst others PTH1-30, PTH2-7 34, PTH3-34 and PTH4-34. Moreover, chemical modifications of certain amino acids of PTH1-34, 8 e.g. oxidation of methionine residues or deamidation of asparagine residues, give rise to 9 additional variants.
However, as homogeneity of a product intended to be used as a therapeutic is of utmost 11 importance for safety reasons, any kind of truncated or chemically modified form of PTH1-34 in 12 the final product is clearly undesirable. Accordingly, current production processes normally 13 include one or more chromatography steps that aim at the purification of the PTH1-34 polypeptide.
14 However, it has been found to be problematic to separate PTH1-34 from some of its variants and fragments without significant product loss. In particular, it was observed that the fragment PTH2-16 .. 34 shows essentially the same retention properties in ion exchange chromatography as PTH1-17 34. Consequently, the PTH2-34 fragment is co-eluted with PTH1-34 which makes PTH1-34 18 purification challenging. Moreover, alternative chromatographic techniques, e.g. reversed phase 19 chromatography, were tested but did not show any potential for a preparative separation of truncated and full-length teriparatide.
21 In light of the above, new methods are needed which are effective in separating PTH1-34 from its 22 fragments and chemically modified variants. The method of the present invention provides for the 23 .. effective purification of the PTH1-34 polypeptide by cation exchange chromatography using 24 .. inversely directed gradients of pH and ionic strength. As discussed in more detail below, the method is particularly suitable for separating PTH1-34 from PTH2-34.
26 .. BRIEF DESCRIPTION OF THE FIGURES
27 Fig. 1 shows the primary and secondary structure of the parathyroid hormone fragment PTH1-34.
28 Fig. la shows the amino acid sequence and the distribution of charges in PTH1-34. Fig. lb shows 29 the secondary structure of PTH1-34.
CPST Doc: 375089.2 2 Date recue / Date received 2021-11-09 CA 2,908,295 Blakes Ref.: 12770/00001 1 Fig. 2 shows the attempt to separate PTH1-34 from PTH2-34 by cation exchange chromatography 2 at analytical scale using a salt gradient. Column volume (CV): 1 mL.
Resin: FractogelTM S03- (S).
3 Eluent A: 20 mM KH2PO4, pH 6.5. Eluent B: 20 mM KH2PO4, 500 mM KCI, pH
6.5. Loading: 1 4 mg/mL resin. Gradient 0-100% eluent B in 60 CV. Flow rate: 120 cm/h.
Fig. 3 shows the chromatographic separation of PTH1-34 and PTH2-34 using a cation exchange 6 column with a column volume of 29 mL. Eluent A: 20 mM Tris, 20 mM Bis-Tris, pH 6.5. Eluent B:
7 20 mM Tris, 20 mM Bis-Tris, 4 mM KCI, pH 8.2. Loading: 4.5 mg/mL resin.
Step elution with 13.5 8 column volumes of 100% elution buffer. Resin: Fractogel S03- (S). Flow rate: 150 cm/h.
9 Fig. 4 shows the chromatographic separation of PTH1-34 and PTH2-34 at preparative scale using a cation exchange column with a column volume of 26.3 L. Eluent A: 20 mM Tris, 20 mM
11 potassium dihydrogen phosphate, pH 6.5. Eluent B: 20 mM Tris, 16 mM
dipotassium hydrogen 12 phosphate, 4 mM potassium dihydrogen phosphate, pH 8.9. Loading: 3.5 g/L
resin. Resin:
13 Fractogel 503- (S). Flow rate: 100 cm/h.
14 Fig. 5 shows the results of the reversed-phase ultra high performance liquid chromatography (RP-uHPLC) analysis of fractions collected from the cation exchange chromatography run depicted in 16 Figure 4.

