EP2220108A2

EP2220108A2 - Protein purification and endotoxin removal

Info

Publication number: EP2220108A2
Application number: EP08849938A
Authority: EP
Inventors: Rikke Christina Nielsen
Original assignee: Novo Nordisk AS
Current assignee: Novo Nordisk AS
Priority date: 2007-11-15
Filing date: 2008-11-14
Publication date: 2010-08-25
Also published as: WO2009063069A3; WO2009063069A2

Abstract

This invention relates to a process for purifying peptides, in particular but not exclusively, to a process for removing endotoxins from a peptide solution, to a kit comprising reagents for said process and to the purified peptide obtained by said process.

Description

ENDOTOXIN REMOVAL

FIELD OF THE INVENTION

This invention relates to a process for purifying peptides, for instance to a process for removing endotoxins from a peptide solution resulting from bacterial expression, to a kit comprising reagents for said process and to the purified peptide obtained by said process.

BACKGROUND OF THE INVENTION

Recombinant expression of polypeptides, or simply peptides, is a technology of widespread and increasing use and the art is still being developed intensely.

After expression, the recombinant peptide often needs to be purified, particularly if the peptide is to be used for instance in a pharmaceutical formulation.

Purification often involves several chromatography and filtration steps, some are described in for instance EP1586580A2, WO0215927A1 and US20030229212A1.

Endotoxins are complex lipopolysaccharide molecules associated with the outer membrane of gram-negative bacteria, e.g. E. coli. They are frequent contaminants in proteins or peptides prepared from such bacteria.

Endotoxins are usually toxic to most mammals. Exposure to concentrations of even a few nanograms of endotoxins per kilogram body weight may cause a range of reactions, including fever, hypotension and shock. As a result, it is essential to remove endotoxins from any system or substance that will come in to contact with patients during medical therapies. Removal of endotoxins during purification of polypeptides and peptides is a difficult process. A number of purification protocols, such as affinity chromatography, are unable to remove endotoxins satisfactorily without excessive loss of the protein or peptide. In addition, purification processes that utilise a number of phases require extensive manipulation of the sample between each stage, which reduces the speed and efficiency of these processes. US 2004/0198957 (Way et al) discloses one method for removing endotoxins from protein solutions which comprises the use of hydrophobic charge induction chromatography sorbents. Hou KC et al., Biochim Biophys Acta. 1073(1), 149-54 (1991) describes the effect of hydrophobic interaction on endotoxin adsorption by polymeric affinity matrix. One resin used for chromatography is the multimodal, or mixed-mode, ion-exchange resin. Purification of polypeptides using this resin is described in for instance Neidhardt EA et al., J Chromatogr. 590(2), 255-61 (1992). There is a need for a new process that can efficiently remove endotoxins from proteins or peptides prepared from bacteria, particularly without extensive manipulation of the sample between each step.

SUMMARY OF THE INVENTION In one aspect, the present invention provides a method for purifying a peptide having a pi below 6.5, which peptide is recombinantly expressed in a host cell, which purification method comprises the steps of: (i) recovering the peptide in a biologically active form from inclusion bodies produced in the recombinant expression, (ii) if necessary, adjusting the conductivity of the solution comprising the recovered recombinant peptide to between 0 and 4 mS/cm,

(a) subjecting the solution from step ii) to a first chromatographic separation with an anionic exchanger and collecting an eluate comprising the recombinant peptide; and

(b) performing a second chromatographic separation on the eluate from step (a) with a multimodal exchanger and collecting an eluate comprising the recombinant peptide.

In one aspect, the present invention provides a method for preparing a pharmaceutical formulation of a peptide having a pi below 6.5, which peptide is recombinantly expressed in a host cell, which method comprises the steps of: (i) recovering the peptide in a biologically active form from inclusion bodies produced in the recombinant expression,

(ii) if necessary, adjusting the conductivity of the solution comprising the recovered recombinant peptide to between 0 and 4 mS/cm, (a) subjecting the solution from step ii) to a first chromatographic separation with an anionic exchanger and collecting an eluate comprising the recombinant peptide; (b) performing a second chromatographic separation on the eluate from step (a) with a multimodal exchanger and collecting an eluate comprising the recombinant peptide.

In one aspect, the present invention provides a polypeptide obtained by a process described herein.

In one aspect, the present invention provides a recombinant peptide purification kit, which comprises an anionic exchange resin, a multimodal exchange resin and instructions to use said kit in accordance with a method according to the present invention.

In one aspect, the present invention provides a pharmaceutical formulation prepared in accordance with a method according to the present invention. DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides a method for purifying a peptide having a pi below 6.5, which peptide is recombinantly expressed in a host cell, wherein the recombinant peptide is produced in inclusion bodies, which purification method comprises the steps of:

(i) recovering the peptide in a biologically active form from inclusion bodies produced in the recombinant expression, (ii) if necessary, adjusting the conductivity of the solution comprising the recovered recombinant peptide to between 0 and 4 mS/cm, (a) subjecting the solution from step ii) to a first chromatographic separation with an anionic exchanger and collecting an eluate comprising the recombinant peptide; and (b) performing a second chromatographic separation on the eluate from step (a) with a multimodal exchanger and collecting an eluate comprising the recombinant peptide.