18 The present invention is based on the surprising insight that teriparatide (also referred to herein 19 as PTH1-34) can be effectively purified in an ion exchange chromatography process by increasing the pH and simultaneously decreasing the ionic strength in the fluid used for eluting the 21 polypeptide from the cation exchange material. The use of such inversely directed gradients has 22 not been disclosed in the prior art. Instead, it was basic knowledge in the field of ion exchange 23 chromatography that polypeptides, which have bound to an ion exchanger, can be eluted by 24 increasing the ionic strength in the eluent, e.g., by increasing the salt concentration in the eluent (S. Yamamoto, K. Nakanishi, R. Matsuno (1988), "Ion-Exchange Chromatography of Proteins", 26 Chromatographic Science Series, Vol. 43, Marcel Dekker, New York; C. T.
Mant et al. (2007), 27 "HPLC Analysis and Purification of Peptides", Methods in Molecular Biology, Vol. 386, 3-55).
28 Decreasing the ionic strength in the eluent is not a usual step in ion exchange chromatography.
29 It is therefore surprising that a dual gradient as applied in the method of the present invention is suitable for providing highly purified PTH1-34.
CPST Doc: 375089.2 3 Date recue / Date received 2021-11-09 CA 2,908,295 Blakes Ref.: 12770/00001 1 In a first aspect, the present invention therefore relates to a method for purifying PTH1-34 by ion 2 exchange chromatography, said method comprising the steps of:
3 (a) contacting a fluid containing PTH1-34 with a cation exchange material under conditions 4 that allow for the reversible binding of PTH1-34 to said cation exchange material;
(b) optionally washing the cation exchange material to remove unbound material;
6 (c) eluting PTH1-34 by increasing the pH and decreasing the ionic strength.
7 PTH1-34 and impurities derived thereof 8 The present method has been found to be effective for purifying therapeutically active PTH1-34 9 which is present in a fluid from any undesired PTH1-34 variants and fragments which might also be present in said fluid. These fragments may include, for example, truncated forms of PTH1-34 11 which can be regarded as product related impurities resulting from the polypeptide production 12 process. For example, the most common way of producing PTH1-34 includes heterologous 13 expression of the polypeptide in E. coil host cells. It was found that heterologous expression of 14 PTH1-34 may give rise to N-terminally and C-terminally truncated forms of PTH1-34 which are unacceptable and have to be removed from the product for safety reasons.
16 In particular, it has been observed that PTH2-34, a truncated form of PTH1-34 which lacks the N-17 terminal serine residue, is regularly present after heterologous expression. The overall amount of 18 the truncated PTH2-34 in the product solution obtained is up to 1%
(w/w). Other possible 19 truncated forms of PTH1-34 might lack the first 2 or 3 amino acids at the N-terminus. These variants are referred to herein as PTH3-34 and PTH4-34, respectively. In a preferred embodiment 21 of the invention, the ion exchange chromatography method provided herein is applied for the 22 purpose of removing one or more of the N-terminally truncated fragments PTH2-34, PTH3-34 and 23 PTH4-34. In a particular preferred embodiment, the claimed method is directed to the removal of 24 the truncated fragment PTH2-34.
Apart from N-terminally truncated fragments of PTH1-34, the fluid to be subjected to the 26 purification method of the present invention may also contain C-terminally truncated forms of 27 PTH1-34. For example, fragments which lack 1, 2, 3 or 4 of the amino acids located at the C-28 terminus in PTH1-34 may occur. Therefore, in another preferred embodiment, the ion exchange CPST Doc: 375089.2 4 Date recue / Date received 2021-11-09 CA 2,908,295 Blakes Ref.: 12770/00001 1 chromatography method provided herein is applied for the purpose of removing one or more of 2 the C-terminally truncated polypeptides PTH1-33, PTH1-32, PTH1-31 and PTH1-30. In a 3 particularly preferred embodiment, the claimed method is applied for removing PTH1-30.
4 In a still further embodiment of the present invention, the new method is carried out for the purpose of removing chemically modified variants of PTH1-34 comprising modifications at one or more 6 amino acid side chains. These variants may arise as product related impurities in the production 7 of PTH1-34. A number of variants of PTH1-34 have been identified which differ from the original 8 polypeptide in that certain amino acid residues have been oxidized, deaminated and/or modified 9 by succinimide formation. For example, a variant has been identified which has a deaminated asparagine residue in position 16 of PTH1-34. Another variant has oxidized methionine residues 11 in position 8 and/or 18 of PTH1-34. Like with the truncated fragments, it is required for safety 12 reasons to remove these variants from the final PTH1-34 product.
13 The PTH1-34 to be purified with the method of the invention preferably comprises or consists of 14 the amino acid sequence set forth in SEQ ID NO:1. The polypeptide depicted in SEQ ID NO:1 corresponds to the 34 N-terminal amino acids of the naturally occurring human parathyroid 16 hormone. For comparison, the amino acid sequence of the full-length human parathyroid hormone 17 is shown in SEQ ID NO:2. Figure la shows the distribution of the charged amino acids within the 18 PTH1-34 sequence of SEQ ID NO:1. Under physiological conditions, the PTH1-34 sequence of 19 SEQ ID NO:1 comprises two alpha-helical regions that are connected via a loop region (see Figure 1 b). No tertiary structure had been observed for the molecule under physiological 21 conditions.
22 Also encompassed by the term "PTH1-34" are homologs of the sequence set forth in SEQ ID
23 NO:1. As used herein, homologs of the PTH1-34 of SEQ ID NO:1 are polypeptides which differ 24 from the polypeptide in SEQ ID NO:1 in a limited number of amino acids, e.g. in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acids. Preferably, the homolog differs in no more than 15 amino 26 acid from the polypeptide depicted in SEQ ID NO:l. For example, the polypeptide to be purified 27 by the method of the invention can be a polypeptide which differs from PTH1-34 as shown in SEQ
28 ID NO:1 in that no more than 1, 2, 3,4, or 5 amino acids have been substituted by other amino 29 acids. Where an amino acid substitution is made, the substitution preferably is a conservative amino acid substitution, i.e. a substitution of one amino acid by another amino acid of similar 31 polarity which can act as a functional equivalent. Preferably, the amino acid used as a substitute CPST Doc: 375089.2 5 Date recue / Date received 2021-11-09 CA 2,908,295 Blakes Ref.: 12770/00001 1 is selected from the same group as the amino acid residue to be substituted. For example, a 2 hydrophobic residue can be substituted with another hydrophobic residue, or a polar residue can 3 be substituted with another polar residue having the same charge.
Functionally homologous 4 amino acids which may be used for a conservative substitution comprise, for example, non-polar amino acids such as glycine, valine, alanine, isoleucine, leucine, methionine, proline, 6 phenylalanine, and tryptophan. Examples of uncharged polar amino acids comprise serine, 7 threonine, glutamine, asparagine, tyrosine and cysteine. Examples of charged polar (basic) amino 8 acids comprise histidine, arginine and lysine. Examples of charged polar (acidic) amino acids 9 comprise aspartic acid and glutamic acid.
It is particularly preferred that the PTH1-34 homolog which may comprise 1, 2, 3, 4, or 5 amino 11 acid substitutions compared with the PTH1-34 of SEQ ID NO:1 comprises no chemical modifica-12 tions, such as oxidations, deamidations, cyclizations, and the like. It is furthermore preferred that 13 the homolog comprises or consists of 34 amino acids.