In one aspect, the present invention provides a method for preparing a pharmaceutical formulation of a peptide having a pi below 6.5, which peptide is recombinantly expressed in a host cell, wherein the recombinant peptide is produced in inclusion bodies, which method comprises the steps of:

(i) recovering the peptide in a biologically active form from inclusion bodies produced in the recombinant expression, (ii) if necessary, adjusting the conductivity of the solution comprising the recovered recombinant peptide to between 0 and 4 mS/cm,

(a) subjecting the solution from step ii) to a first chromatographic separation with an anionic exchanger and collecting an eluate comprising the recombinant peptide;

In one embodiment, the pharmaceutical formulation is a lyophilized pharmaceutical formulation, which is made by lyophilizing the eluate from step (b)

In one embodiment, the eluate from step (b) is used as the pharmaceutical formulation. It is known that peptides prepared by recombinant protein production processes, particularly in prokayotic cells, often accumulate as insoluble aggregates known as inclusion bodies. In one embodiment, step (i) comprises the steps of (ia) solubilising the insoluble aggregate; and (ib) refolding the recombinant peptide to a biologically active conformation. The solubilising step (ia) is achieved using a solubilising agent, which disrupts the peptide's conformation and "unfolds" the peptide to a degree that depends on the strength of the solubilising agent. The greater the extent of the unfolding, the less degree of biological activity the protein likely displays. Thus, the resulting solubilized peptide solution obtained following step (ia) comprises the peptide in some stage of unfolding.

In one embodiment, the solubilisation agent comprises a guanidinium salt, urea, a detergent, or other organic solvent. In one embodiment, the solubilisation agent comprises urea.

Typically, the refolding step (ib) involves diluting out the solubilising agent with a large volume of diluent, generally a refolding buffer. When the concentration of solubilizing agent is reduced to a particular dilution level by the refolding buffer, the peptide spontaneously refolds into a soluble, biologically active conformation.

In one embodiment, the refolding buffer comprises arginine salts and/or Tris.

In one embodiment, the process additionally comprises a washing step before step (ia). The washing step helps to remove some undesirable materials from the solution. In one embodiment, the washing step is achieved using a suitable washing regime (e.g. rough mixing) in a suitable buffer, such as Na₂HPO₄. In one embodiment, the washing step is accompanied by centrifugation. The washing step may be repeated one or more times.

In one embodiment, the cell is a prokaryotic cell. In one embodiment, said prokaryotic cell is Escherichia coli.

In one aspect, the present invention provides a method for purifying a peptide having a pi below 6.5, which peptide is recombinantly expressed in a host cell, wherein the recombinant peptide is produced in soluble form, which purification method comprises the steps of: (i) obtaining a solution comprising the peptide from the recombinant expression,

(ii) if necessary, adjusting the conductivity of said solution to between 0 and 4 mS/cm,

In one aspect, the present invention provides a method for preparing a pharmaceutical formulation of a peptide having a pi below 6.5, which peptide is recombinantly expressed in a host cell, wherein the recombinant peptide is produced in soluble form, which method comprises the steps of: (i) obtaining a solution comprising the peptide from the recombinant expression,

(a) subjecting the solution from step ii) to a first chromatographic separation with an anionic exchanger and collecting an eluate comprising the recombinant peptide; (b) performing a second chromatographic separation on the eluate from step (a) with a multimodal exchanger and collecting an eluate comprising the recombinant peptide.

In one embodiment, the eluate from step (b) is used as the pharmaceutical formulation, optionally after having been subjected to for instance a sterile filtration.

In one embodiment, said cell is a eukaryotic cell. In one embodiment, said cell is a yeast cell. In one embodiment, said cell is Saccharomyces cerevisiae. In one embodiment, said cell is a human cell. In one embodiment, said cell is a CHO cell.

For use of human cells, the conductivity adjustment in step (ii) may additionally be aimed at adjusting the salt levels of the solution to about 10-20 mM depending on the pi of the peptide and the pH of the buffer.

The term "peptide" as used herein means a compound composed of at least five constituent amino acids connected by peptide bonds. The term "peptide" may be used interchangably with the term "polypeptide". The constituent amino acids may be from the group of the amino acids encoded by the genetic code and they may be natural amino acids which are not encoded by the genetic code, as well as synthetic amino acids. Natural amino acids which are not encoded by the genetic code are e.g. hydroxyproline, y- carboxyglutamate, ornithine, phosphoserine, D-alanine and D-glutamine. Synthetic amino acids comprise amino acids manufactured by chemical synthesis, i.e. D-isomers of the amino acids encoded by the genetic code such as D-alanine and D-leucine, Aib (a-aminoisobutyric acid), Abu (a-aminobutyric acid), Tie (tert-butylglycine), β-alanine, 3-aminomethyl benzoic acid and anthranilic acid.

The term "peptide" encompasses the terms polypeptides and proteins, which may consists of two or more polypeptides held together by covalent interactions, such as for instance cysteine bridges, or non-covalent interactions. The term peptide includes any suitable peptide and may be used synonymously with the terms polypeptide and protein, unless otherwise stated or contradicted by context; provided that the reader recognize that each type of respective amino acid polymer-containing molecule may be associated with significant differences and thereby form individual embodiments of the present invention (for example, a peptide such as an antibody, which is composed of multiple polypeptide chains, is significantly different from, for example, a single chain antibody, a peptide immunoadhesin, or single chain immunogenic peptide). Therefore, the term peptide herein should generally be understood as referring to any suitable peptide of any suitable size and composition (with respect to the number of amino acids and number of associated chains in a protein molecule). Moreover, peptides described herein may comprise non-naturally occurring and/or non-L amino acid residues, unless otherwise stated or contradicted by context.

The term "peptide", unless otherwise stated or contradicted by context, (and if discussed as individual embodiments of the term(s) polypeptide and/or protein) also encompasses derivatized peptide molecules. Briefly, in the context of the present invention, a derivative is a peptide in which one or more of the amino acid residues of the peptide have been chemically modified (for instance by alkylation, acylation, ester formation, or amide formation) or associated with one or more non-amino acid organic and/or inorganic atomic or molecular substituents (for instance a polyethylene glycol (PEG) group, a lipophilic substituent (which optionally may be linked to the amino acid sequence of the peptide by a spacer residue or group such as β-alanine, γ-aminobutyric acid (GABA), L/D-glutamic acid, succinic acid, and the like), a fluorophore, biotin, a radionuclide, etc.) and may also or alternatively comprise non-essential, non-naturally occurring, and/or non-L amino acid residues, unless otherwise stated or contradicted by context (however, it should again be recognized that such derivatives may, in and of themselves, be considered independent features of the present invention and inclusion of such molecules within the meaning of peptide is done for the sake of convenience in describing the present invention rather than to imply any sort of equivalence between naked peptides and such derivatives). Non-limiting examples of such amino acid residues include for instance 2-aminoadipic acid, 3-amino- adipic acid, β-alanine, β-aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid,

6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-di- aminopropionic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, allohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, N-methylglycine, N-methyl- isoleucine, 6-N-methyllysine, N-methylvaline, norvaline, norleucine, ornithine, and statine halogenated amino acids. It is to be understood that this derivatization is not a derivatization of the present invention, but rather a derivatization already present on the growth hormone compound before the conjugation of the present invention, or a derivatization performed after the conjugation of the present invention. The term "(parent peptide) analogue" or "analogue of a (parent peptide)" - wherein (parent peptide) indicates a given specific peptide (or peptide class) - as used herein refers to a peptide, which has a sequence, which is similar or has a certain percent identity to the (parent peptide), wherein the analogue has an amino acid sequence, which is a mutated version of the amino acid sequence of the (parent peptide). Such analogues are typically created by site-directed mutagenesis of a nucleic acid encoding the (parent peptide), but can also be generated using other techniques for nucleic acid manipulation or genetic engineering. In one embodiment, a (parent peptide) analogue has retained one or more capabilities of the (parent peptide), such as for instance the capability of binding to one or more of the relevant (parent peptide) receptors.

The method is particularly useful for purification of peptides, that do not undergo irreversible conformation changes in the pH ranges used in the current method, which generally is a pH from about 6.5 to about 8.0.

The method is particularly useful for the purification of recombinant therapeutic peptides, as the eluation in step (b) can be performed using a pharmaceutically acceptable buffer, and that the eluate from step (b) in that case can be used directly as the pharmaceutical formulation or can be lyophilized directly for the preparation of a lyophilized pharmaceutical formulation.

The term "pharmaceutically acceptable" as used herein means suited for normal pharmaceutical applications, i.e. giving rise to no unacceptable adverse events in patients etc.