14 According to the invention, the PTH1-34 polypeptide which is to be subjected to the method of the invention can be derived from any suitable method which is known in the art for producing 16 polypeptides. The polypeptide may have been prepared both by ribosomal and non-ribosomal 17 methods. For example, the PTH1-34 may have been chemically synthesized by solid phase or 18 liquid phase methods. Protocols for solution-phase chemical synthesis of peptides have been 19 described (see, e.g., Andersson et al., Biopolymers 55:227-250, 2000).
For solid phase synthesis the basic technique described by Merrifield (J. Am. Chem. Soc., 1964, 85, 2149-2154) may have 21 been used. In that approach, the growing peptide is anchored on an insoluble resin, and unreacted 22 soluble reagents are removed by filtration or washing steps without manipulative losses. The solid 23 phase synthesis technique of Merrifield has been improved in the last decade and nowadays 24 allows the synthesis of polypeptides with up to 50 amino acids. The chemical synthesis of PTH1-34 fragments was disclosed, e.g. in the US patents 4,427,827 and US 4,105,602.
26 Alternatively, the PTH1-34 polypeptide to be purified according to the invention can be a 27 recombinantly produced PTH1-34, i.e. the polypeptide has been produced by biotechnological 28 processes that involve genetically modified cells or organisms.
Typically, a nucleotide sequence 29 encoding the PTH1-34 polypeptide is cloned into a suitable vector which provides for the express-ion or overexpression of the polypeptide in a host cell. The vector is introduced into a prokaryotic 31 or eukaryotic host cell, and the cell is cultured under conditions suitable for the expression of the CPST Doc: 375089.2 6 Date recue / Date received 2021-11-09 CA 2,908,295 Blakes Ref.: 12770/00001 1 polypeptide. Normally, the polypeptide to be expressed will be under the control of an inducible 2 promoter so that expression may be initiated by the addition of an inducer, e.g. IPTG, to the 3 culture medium. After incubation of the cells to allow expression, the cells are usually harvested 4 and homogenized to release the recombinant PTH1-34 polypeptide. Finally, the polypeptide is recovered by one or more purification steps. In accordance with the present invention, it is 6 particularly preferred that the PTH1-34 polypeptide is a polypeptide that has been recombinantly 7 produced by heterologous expression in host cells.
8 To simplify its subsequent enrichment and purification, the recombinant PTH1-34 may be 9 prepared in form of a fusion polypeptide. As used herein, a fusion polypeptide refers to a fusion of the amino acid sequence of PTH1-34 to a second amino acid sequence. The second amino 11 acid sequence may be, for example, an affinity tag, i.e. an amino acid sequence which is either 12 N-terminally or C-terminally fused to the PTH1-34 polypeptide, and which has a strong affinity to 13 another compound or material, thereby allowing enrichment and/or purification of the fusion 14 polypeptide as a whole. The affinity tag may be, for example, a poly-histidine sequence comprising 6-12 histidines which specifically interact with a nickel ion chelate matrix or alternative 16 IMAC resins. Alternatively, the affinity tag may be a glutathione-S-transferase which allows the 17 purification via a glutathione matrix. Further affinity tags are well-known in the art. An affinity tag 18 sequence can be removed from the PTH1-34 sequence after purification with the affinity matrix, 19 for example, by providing a proteolytic cleavage site between PTH1-34 and the affinity tag.
The recombinant PTH1-34 may be expressed in high amounts in the bacterial host cells so that 21 inclusion bodies are formed in which the recombinant polypeptides are present in partially folded 22 form and aggregate through non-covalent hydrophobic or ionic interactions. Where the method 23 for producing the recombinant PTH1-34 provides the polypeptide in inclusion bodies, it will 24 preferably also include a step in which the polypeptides in the inclusion bodies are solubilised and refolded. Methods for solubilisation and refolding of polypeptides from inclusion bodies are well 26 known in the art.
27 Chromatographic device and cation exchange material 28 The present method is preferably carried out by use of a FPLC (fast protein liquid 29 chromatography) device, i.e. an automated device which comprises a column which includes the cation exchange material as the stationary phase, one or more pumps which provide a pressure 31 that is sufficient for causing the mobile phase (i.e. the buffers used for binding, washing and CPST Doc: 375089.2 7 Date recue / Date received 2021-11-09 CA 2,908,295 Blakes Ref.: 12770/00001 1 elution) to flow through the cation exchange material, an apparatus which collects the fractions 2 eluted from the column, and a detector that is capable of detecting the PTH1-34 which is eluted 3 from the column. Detection can be achieved by measuring the absorption at a wavelength of e.g.
4 280 nm. In addition, the changes in pH and conductivity can also be monitored in the fractions. A
considerable number of automated FPLC devices which are suitable for cation exchange 6 chromatography are commercially available from different manufacturers.
Non-limiting examples 7 for such chromatographic devices include the AKTApurifier, AKTAavant, AKTApilot, 8 AKTAexplorer or AKTAprocess systems (GE Healthcare, the K-Prime 40 systems (Merck 9 Millipore), the NCG systems (Bio-Rad), and similar devices. The use of these automated devices is, however, not mandatory for carrying out the method of the present invention.
11 The method of the invention can make use of any cation exchange material that has been 12 described in the art as being useful for the purification of polypeptides. As used herein, the cation 13 exchange material is an insoluble polymeric matrix which comprises on its surface negatively 14 charged functional groups that are capable of attracting cations or positively charged molecules.
The cation exchange material can be in the form of porous beads having, e.g., a size of between 16 10-100 pm, preferably 20-80 pm. A number of different cation exchange materials are known in 17 the art, including cross-linked natural polymers, organic polymers and inorganic materials. While 18 carboxymethyl groups are commonly used in weak cation exchangers, strong cation exchangers 19 often include sulfopropyl groups. When performing the method of the present invention, it is preferred that the cation exchanger is present in the form of a packed column suitable for use in 21 an automated FPLC device, such as the Akta Purifier 100 FPLC System.
22 In another preferred embodiment of the invention the cation exchange material used in the 23 purification method is a strong cation exchanger which comprises sulfoethyl, sulfopropyl, 24 sulfobutyl and/or sulfoisobutyl as functional groups. Cation exchanger materials which comprise a mixture of one or more of the above groups may likewise be used in the method of the invention.
26 Further, the cation exchange material can be based on any common polymeric material which 27 has been described for use in this particular type of chromatography.
For example, the polymeric 28 cation exchange material can comprise cross-linked polystyroldivinylbenzol, polymethacrylate, 29 styrene-divinylbenzene, cross-linked Agarose, silica-based matrices, polyvinyl ether, cellulose, and dextran.
CPST Doc: 375089.2 8 Date recue / Date received 2021-11-09 CA 2,908,295 Blakes Ref.: 12770/00001 1 Cation exchange materials or columns containing these materials can be purchased from different 2 providers and include, e.g. the Unosphere S 70 resin (Biorad, Munich, Germany), the Nuvia TM S
3 resin (Biorad, Munich, Germany), the Poros HS20 or HS50 resin (Life Technologies, Darmstadt, 4 Germany), the Source 15S or 30S resin (GE Healthcare, Freiburg, Germany), the Polysulphoethyl A resin (PolyLC, Columbia, USA), the Ceramic HyperD F(S) resin (Pall, Dreieich, Deutschland), 6 the BakerbondTM PolyCSX-35 Bioseparation Media (Avantor Performance Materials, Center 7 Valley, USA), the Eshmuno S and CPX resins (Merck Millipore, Darmstadt, Germany), the 8 Toyopearl resins SP-650M and GigaCap S-650M (Tosoh Bioscience LLC, King of Prussia, USA) 9 and other cation exchange stationary phases regularly used for purifying proteins.
The use of one of the following resins is particularly preferred in the method of the invention: the 11 Eshmuno S resin, the Eshmuno CPX resin, the Poros HS20 resin, and the Toyopearl Tm 12 GigaCapTM S-650M resin. A further particularly preferred cation exchange resin material for use 13 in the method of the invention is the Fractogele resin developed by Merck Millipore. The 14 Fractogele resin uses the so-called "tentacle technology" which means that the surface of the resin beads comprises long polymer chains which provide for an improved accessibility of the 16 proteins to be purified to the functional groups of the resin material.
The method of the invention 17 is preferably carried out with the Fractogele resin EMD SE Hicap (M), the Fractogele resin EMD
18 503- (S), or the Fractogele resin EMD 503- (M). Use of the Fractogele resin EMD 503- (S) is 19 particularly preferred according to the invention.
The cation exchange columns used in the purification method disclosed herein can be of any size.
21 Given that the method of the invention aims at the purification of PTH1-34 from production 22 batches, the use of preparative columns which allow for the binding of high amounts of PTH1-34 23 is particularly preferred. Preparative columns having a bed volume of 1-100 L can be used, 24 whereas bed volumes of 10-50 L are particularly preferred, for example 45 L. For example, the preparative columns used in the purification method of the present invention can have a bed 26 volume of at least 1 L, at least 2 L, at least 3 L, at least 4 L, at least 5 L, at least 10 L, at least 20 27 L, at least 25 L, or more. Usually, preparative columns useful in the method of the invention will 28 have a diameter of between 1-100 cm, whereas a diameter of between 20-80 cm, or 40-60 cm is 29 particularly preferred.
Analytical columns having bed volumes of 1-5 mL may be used for further optimizing the 31 conditions of the cation exchange purification method of the invention, for example, to adjust the CPST Doc: 375089.2 9 Date recue / Date received 2021-11-09 CA 2,908,295 Blakes Ref.: 12770/00001 1 method to other polypeptides. It should, however, be noted that conditions that were found highly 2 useful for an analytical column might not equally be transferable to the conditions on a preparative 3 column (see Examples below).
4 Sample and column preparation The method of the invention uses as a starting material a fluid that contains PTH1-34. The fluid 6 can be the supernatant of a cell culture that was used for heterologous expression of the 7 polypeptide. For example, it is possible to subject the supernatant comprising the soluble PTH1-8 34 polypeptides and other soluble components of the host cells directly to the ion exchange 9 chromatography method of the invention, either with or without prior dilution. It is, however, preferred that before applying the purification method of the invention the supernatant obtained 11 from cell culture is further processed, e.g. by centrifugation, filtration, size-exclusion chromato-12 graphy, affinity chromatography, RP-HPLC, and the like, to remove undesired substances, e.g.
13 polynucleotides and/or polysaccharides, to cleave off an affinity tag or another peptide or 14 polypeptide that is fused to PTH1-34, and/or to enrich the PTH1-34 polypeptide. The fluid to be used in the method of the invention preferably comprises the PTH1-34 polypeptide in substantially 16 enriched form which means that at least 50% of the polypeptide compounds dissolved in the fluid 17 is PTH1-34.
18 In one embodiment, the fluid used as a starting material in the method of the invention is obtained 19 from a prior reversed phase chromatography run, i.e. the fluid is the eluate of reversed phase chromatography. This eluate will contain significant amounts of organic solvents, such as 21 acetonitrile, and it might be necessary to dilute it with a suitable buffer before applying it to the 22 cation exchange material. Conveniently, the buffer used for dilution can be the same buffer that 23 was used for equilibrating the cation exchange material. The eluate from the reversed phase 24 chromatography can be diluted by factor 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. A dilution by factor 2-4 has been proven particularly useful. Where dilution is not possible, e.g., due to an excessive 26 amount of acetonitrile in the fluid, a complete buffer exchange may be performed, which means 27 that PTH1-34 is transferred from the reversed phase eluate to a buffer that is adjusted to 28 subsequent ion exchange chromatography. A buffer exchange can be easily accomplished, e.g.
29 by tangential flow filtration, size exclusion chromatography, diafiltration or dialysis. It is preferred that PTH1-34 is transferred to the buffer which is used during binding of the polypeptide to the 31 cation exchange material (referred to in the following as "binding buffer").
CPST Doc: 375089.2 10 Date recue / Date received 2021-11-09 CA 2,908,295 Blakes Ref.: 12770/00001 1 Before application of the fluid containing the PTH1-34 polypeptide to the cation exchange 2 material, the cation exchange material will usually be equilibrated by washing the column with 1-3 50, preferably 1-25, and more preferably 1-10 column volumes of an equilibration buffer to provide 4 conditions that promote binding of the polypeptide to the cation exchanger. Preferably the equilibration buffer and the binding buffer are identical. After equilibration, the fluid containing the 6 PTH1-34 polypeptide is contacted with the cation exchange material.
7 Contacting PTH1-34 with the cation exchange material 8 According to step (a) of the above method, the fluid containing PTH1-34 is contacted with the 9 cation exchange material under conditions that provide for the binding of the polypeptide to the polymeric matrix of the cation exchanger. This means that the conditions in terms of temperature, 11 pressure and pH are such that PTH1-34 can displace the cations that have bound to the cation 12 exchange material after equilibration. In particular, the binding of the polypeptide to the cation 13 exchange material is facilitated by selection of an appropriate pH of the binding buffer.
14 For identifying a suitable pH that allows binding of the PTH1-34 to a particular cation exchange material, the polypeptide can be contacted with the cation exchanger of choice under different pH
16 conditions while detecting the amount of the polypeptide in the flow-through. In this way, a suitable 17 pH range for binding of the polypeptide to the cation exchanger can be readily determined. It is 18 preferred according to the invention that the pH is selected such that the PTH1-34 is present in 19 the form of a positively charged polypeptide which supports its binding to the cation exchanger.