In one embodiment, the peptide is a cytokine compound. The term "cytokine" as used herein has the meaning commonly known in the art, generally speaking small proteins or biological factors (for instance in the range of 5-30 kD) that are released by cells and have specific effects on cell-cell interaction, communication and behaviour of other cells. Any of several regulatory proteins, such as the interleukins, chemokines, and lymphokines, that are released by cells of the immune system and act as intercellular mediators in the generation of an immune response are included in the defition of the term "cytokine". The term "cytokine compound" is intended to mean compounds, which are cytokines or analogues of cytokines, or fragments of cytokines or analogues of cytokine, or derivatives of either of these. The term "cytokine analogue" or "analogue of cytokines" as used herein refers to a peptide, which has a sequence, which is similar or has a certain percent identity to a given cytokine, wherein the analogue has an amino acid sequence, which is a mutated version of the amino acid sequence of the cytokine. Such analogues are typically created by site-directed mutagenesis of a nucleic acid encoding the cytokine, but can also be generated using other techniques for nucleic acid manipulation or genetic engineering. In one embodiment, a cytokine compound has retained one or more capabilities of the parent cytokine, such as for instance the capability of binding to one or more of the relevant cytokine receptors. In one embodiment, the peptide is a hormone compound. The term "hormone" as used herein has the meaning commonly known in the art, generally speaking extracellular signalling peptides nessecary for cell-to-cell communication throughout the body. The term "hormone compound" is intended to mean compounds, which are hormones or analogues of hormones, or fragments of hormones or hormone analogues, or derivatives of either of these. The term "hormone analogue" or "analogue of hormones" as used herein refers to a peptide, which has a sequence, which is similar or has a certain percent identity to a given hormone, wherein the analogue has an amino acid sequence, which is a mutated version of the amino acid sequence of the hormone. Such analogues are typically created by site- directed mutagenesis of a nucleic acid encoding the hormone, but can also be generated using other techniques for nucleic acid manipulation or genetic engineering. In one embodiment, a hormone compound has retained one or more capabilities of the parent hormone, such as for instance the capability of binding to one or more of the relevant hormone receptors.

In one embodiment, the peptide is a chemokine compound. The term "chemokine" as used herein has the meaning commonly known in the art, generally speaking a family of small cytokines, or proteins secreted by cells. Proteins are classified as chemokines according to shared structural characteristics such as small size (they are all approximately 8-10 kilodaltons in size), and the presence of four cysteine residues in conserved locations that are key to forming their 3-dimensional shape. The term "chemokine compound" is intended to mean compounds, which are chemokines or analogues of chemokines, or fragments of chemokines or chemokine analogues, or derivatives of either of these. The term "chemokine analogue" or "analogue of chemokines" as used herein refers to a peptide, which has a sequence, which is similar or has a certain percent identity to a given chemokine, wherein the analogue has an amino acid sequence, which is a mutated version of the amino acid sequence of the chemokine. Such analogues are typically created by site-directed mutagenesis of a nucleic acid encoding the chemokine, but can also be generated using other techniques for nucleic acid manipulation or genetic engineering. In one embodiment, a chemokine compound has retained one or more capabilities of the parent chemokine, such as for instance the capability of binding to one or more of the relevant chemokine receptors. In one embodiment, the peptide is a interleukine compound. The term "interleukin" as used herein has the meaning commonly known in the art, generally speaking a group of cytokines (secreted signaling molecules) that were first seen to be expressed by white blood cells as a means of communication. It has since been found that interleukins are produced by a wide variety of bodily cells. Interleukins are named as "Interleukin" followed by a number. The term "interleukin compound" is intended to mean compounds, which are interleukins or analogues of interleukins, or fragments of interleukins or interleukin analogues, or derivatives of either of these. The term "interleukin analogue" or "analogue of interleukins" as used herein refers to a peptide, which has a sequence, which is similar or has a certain percent identity to a given interleukin, wherein the analogue has an amino acid sequence, which is a mutated version of the amino acid sequence of the interleukin. Such analogues are typically created by site-directed mutagenesis of a nucleic acid encoding the interleukin, but can also be generated using other techniques for nucleic acid manipulation or genetic engineering. In one embodiment, an interleukin compound has retained one or more capabilities of the parent interleukin, such as for instance the capability of binding to one or more of the relevant interleukin receptors.

In one embodiment, the peptide is an immunoglobulin compound. The term "immunoglobulin" refers to a molecule belonging to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four inter-connected by disulfide bonds. The structure of immunoglobulins has been well characterized. See for instance Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N. Y. (1989)). Briefly, each heavy chain typically is comprised of a heavy chain variable region (abbreviated herein as V_H) and a heavy chain constant region. The term "immunoglobulin compound" is intended to mean compounds, which are immunoglobulins or analogues of immunoglobulins, or fragments of immunoglobulins or immunoglobulin analogues, or derivatives of either of these. The term "immunoglobulin analogue" or "analogue of immunoglobulins" as used herein refers to a peptide, which has a sequence, which is similar or has a certain percent identity to a given immunoglobulin, wherein the analogue has an amino acid sequence, which is a mutated version of the amino acid sequence of the immunoglobulin, and wherein the analogue has maintain the overall immunoglobulin structure. Such analogues are typically created by site-directed mutagenesis of a nucleic acid encoding the immunoglobulin, but can also be generated using other techniques for nucleic acid manipulation or genetic engineering. In one embodiment, an immunoglobulin compound has retained one or more capabilities of the parent immunoglobulin, such as for instance the capability of binding to one or more of the relevant immunoglobulin receptors.

In one embodiment, the peptide is an antibody compound. The term "antibody" as used herein has the meaning commonly known in the art, generally speaking designating an immunoglobulin molecule or a fragment of an immunoglobulin molecule, which has the ability to specifically bind to a given antigen under typical physiological conditions for significant periods of time. The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The term "antibody compound" is intended to mean compounds, which are antibodies or analogues of antibodies, or fragments of antibodies or antibody analogues, or derivatives of either of these. The term "antibody analogue" or "analogue of antibodies" as used herein refers to a peptide, which has a sequence, which is similar or has a certain percent identity to a given antibody, wherein the analogue has an amino acid sequence, which is a mutated version of the amino acid sequence of the antibody. Such analogues are typically created by site- directed mutagenesis of a nucleic acid encoding the antibody, but can also be generated using other techniques for nucleic acid manipulation or genetic engineering. In one embodiment, an antibody analogue has retained one or more capabilities of the parent antibody, such as binding to the antigen.

In one embodiment, the peptide is a PRL-like (prolactin-like) cytokine having a pi below 6.5. pi may be calculated as described in Gasteiger E. et al., The Proteomics

Protocols Handbook, Humana Press (2005) pp. 571-607. A PRL-like cytokine is a naturally occurring polypeptide ligand which are structurally similar to prolactin having four amphiphatic alpha helices, wherein said natural polypeptide ligand binds to two receptor polypeptides located on the surface of mammalian cells forming a 1 :2 complex between the ligand and the receptor polypeptides. Binding of the polypeptide ligand to the receptor polypeptides is through a first polypeptide binding site and a second polypeptide binding site, both binding sites located on the polypeptide ligand. The receptor polypeptides may be same or different . Examples of PRL-like cytokines are prolactin, growth hormone, placental lactogen, interleukin 6, 31 , and 32. In one embodiment, the peptide is a prolactin compound, a growth hormone compound or a placental lactogen compound.

In one embodiment, the peptide is human prolactin. The sequence of human prolactin is given in SEQ ID No. 1. In one embodiment, the peptide is a human prolactin analogue. In one embodiment, the peptide is a human prolactin analogue, which has the capability of binding to the prolactin receptor. In one embodiment, such prolactin analogue has an amino acid sequence having at least 80% identity to SEQ ID No. 1. In one embodiment, the prolactin analogue has an amino acid sequence having at least 85%, such at least 90%, for instance at least 95%, such as for instance at least 99% identity to SEQ ID No. 1.

The term "identity" as known in the art, refers to a relationship between the sequences of two or more peptides, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between peptides, as determined by the number of matches between strings of two or more amino acid residues. "Identity" measures the percentage of identical matches between the two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., "algorithms"). Identity of related peptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1 , Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991 ; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988). Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are described in publicly available computer programs. Preferred computer program methods to determine identity between two sequences include the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res. 12, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. MoI. Biol. 215, 403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well known Smith Waterman algorithm may also be used to determine identity.

In one embodiment, the prolactin analogue has an amino acid sequence, which is at least 80% similar to SEQ ID No. 1. In one embodiment, the prolactin analogue has an amino acid sequence, which is at least 85%, such at least 90%, for instance at least 95%, such as for instance at least 99% similar to SEQ ID No. 1.

The term "similarity" is a concept related to identity, but in contrast to "identity", refers to a sequence relationship that includes both identical matches and conservative substitution matches. If two peptide sequences have, for example, (fraction (10/20)) identical amino acids, and the remainder are all non-conservative substitutions, then the percent identity and similarity would both be 50%. If, in the same example, there are 5 more positions where there are conservative substitutions, then the percent identity remains 50%, but the percent similarity would be 75% (fraction (15/20)). Therefore, in cases where there are conservative substitutions, the degree of similarity between two peptides will be higher than the percent identity between those two peptides.

Conservative modifications of a peptide will produce peptides having functional and chemical characteristics similar to those of the parent peptide. In contrast, substantial modifications in the functional and/or chemical characteristics of peptides as compared to the parent peptide may be accomplished by selecting substitutions in the amino acid sequence that differ significantly in their effect on maintaining (a) the structure of the molecular backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. For example, a "conservative amino acid substitution" may involve a substitution of a native amino acid residue with a nonnative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Furthermore, any native residue in the peptide may also be substituted with alanine, as has been previously described for "alanine scanning mutagenesis" (see, for example, MacLennan et al., Acta Physiol. Scand. Suppl. 643, 55-67 (1998); Sasaki et al., Adv. Biophys. 35, 1-24 (1998), which discuss alanine scanning mutagenesis).

Desired amino acid substitutions (whether conservative or non-conservative) may be determined by those skilled in the art at the time such substitutions are desired. For example, amino acid substitutions can be used to identify important residues of the peptides according to the invention, or to increase or decrease the affinity of the peptides described herein for the receptor in addition to the already described mutations.

Naturally occurring residues may be divided into classes based on common side chain properties:

1) hydrophobic: norleucine, Met, Ala, VaI, Leu, lie; 2) neutral hydrophilic: Cys, Ser, Thr, Asn, GIn;

3) acidic: Asp, GIu;

4) basic: His, Lys, Arg;

5) residues that influence chain orientation: GIy, Pro; and

6) aromatic: Trp, Tyr, Phe. In making such changes, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (- 0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (- 3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art. Kyte et al., J. MoI. Biol., 157, 105-131 (1982). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within .±2 is preferred, those that are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. The greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine ('3.O); aspartate (+3.0±1 ); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (- 0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those that are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. The term "prolactin compound" as used herein is intended to mean a compound, which is human prolactin or a human prolactin analogue.