To provide positively charged PTH1-34 polypeptide molecules the pH of the buffer used in the 21 binding step should preferably be below the isoelectric point of the polypeptide. The PTH1-34 set 22 forth in SEQ ID NO:1 has an isoelectric point of 8.3. Thus, where it is intended to purify the PTH1-23 34 of SEQ ID NO:1 with the method of the present invention, the pH in the binding step should 24 be selected to be below 8.3.
Preferably, the binding buffer used for application of the PTH1-34 polypeptide of SEQ ID NO:1 to 26 the cation exchange material will be adjusted to a pH of 8.0 or less. It will be particularly preferred 27 that the pH of the binding buffer will be at least 0.5 pH units, more preferably at least 1.0, at least 28 1.5, or at least 2.0 pH units below the isoelectric point of the polypeptide to be purified. Where the 29 method of the invention is performed with the PTH1-34 set forth in SEQ
ID NO:1, binding of the polypeptide to the cation exchange material is preferably performed in binding buffer having a pH
31 below 7.8, below 7.3, below 6.8, and more preferably below 6.3, or even below 6Ø For example, CPST Doc: 375089.2 11 Date recue / Date received 2021-11-09 CA 2,908,295 Blakes Ref.: 12770/00001 1 the PTH1-34 of SEQ ID NO:1 is bound to the cation exchange material using binding buffer at a 2 pH of between 6.0 and 8.0, preferably at a pH of between 6.5 and 7.5, and more preferably at a 3 .. pH of between 6.8 and 7.2. In cases where a homolog of PTH1-34 is used which differs from the 4 PTH1-34 in SEQ ID NO:1 by 1, 2 or 3 amino acid substitutions, it may be possible that the .. isoelectric point of said modified polypeptide is slightly different. The skilled person will be readily 6 able to determine the isoelectric point of the homolog by use of routine methods well known in 7 the art.
8 The present invention is amongst others based on the insight that chromatographic separation of 9 .. PTH1-34 from its fragments and variants can be improved by eluting PTH1-34 from the cation exchange material by increasing the pH and simultaneously lowering ionic strength in the mobile 11 phase. This can be conveniently be accomplished by using an eluent which, compared to the 12 binding or washing buffer, has a higher pH and a lower conductivity.
Thus, the binding buffer used 13 .. for binding PTH1-34 to the cation exchange resin in the method of the invention will exhibit an 14 .. ionic strength which is higher than that of the eluent. This is unusual for cation exchange chromatography, because the ionic strength in the eluent is normally gradually increased to 16 support displacement of polypeptides from a cation exchanger. The conductivity (as expressed in 17 .. mS/cm) of the buffers used in the ion exchange chromatography process is a suitable measure 18 that reflects ionic strength of said buffer. Accordingly, the terms "conductivity" and "ionic strength"
19 .. are used interchangeably herein.
.. It is preferred that the binding buffer has a conductivity that is at least 30% higher, at least 50%
21 higher, at least 75% higher, at least 100% higher, at least 150% higher, at least 200% higher, or 22 .. at least 300% higher than the conductivity of the corresponding eluent.
The conductivity of the 23 binding buffer will preferably be in the range 1.5-3.0 mS/cm, for example, more than 1.5 mS/cm, 24 more than 1.6 mS/cm, more than 1.7 mS/cm, more than 1.8 mS/cm, more than 1.9 mS/cm, more than 2.0 mS/cm, more than 2.1 mS/cm, more than 2.2 mS/cm, more than 2.3 mS/cm, more than 26 .. 2.4 mS/cm, more than 2.5 mS/cm, more than 2.6 mS/cm, more than 2.7 mS/cm, more than 2.8 27 mS/cm, or more than 2.9 mS/cm. Preferably, the conductivity of the binding buffer will be at least 28 0.5 mS/cm higher than the conductivity of the corresponding eluent, more preferably at least 0.6 29 mS/cm, at least 0.7 mS/cm, at least 0.8 mS/cm, at least 0.9 mS/cm, at least 1.0 mS/cm, at least 1.1 mS/cm, at least 1.2 mS/cm, at least 1.3 mS/cm, at least 1.4 mS/cm, at least 1.5 mS/cm, at 31 .. least 1.6 mS/cm, at least 1.7 mS/cm, at least 1.8 mS/cm, at least 1.9 mS/cm, or at least 2.0 32 mS/cm.
CPST Doc: 375089.2 12 Date recue / Date received 2021-11-09 CA 2,908,295 Blakes Ref.: 12770/00001 1 In a preferred aspect, the conductivity of the binding buffer is in the range between 2.0 and 3.0 2 mS/cm, and the conductivity of the eluent is in the range between 0.5 and 1.5 mS/cm. In an even 3 more preferred aspect, the conductivity of the binding buffer is in the range between 2.5 and 2.8 4 mS/cm, and the conductivity of the eluent is in the range between 1.0 and 1.3 mS/cm.
Several buffers can be used in the method of the present invention. It is preferred according to 6 the invention that the binding buffer and the elution buffer are prepared with the same buffer 7 substance. The buffer compound used for preparing the binding buffer and the elution buffer is 8 selected such that its pH range covers the isoelectric point of the PTH1-34 polypeptide, i.e. the 9 pK, value of the buffer substance is not more than one pH unit higher or lower than the isolelectric point of the PTH1-34 polypeptide. Where the PTH1-34 polypeptide to be purified is the 11 polypeptide of SEQ ID NO:1, the buffer will be selected such that its pH
range covers pH 8.3, i.e.
12 the buffering compound has a pK, of between 7.3 and 9.3. A pH range in the area of the isoelectric 13 point of the PTH1-34 has the particular advantage that the preparation of the binding buffer, which 14 preferably has a pH of below 7.5, and more preferably a pH around 6.5, will require considerable amounts of acid, such as HCI, for pH adjustment. The addition of HCI or other acids to the binding 16 buffer will increase the ionic strength of the binding buffer relative to the elution buffer, the latter 17 of which has a higher pH and therefore requires only slight or no pH
adjustment.
18 Suitable buffer substances having a pK, in the range of 8.3 include 19 Tris(hydroxymethyl)aminomethane (referred to as "Tris" herein), triethanolamine, and borate buffer. Additional buffers that have been found to be suitable for carrying out the method of the 21 invention include a barbital buffer having a pH in the range of, e.g., 6.8-9.2, and a glycylglycine 22 buffer having a pH in the range of, e.g., 7.3-9.3. The use of Tris buffer is preferred herein, because 23 it was unexpectedly found to result in a particularly good separation of PTH1-34. The use of Tris 24 buffer was not an obvious measure in the method of the invention, because Tris is commonly recommended in the literature for use in anion exchange chromatography. Tris is a cationic buffer 26 which is in principle able to bind to the functional groups of the cation exchange resin, thereby 27 disturbing reproducible separation. In the method of the present invention, however, it was 28 observed that the use of Tris does not negatively influence the purification of PTH1-34 via cation 29 exchange chromatography.
The buffering compound will be present in the binding buffer in an amount of 1-200 mM, preferably 31 10-100 mM, and more preferably 20-80 mM. Where Tris is used in the binding buffer, the preferred CPST Doc: 375089.2 13 Date recue / Date received 2021-11-09 CA 2,908,295 Blakes Ref.: 12770/00001 1 concentration will be in range of 20-40 mM, wherein 20 mM is particularly preferred. Additional 2 salts, such as NaCI or KH2PO4, may be used as additives in amounts of 1-100 mM, preferably 3 20-80 mM, to adjust the conductivity of the buffer where necessary. Other buffering compounds, 4 such as Bis-Tris may also be added to the Tris buffer. Non-limiting examples for a Tris-based binding buffer which is suitable to be used in the purification of the PTH1-34 polypeptide of SEQ
6 ID NO:1 include:
7 20 mM Tris, pH 6.5-7.5 8 30 mM Tris, pH 6.5-7.5 9 40 mM Tris, pH 6.5-7.5 20 mM Tris, 20 mM KH2PO4, pH 6.5-7.5 11 30 mM Tris, 30 mM KH2PO4, pH 6.5-7.5 12 40 mM Tris, 40 mM KH2PO4, pH 6.5-7.5 13 20 mM Tris, 20 mM Bis-Tris, pH 6.5-7.5 14 30 mM Tris, 30 mM Bis-Tris, pH 6.5-7.5 40 mM Tris, 40 mM Bis-Tris, pH 6.5-7.5 16 Based on his experience and the information and the particular examples provided herein, the 17 skilled person will be readily able to prepare additional binding buffers that are suitable for being 18 used in the purification process of the present invention.
19 The contacting of the PTH1-34 polypeptide with the cation exchange material can be performed in different ways, depending on the particular equipment which is used for carrying out the method 21 of the invention. Where an FPLC device is used for the purification process of the invention, the 22 PTH1-34 polypeptide can be contacted with the cation exchange material conveniently by 23 injecting the PTH1-34 containing fluid (either diluted with binding buffer or not) into an injection 24 loop which is then introduced into the flow of the binding buffer. The flow rate during binding will be in the range of 50-400 cm/h, preferably 100-300 cm/h, more preferably 150-200 cm/h.
26 When using preparative columns having a having a bed volume size of 1-50 L, the PTH1-34 will 27 be applied to the column such that the overall protein load is in the range of 2.0-4.5 g/L. In 28 approaches using columns having a bed volume size of 10-250 mL, the overall protein load is in 29 the range of 10-20 mg/ mL. When using analytical columns having a bed volume size of less than CPST Doc: 375089.2 14 Date recue / Date received 2021-11-09 CA 2,908,295 Blakes Ref.: 12770/00001 1 10 mL, e.g., 5 mL or 1 mL, the overall protein load will normally be in the range of 0.1-5 mg/ mL, 2 e.g. about 1 mg/ mL.
3 Washing the cation exchange resin 4 After protein binding the cation exchange material is optionally washed to remove unbound material. For this purpose, the cation exchange material is preferably washed with several column 6 volumes of washing buffer. According to the invention, the washing buffer may be identical to the 7 binding buffer in terms of its pH or ionic strength. The washing buffer may contain additional 8 compounds not included in the binding buffer. In any case, however, the pH and ionic strength of 9 the washing buffer should be adjusted to avoid any premature elution of the PTH1-34 polypeptide from the cation exchange material.
11 Depending on the size of the column used in the method of the invention, it will be useful to wash 12 the column after polypeptide binding with 1-25 column volumes, preferably 1-15 column volumes, 13 and more preferably 1-10 column volumes. In an even more preferable embodiment of the 14 invention, the cation exchange resin is washed after PTH1-34 binding until the detected UV
absorption, pH and conductivity signals remain constant.
16 Eluting the PTH1-34 polvpeptide 17 After PTH1-34 binding to the cation exchange material and (where applicable) washing said 18 material, the PTH1-34 polypeptide is eluted. According to the invention, elution is effected by 19 increasing the pH and decreasing the ionic strength. Compared with the binding buffer, the eluent will thus have a higher pH which is close to the isoelectric point of the PTH1-34 polypeptide. By 21 increasing the pH, the overall positive charge of the PTH1-34 polypeptide is reduced and the 22 polypeptide will ultimately be released from the cation exchange material. Preferably, the eluent 23 is an elution buffer, i.e. it comprises at least one buffering compound.
24 The increase in pH can be accomplished in different ways. For example, a gradient elution can be conducted in which the eluent is gradually mixed with the binding or washing buffer. In this 26 way, the pH of the mobile phase flowing over the cation exchange material gradually increases 27 so that different polypeptides, which have bound to the cation exchange material, are released in 28 accordance with their interaction strength. The gradient used can be a convex, concave or linear 29 gradient, but linear gradients are particularly preferred. The steepness of the gradient can be varied as needed. Normally, linear gradients with a steepness of 1-5% elution buffer per column CPST Doc: 375089.2 15 Date recue / Date received 2021-11-09 CA 2,908,295 Blakes Ref.: 12770/00001 1 volume are useful for carrying out the method of the invention.
Alternatively, an isocratic step 2 elution can be performed by switching from binding or wash buffer directly to 100% eluent without 3 any gradient. In an isocratic step elution, the composition of the eluent remains constant during 4 elution, because the eluent is not gradually mixed with binding or washing buffer in the course of elution.
6 The eluent used for releasing the PTH1-34 polypeptide from the cation exchange material will be 7 adjusted to a pH of 8.0 or higher. It will be preferred that the pH of the eluent will be 0.1 pH units, 8 0.2 pH units, or 0.3 pH units higher or lower than the isoelectric point of the PTH1-34 polypeptide 9 to be purified. Where the method of the invention is performed with the PTH1-34 of SEQ ID NO:1, the eluent preferably has a pH of at least 8.0 or more, at least 8.1 or more, at least 8.2 or more, 11 at least 8.3 or more, at least 8.4 or more, at least 8.5 or more, or at least 9.0 or more. In cases 12 where a homolog of PTH1-34 is used which differs from the PTH1-34 in SEQ
ID NO:1 by 1, 2 or 13 3 amino acid substitutions, it may be possible that the isoelectric point of said modified polypeptide 14 is slightly different. Therefore, the optimum pH to be used during the elution step might slightly vary from that determined for the PTH1-34 of SEQ ID NO:1.
16 As stated above in the context with the binding buffer, the eluent used for elution of PTH1-34 will 17 have an ionic strength which is significantly lower than that of the binding and/or washing buffer.
18 Preferably, the eluent will have a conductivity in the range 0.1-1.4 mS/cm, for example, less than 19 1.4 mS/cm, less than 1.3 mS/cm, less than 1.2 mS/cm, less than 1.1 mS/cm, less than 1.0 mS/cm, less than 0.9 mS/cm, less than 0.8 mS/cm, less than 0.7 mS/cm, less than 0.6 mS/cm, less than 21 0.5 mS/cm, less than 0.4 mS/cm, less than 0.3 mS/cm, or less than 0.2 mS/cm. In another aspect, 22 the conductivity of the eluent is in the range between 0.2 and 1.4 mS/cm, preferably between 0.5 23 and 1.3 mS/cm.
24 The buffer used for preparing the eluent will preferably be the same as the one used for binding and/or washing buffer, wherein Tris, triethanolamine, and borate buffer are preferred for use. The 26 use of Tris buffer is particularly preferred.
27 The buffering substance will be present in the eluent in an amount of 1-200 mM, preferably 10-28 100 mM, and more preferably 20-80 mM. Where Tris is used in the eluent, the preferred 29 concentration will be in range of 20-40 mM. An amount of 20 mM Tris in the eluent is particularly preferred. Additional salts, such as NaCI or KH2PO4, may be used as additives in amounts of 1-CPST Doc: 375089.2 16 Date recue / Date received 2021-11-09 CA 2,908,295 Blakes Ref.: 12770/00001 1 100 mM, preferably 20-80 mM. Other buffering compounds, such as Bis-Tris may also be added 2 to the Tris buffer. Non-limiting examples for a Tris-based eluent which is suitable to be used in 3 the purification of the PTH1-34 polypeptide of SEQ ID NO:1 include:
4 20 mM Tris, pH 9.0 30 mM Tris, pH 9.0 6 40 mM Tris, pH 9.0 7 20 mM Tris, 3 mM KH2PO4, 13 mM K2HPO4, pH 9.0 8 30 mM Tris, 4 mM KH2PO4, 14 mM K2HPO4, pH 9.0 9 40 mM Tris, 5 mM KH2PO4, 15 mM K2HPO4, pH 9.0 20 mM Tris, 20 mM Bis-Tris, 5 mM KCI, pH 8.2 11 20 mM Tris, 20 mM Bis-Tris, 5 mM KCI, pH 8.3 12 20 mM Tris, 20 mM Bis-Tris, 5 mM KCI, pH 9.0 13 20 mM Tris, 20 mM Bis-Tris, 5 mM KCI, pH 10.0 14 The elution will be performed by applying 10-50 column volumes to the cation exchange material.