The term "growth hormone compound" as used herein is intended to mean a compound, which is human growth hormone or a human growth hormone analogue.

The term "placental lactogen compound" as used herein is intended to mean a compound, which is placental lactogen or a human placental lactogen analogue.

The term "anionic exchanger" as used herein refers to a specific form of ion exchange chromatography which involves the use of an anion exchanger that exchanges negatively charged ions (anions). The term "multimodal exchanger" or "mixed mode exchanger" as used herein refers to a specific form of ion exchange chromatography which involves the use of an exchanger capable of both ionic and non-ionic interactions. For example, the multimodal ion exchanger may possess the properties of a cation exchanger and possess the ability to interact via hydrogen bonding and/or hydrophobic interaction. Furthermore, the multimodal ion exchanger may possess the properties of an anion exchanger and possess the ability to interact via hydrogen bonding and/or hydrophobic interaction.

Multimodal anion-exchangers have been disclosed for instance in WO9729825 providing interactions based on charges and hydrogen-bonding involving oxygen and amino nitrogen on 2-3 carbons' distance from positively charged amine nitrogen. Multimodal cation- exchangers have been suggested in WO9965607. Furthermore, WO9729825 and WO 9965607 describe anion and cation-exchange ligands. Examples of multimodal exchangers, which may be used in the methods and kits of the present invention, are also described in for instance US patent applications US10/489,468 (issued as US7067059) and US10/547,567 (issued as US7320754).

It is believed that the anionic exchanger interacts with the charged sugar of the endotoxins and removes them from the solution. The multimodal exchanger interacts with the hydrophobic part of the endotoxins and removes them from the solution. There is thus a synergistic arrangement provided by the combination of the two forms of chromatographic separation. Advantageously, the removal process removes endotoxins through both charge and hydrophobic interactions. Thus the process is extremely efficient in removing endotoxins, and is able to reduce the endotoxin content to a level where the peptide solution can be used directly for animal experiments (e.g. below 5.0 EU/mg). Furthermore, the conditions in the anionic exchanger and the multimodal exchanger are well aligned, which means that the number of steps between contacting the anionic exchanger and contacting the multimodal exchanger can be reduced. Suitable anion exchangers are anion exchangers, which binds to the negative charged sugar on endotoxins/lipopolysaccarides.

In one embodiment, the anionic exchanger used in step (a) is a strong anionic exchanger. A strong ion exchanger has functional groups, which show no loss or gain of charge with varying pH, while a weak functional group's ion exchange capacity can vary with pH. In one embodiment, the anionic exchanger used in step (a) binds to negative charged sugar residues on endotoxins or lipopolysaccarides.

Examples of suitable anionic exchangers include: Q-Sepharose Fast Flow™ Q-Sepharose High Performance™

Source 30Q™

Toyopearl SuperQ TM

UNOsphere Q™ CaptoQ™

In one embodiment, the anionic exchanger comprises a quaternary amine resin. In one embodiment, the anionic exchanger comprises Q SEPHAROSE FAST FLOW™ (Amersham Biosciences).

The term "buffer" as used herein refers to a chemical compound that reduces the tendency of pH of a solution such as chromatographic solutions to change over time as would otherwise occur. Buffers include the following non-limiting examples: sodium acetate, sodium carbonate, sodium citrate, glycylglycine, glycine, histidine, lysine, sodium phosphate, borate, Trishydroxymethyl-aminomethane, ethanolamine and mixtures thereof.

Examples of buffers which may be used in step (a) include triethanolamine, Tris (tris(hydroxymethyl)methylamine), TAPS (3-{[tris(hydroxymethyl)methyl]amino}propane- sulfonic acid), Bicine (N,N-bis(2-hydroxyethyl)glycine), Tricine (N-tris(hydroxymethyl)methyl- glycine), HEPES (4-2-hydroxyethyl-1 -piperazineethanesulfonic acid), TES (2-{[tris(hydroxy- methyl)methyl]amino}ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), Cacodylate (dimethylarsinic acid), MES (2-(N-morpholino)ethanesulfonic acid) or acetate.

In one embodiment, the buffer used in step (a) comprises triethanolamine buffer.

In one embodiment, the buffer used in step (a) has a pH which is +/-1 of the pi of the peptide to be purified. In one embodiment, chromatographic separation may be achieved in step (a) by using a salt gradient, that is by increasing the ionic strength of the buffer. In one embodiment, the buffer used in step (a) comprises any suitable salt, e.g. ammonium bicarbonate, KCI or NaCI. In one embodiment, the buffer used in step (a) comprises from 0 mM to 50OmM NaCI (e.g. 0 mM to 360 mM).