If a linear gradient from 0-100% eluent is used, the gradient may be applied in 10-50 column 16 volumes. Gradient volumes of 10-20 column volumes are expected to provide the best 17 compromise between peak dilution and resolution.
18 Preferred modes for carrying out the purification 19 The purification method of the invention is able to separate therapeutically active PTH1-34 from chemically modified by-products and fragments. It is preferred that the eluate obtained in step (c) 21 contains less than 5% impurities, i.e. proteinaceous compounds which are not unmodified PTH1-22 34. Specifically, it is preferred that the eluate obtained in step (c) contains less than 5% (w/w) of 23 the PTH2-34 fragment and/or less than 5% deamidated PTH1-34. Even more preferably, the 24 eluate obtained in step (c) contains less than 0.5% (w/w) of the PTH2-34 fragment and/or less than 0.5% deamidated PTH1-34. In a most preferred embodiment of the method, the eluate 26 obtained in step (c) contains no detectable PTH2-34.
27 According to a particularly preferred embodiment of the invention, the PTH1-34 to be purified is 28 the polypeptide depicted in SEQ ID NO:1, and the purification method is performed with a buffer 29 system comprising eluent A: 20 mM Tris (with or without 20mM Bis-Tris), pH 6.5-7.0, and eluent CPST Doc: 375089.2 17 Date recue / Date received 2021-11-09 CA 2,908,295 Blakes Ref.: 12770/00001 1 B: 20 mM Tris (with or without 20mM Bis-Tris), pH 8.0-10Ø It is moreover preferred to initiate 2 elution by performing a step elution with 75-100% eluent B.
3 According to the invention, it is possible to add further compounds to eluent A or B that may prove 4 advantageous for a number of embodiments of the invention. For example, the elution buffer may comprise 250 mM sucrose, Tween 20 (1%), urea (1-2 M) and/or arginine (50-100 mM).
6 Preparation of the PTH1-34 polypeptide 7 The purification method described above can be part of a process for the preparation of PTH1-8 34. This means that the purification method of the present invention can be implemented into a 9 .. manufacturing process which includes the heterologous expression of the polypeptide and several downstream processing steps which are directed to the refolding and purification of the 11 .. polypeptide.
12 .. Thus, in another aspect, the present invention also relates to a method for preparing PTH1-34, in 13 .. particular the PTH1-34 of SEQ ID NO:1, comprising the steps of 14 (a) recombinantly expressing PTH1-34 in a host cell;
(b) optionally disrupting the host cells to release the recombinantly expressed PTH1-34;
16 (c) purifying the PTH1-34 by a purification method as described in more detail above.
17 .. In step (a) of the above manufacturing method, the PTH1-34, or a homolog thereof as described 18 elsewhere herein, is recombinantly expressed in a host cell, such as a prokaryotic or eukaryotic 19 host cell. In a preferred embodiment of the manufacturing method of the invention, the PTH1-34 is expressed in bacterial host cells, e.g. in bacteria of the genus Escherichia. The manufacturing 21 .. method may use, for example, common bacterial strains of E. coli for expression of high amounts 22 .. of the PTH1-34 polypeptide.
23 After expression of the polypeptide in the cells, it may be necessary to disrupt the cells to release 24 the recombinant polypeptide from the cytoplasm. Alternatively, where the polypeptide is secreted .. by the host strain in the supernatant, e.g. as a result of the use of appropriate signal peptides, it 26 will not be necessary to disrupt the cells. Instead, the recombinant polypeptide can be directly 27 obtained from the culture supernatant. The recombinant polypeptide can be further processed, 28 e.g. by removal of the signal peptide.
CPST Doc: 375089.2 18 Date recue / Date received 2021-11-09 CA 2,908,295 Blakes Ref.: 12770/00001 1 In step (c) of the above manufacturing method, the PTH1-34 is purified by employing the cation 2 exchange chromatography method referred to above.
3 Preferably, the method for preparing PTH1-34 also comprises one or more additional 4 chromatography steps, such as reversed phase chromatography, which are performed prior to or subsequent to step (c).
CPST Doc: 375089.2 19 Date recue / Date received 2021-11-09 CA 2,908,295 Blakes Ref.: 12770/00001 2 Example 1: Purification of PTH1-34 on an analytical column 3 The conditions for separating PTH1-34 from the N-terminally truncated PTH2-34 were analyzed 4 on an analytic Fractogel S03- (S) cation exchange column with a volume of 1 mL (diameter 0.8 mm, height: 20 mm). Eluate from a reversed phase chromatography containing approximately 0.6 6 mg/ mL PTH1-34 was applied to the column at a load of approximately 1 mg/
mL of the resin.
7 Binding of the polypeptides to the column was performed in Eluent A (also used as binding buffer) 8 consisting of 20 mM KH2PO4, pH 6.5. Elution was performed by use of Eluent B consisting of 20 9 mM KH2PO4, 500 mM KCI, pH 6.5. The eluent was applied in a linear gradient of 0-100% eluent in 60 column volumes. The flow rate was 120 cm/h. Fractions of 1 mL were collected. For each 11 of the collected samples, the extinction at 220 nm was measured.
12 The result of the chromatography is depicted in Figure 2. It can be seen that PTH1-34 and PTH2-13 34 co-eluted in the same peak. It was evidently not possible under the conditions applied to 14 separate PTH1-34 and PTH2-34 to a sufficient extent. There was no binding selectivity of either PTH1-34 or PTH2-34 to the Fractogel S03- (S) cation exchange column.
Accordingly, efforts were 16 made to provide an improved method for purifying PTH1-34 and obtaining increased yields.
17 Example 2: Purification of PTH1-34 on a 29 mL column 18 To define robust and reliable scale up conditions, a number of different conditions were tested 19 using a Fractogel S03- (S) cation exchange column with a volume of 29 mL. Improvements were observed when using an increasing pH instead of an increasing salt concentration for elution. To 21 provide for a low ionic strength during elution, the buffers were prepared such that eluent A (which 22 corresponds to the binding buffer) has a high ionic strength and eluent B has a significantly lower 23 ionic strength. Tris buffer was found to be particularly useful for this approach. The following 24 conditions were tested:
CPST Doc: 375089.2 20 Date recue / Date received 2021-11-09 CA 2,908,295 Blakes Ref.: 12770/00001 Gradient Loading Step Yield ro [nigicm2 ro]
Process run Buffer Conditions BICV]
and nig/rnL
Resin) Fluent A 20 mM Tris 20 mM
Bis-Tris pH 65 2;7 Cond; 2.7 matm lEluent B: 20 mriA Tris 20 mM (40% wash; 40- 60/6 76 100% Bis Tris 5 mM KCI pH 10.0 elution) Gond_ 0.8 mS/cm lEluent A 20 rail Tris 20 mM
Bis-Tris pH 6;5 2_5 Gond_ 2.7 mS/cm P226 65 /4_5 73 lEluent B: 20 mM Iris 20 mM (45% wash; 45-100% Bis Tris 5 mM KCI pH 10.0 elution) Cond_ 0.8 mS/cm lEluent A 20 mM Tris 20 mM
Bis-Tris pH 65 Cond_ 2.7 mS/cm (0% wash 75%
65 /4_5 92 lEluent B; 20 rail Tris 20 mM elution) Bis Tris 5 mM KCI pH 10.0 Cond. 0.8 mS/cm lEluent A 20 rail Tris 20 mM
Bis-Tris pH 6;5 'Gond_ 2.7 mS/cm (0% wash; 100%
P243 65 /4;5 88 lEluent B: 20 rail Tris 20 mM elution) Bis Tris 4 mM KCI pH 8_3 Cond. 1.3 mS/cm lEluent A 20 rail Tris 20 mM
Bis-Tris pH 6;5 P250 _ Gond 2.7 mS/cm (0% wash 100%
Fluent B: 20 mM Tris 20 mM elution) Bis Iris 4 mM KCI pH 82 Cond. 1.4 mS/cm lEluent A 20 rail Tris 20 mM
Bis-Tris pH 6;5 Cond. 2.7 mS/cm (0% wash 100%
P251 65 /4;5 75 Fluent B: 20 rail Tris 20 mM elution) Bis Tris 4 mM KCI pH 8.1 Cond. 1.3 mS/cm 2 Eluate from a reversed phase RP chromatography containing approximately 0.6 mg/mL PTH1 -34 3 was diluted by factor 2 to reduce the acetonitrile content. The RP el uate was applied to the column 4 at loads of 4.5-6 mg/mL resin. The column was washed with 3 column volumes binding buffer