In one embodiment, the multimodal exchanger comprises a negatively charged multimodal exchanger. In one embodiment, the multimodal exchanger comprises a negatively charged multimodal exchanger. In one embodiment, the multimodal exchanger is CAPTO MMC™ (GE healthcare).

In one embodiment, the buffer used in step (b) comprises any buffer suitable for protein formulation or freeze-drying, e.g. phosphate buffered saline, histidine, or histidine and a sugar. In one embodiment, the buffer used in step (b) comprises an ammonium buffer (e.g. 50 mM ammonium acetate buffered to pH 6.5).

In one embodiment, chromatographic separation may be achieved in step (b) by increasing the pH of the buffer (e.g. replacing a first buffer at a given pH with a second buffer at a higher pH). In one embodiment, the second buffer used in step (b) is ammonium bicarbonate (e.g. 5OmM ammonium bicarbonate buffered to pH 8.5).

The chromatographic separations in step (a) and step (b) may be performed by eulation with a single buffer, a series af buffers or by a gradient. This is within the skills of a person skilled in the art while optimizing the conditions for a given protein. In one embodiment, step (a) comprises a strong anionic exchanger and step (b) comprises a negatively charged multimodal exchanger.

In one embodiment, step (a) comprises a Q SEPHAROSE FAST FLOW™ anionic exchanger and step (b) comprises a negatively charged multimodal exchanger.

In one embodiment, step (a) comprises a Q SEPHAROSE FAST FLOW™ anionic exchanger and step (b) comprises a CAPTO MMC™ multimodal exchanger.

In one embodiment, the pH of the peptide solution is adjusted to a pH above the isoelectric point of the peptide prior to step (a). Such an embodiment, results in a negatively charged protein which adsorbs more efficiently to the anion exchange resin. In one embodiment, the pH may be adjusted to between about 6 and about 10 prior to step (a). In one embodiment, the pH may be adjusted to between about 7.5 and about 9 (e.g. 8.5) prior to step (a).

The term "isoelectric point" as used herein means the pH value where the overall net charge of a macromolecule such as a peptide is zero. In peptides there may be many charged groups, and at the isoelectric point the sum of all these charges is zero. At a pH above the isoelectric point the overall net charge of the peptide will be negative, whereas at pH values below the isoelectric point the overall net charge of the peptide will be positive.

In one embodiment, the pH of the peptide solution is adjusted to a pH below the isoelectric point of the peptide solution following step (a) and prior to step (b). In one embodiment where the peptide becomes unstable in solutions with a pH below the isoelectric point, the pH of the peptide solution is adjusted to a pH not more than 1.0 (e.g. about 0.3) above the isoelectric point of the peptide solution following step (a) and prior to step (b).

In one embodiment, the pH may be adjusted to between about 5 and about 7 following step (a) and prior to step (b). In one embodiment, the pH may be adjusted to between about 6 and about 7 (e.g. 6.5). It will be appreciated that solutions, compounds and methods for adjusting the pH in the present invention are well known to the skilled person. In one embodiment, the pH of the buffers used in step (b) may be adjusted using any suitable acid, e.g. sodium citrate.

In one embodiment, the conductivity of the peptide solution is adjusted to between 0 to 8 mS/cm, such as between 0 to 6 mS/cm, following step (a) and prior to step (b). This enables the protein to bind to the column.

In one embodiment, a method according to the present invention additionally comprises a freeze-drying step following purification of the peptide solution in step (b). It will be appreciated that freeze drying may be conducted in accordance with known procedures which will be readily available to the skilled person.

In one aspect, the present invention provides a method for purifying a prolactin compound having a pi below 6.5, which prolactin compound is recombinantly expressed in a host cell, wherein the prolactin compound is produced in inclusion bodies, which purification method comprises the steps of: (i) recovering the prolactin compound in a biologically active form from inclusion bodies produced in the recombinant expression, (ii) if necessary, adjusting the conductivity of the solution comprising the recovered prolactin compound to between 0 and 4 mS/cm,

(a) subjecting the solution from step ii) to a first chromatographic separation with an anionic exchanger and collecting an eluate comprising the prolactin compound; and

(b) performing a second chromatographic separation on the eluate from step (a) with a multimodal exchanger and collecting an eluate comprising the prolactin compound. In one aspect, the present invention provides a method for preparing a lyophilized preparation of a prolactin compound, which prolactin compound is recombinantly expressed in a host cell, wherein the prolactin compound is produced in inclusion bodies, which method comprises the steps of: (i) recovering the prolactin compound in a biologically active form from inclusion bodies produced in the recombinant expression,

(ii) if necessary, adjusting the conductivity of the solution comprising the recovered prolactin compound to between 0 and 4 mS/cm,

(a) subjecting the solution from step ii) to a first chromatographic separation with an anionic exchanger and collecting an eluate comprising the prolactin compound; (b) performing a second chromatographic separation on the eluate from step (a) with a multimodal exchanger and collecting an eluate comprising the prolactin compound; and

(c) lyophilizing the eluate from step (b). The methods are generally performed as described above.