5 (eluent A). Elution from the resin was effected either with isocratic elution or gradient elution.
CPST Doc: 375089.2 21 Date recue / Date received 2021-11-09 CA 2,908,295 Blakes Ref.: 12770/00001 1 Fractions of 10 mL were collected. For each of the collected samples, the extinction at 220 nm 2 was measured.
3 As can be seen from the above table, PTH1-34 yield was significantly increased by elution using 4 a pH increase/ionic strength decrease. Figure 3 exemplarily shows the result of process run P250.
It can be seen that the modified elution profile has the effect that PTH2-34 is eluted as a separate

6 peak which allows for its separation from the PTH1-34 product.

7 Example 3: Purification of PTH1-34 on a 26.3 L column

8 The method of the invention was tested at preparative scale using a Fractogel S03- (S) cation

9 exchange column having a volume of 26.3 L (column diameter: 450 mm, column height 166 mm).
The PTH1-34 preparation used in this chromatographic step was recombinantly expressed PTH1-11 34 that had previously been subjected to reversed-phase (RP) chromatography. The PTH1-34 12 preparation obtained from the RP chromatography step contained approximately 1.2 % PTH2-34.
13 To reduce the acetonitrile concentration, the main fraction obtained from RP chromatography was 14 diluted 1:2 with Eluent A (20 mM Tris, 20 mM potassium dihydrogen phosphate, pH 6.5, conductivity about 4.1 mS/cm) and loaded onto the column. The column was washed with an 16 initial gradient ranging from 0-35% Eluent B (20 mM Tris, 16 mM
dipotassium hydrogen 17 phosphate, 4 mM potassium dihydrogen phosphate, pH 8.9, conductivity about 3.5 mS/cm) in one 18 column volume and, in a subsequent step, at 35% Eluent B for 10 column volumes. The flow rate 19 was adjusted to 100 cm/h. The column was loaded with 3.5 g/L of the polypeptide. Elution was performed with a gradient from 35-100% Eluent B in 22 column volumes.
21 The PTH1-34 product was found to elute in the slowly ascending peak shown in Figure 4, while 22 PTH2-34 eluted in the steeply descending peak area (i.e., the area shown in black in Figure 4).
23 Fractions of the peak were collected for subsequent analysis by ultra high performance liquid 24 chromatography (RP-uH PLC). A first fraction ("fraction 1" comprising 100.7 L) was collected until start of the main fraction. Following the further processed main fraction, further fractions were 26 collected starting from peak maximum. These fractions ("fractions 2-6"), which comprised different 27 volumes (fraction 2: 1.4 L, fraction 3: 3.7 L, fraction 4: 8.1 L, fraction 5: 3.6 L and fraction 6: 9.7 28 L), were collected from the descending peak area.
CPST Doc: 375089.2 22 Date recue / Date received 2021-11-09 CA 2,908,295 Blakes Ref.: 12770/00001 1 The results of the RP-uHPLC are shown in Figure 5. It can be seen there that the initial PTH1-34 2 preparation obtained from RP chromatography contained about 1.2% PTH2-34, while fraction 1 3 collected after cation exchange chromatography contained only about 0.1%
of the truncated 4 polypeptide. Almost the complete PTH2-34 eluted in fractions 3-6, i.e. in the descending peak area. A total of 62.9 g PTH1-34 was loaded to the cation exchange column. The chromatographic 6 process yielded 54.0 g PTH1-34, which represents a recovery of 86%.
7 From these results, it can therefore be concluded that the method of the present invention is highly 8 effective for separating PTH1-34 from PTH2-34 at preparative scale without any significant loss 9 of PTH1-34. The method is therefore suitable for being used in the large-scale production of PTH1-34.
CPST Doc: 375089.2 23 Date recue / Date received 2021-11-09

Claims (20)

1. A method for purifying teriparatide (PTH1-34) by ion exchange chromatography, comprising the steps of (a) contacting a fluid which comprises PTH1-34 with a cation exchange material under conditions that allow for the reversible binding of PTH1-34 to said cation exchange material; and (b) eluting PTH1-34 by increasing the pH and decreasing the ionic strength.

2. The method of claim 1, further comprising the step of washing the cation exchange material to remove unbound material.

3. The method of claim 1 or 2, wherein said PTH1-34 is recombinantly produced PTH1-34.

4. The method of any one of claims 1-3, wherein said PTH1-34 comprises or consists of the amino acid sequence of SEQ ID NO:1 or an amino acid sequence which differs from SEQ
ID NO:1 in the substitution of 1, 2, or 3 amino acids.

5. The method of any one of claims 1-4, wherein said cation exchange material comprises sulfoethyl, sulfopropyl, sulfobutyl or sulfoisobutyl exchanger groups.

6. The method of any one of claims 1-5, wherein said cation exchange material comprises cross-linked polystyroldivinylbenzol, polymethacrylate, polyvinyl ether, cellulose, or d extra n.

7. The method of any one of claims 1-6, wherein the eluate obtained in step (b) contains less than 0.5% (w/w) of the fragment PTH2-34.

8. The method of any one of claims 1-7, wherein binding to said cation exchange material is effected at a pH of between 6.3 and 6.7.

9. The method of any one of claims 1-8, wherein binding to said cation exchange material is effected in the presence of a binding buffer comprising 10-80 mM Tris.

10. The method of claim 9 wherein the binding buffer comprises 20mM Tris.

11. The method of claim 9, wherein elution from said cation exchange material is effected in the presence of an eluent comprising 10-80 mM Tris.

12. The method of claim 11 wherein the eluent comprises 20 mM Tris.

13. The method of any one of claims 1-12, wherein the increase in pH and/or the decrease in ionic strength is effected by using a linear, concave, or convex gradient.

14. The method of any one of claims 1-13, wherein the increase in pH and/or the decrease in ionic strength is effected by using a step elution.

15. The method of any one of claims 1-14, wherein binding to said cation exchange material is effected in the presence of a binding buffer that has an ionic strength in the range of 1.8 to 3.0 m&cm.

16. The method of any one of claims 1-15, wherein elution from said cation exchange material is effected in the presence of an eluent that has an ionic strength in the range of 0.2 to 1.2 mS/cm.

17. A method for preparing teriparatide (PTH1-34), comprising the steps of (a) recombinantly expressing PTH1-34 in a host cell; and (b) purifying the PTH1-34 by a method of any one of claims 1-13.

18. The method of claim 17, further comprising the step of disrupting the host cells to release the recombinantly expressed PTH1-34.

19. The method of claim 18, wherein said host cell is a prokaryotic host cell.

20. The method of any one of claims 18-19, wherein the method comprises one or more additional chromatography steps prior to or subsequent to step (b).