In one embodiment, the chromatographic separation in step (b) is achieved by increasing the pH of the buffer from 6.5 to 8.5 during the separation.

The methods of the present invention are advantageous for instance because you can elute the recombinant peptide in step (b) by using a buffer, which is suitable for lyophilization and proceed directly to lyophilization of the peptide after step (b), which for instance is very useful for therapeutically interesting peptides, which are to be used for preparation of pharmaceutical compositions for administration to mammals.

Another advantage of the methods of the present invention is that there is no need for further upconcentration and/or buffer exchanges. This means that there is no need for using methods such as ultrafiltrering or diafiltration (such as described for instance in US20030229212, for instance after step (b), when purifying a peptide using a method according to the present invention.

In one embodiment, a method according to the present invention does not comprise any additional upconcentration steps after step (b). In one embodiment, a method according to the present invention does not comprise any additional upconcentration steps. In one embodiment, a method according to the present invention does not comprise any additional buffer changing steps after step (b). In one embodiment, a method according to the present invention does not comprise any additional buffer changing steps. This does not exclude steps such as sterile filtration, where no buffer exhance or upconcentration takes place. In one embodiment the level of endotoxins in the eluate in step (b) is acceptable for pharmaceutic use. In one embodiment, the level of endotoxins in the eluate from step (b) is less than 5 EU (endotoxin units) /mg peptide. In one embodiment, it is less than 1 EU/mg peptide.

In one aspect, the present invention provides a pharmaceutical formulation obtained by a method according to the present invention, wherein the peptide to be purified is a therapeutic peptide. The terms "pharmaceutical formulation" or "pharmaceutical composition", which are used interchangeably, as used herein means a product comprising an active therapeutic peptide along with pharmaceutical excipients such as buffer, preservative, and optionally a tonicity modifier and/or a stabilizer. The term "excipient" as used herein means the chemical compounds which are normally added to pharmaceutical compositions, e.g. buffers, tonicity agents, preservatives and the like.

In one embodiment, a pharmaceutical formulation according to the invention is an aqueous formulation and comprises the buffer used in step (b) of a method of the present invention. In one embodiment, a pharmaceutical formulation according to the invention is an aqueous formulation and is the eluate from step (b), which may optionally have been subjected to for instance a sterile filtration. In one embodiment, a pharmaceutical formulation according to the invention is a lyophilized formulation, which has been prepared by lyophilizing the eluate from step (b) of a method of the present invention. In one embodiment, a pharmaceutical formulation according to the invention is an aqueous formulation, which has been prepared by reconstitution of a lyophilized formulation according to the present invention.

A pharmaceutical formulation according to the present invention, may comprise the recombinant peptide present in a concentration from for instance from 10^'15 mg/ml to 200 mg/ml, such as 10 ¹⁰ mg/ml - 5 mg/ml. Optionally, said formulation may comprise one or more further therapeutic agents as described above. The formulation may further comprise a buffer system, preservative(s), tonicity agent(s), chelating agent(s), stabilizers and surfactants.

In one embodiment of the invention the pharmaceutical formulation is an aqueous formulation, i.e. formulation comprising water. Such formulation is typically a solution or a suspension. In one embodiment of the invention the pharmaceutical formulation is an aqueous solution. The term "aqueous formulation" is defined as a formulation comprising at least 50%w/w water. Likewise, the term "aqueous solution" is defined as a solution comprising at least 50%w/w water, and the term "aqueous suspension" is defined as a suspension comprising at least 50%w/w water. In one embodiment the pharmaceutical formulation is a lyophilized (or freeze-dried) formulation, whereto the physician or the patient adds solvents and/or diluents prior to use.

In one embodiment the pharmaceutical formulation is a dried formulation (e.g. freeze-dried or spray-dried) ready for use without any prior dissolution. A pharmaceutical formulation according to the present inventionmay comprise a buffer selected from sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof.

In one embodiment of the invention a pharmaceutical formulation according to the present invention further comprises a pharmaceutically acceptable preservative. In one embodiment of the invention the preservative is selected from the group consisting of phenol, o-cresol, m-cresol, p-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2- phenoxyethanol, butyl p-hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal, bronopol, benzoic acid, imidurea, chlorohexidine, sodium dehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzethonium chloride, chlorphenesine (3p- chlorphenoxypropane-1 ,2-diol) or mixtures thereof. In one embodiment of the invention the preservative is present in a concentration from 0.1 mg/ml to 20 mg/ml. In one embodiment of the invention the preservative is present in a concentration from 0.1 mg/ml to 5 mg/ml. In one embodiment of the invention the preservative is present in a concentration from 5 mg/ml to 10 mg/ml. In one embodiment of the invention the preservative is present in a concentration from 10 mg/ml to 20 mg/ml. Each one of these specific preservatives constitutes an alternative embodiment of the invention. The use of a preservative in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 20^th edition, 2000.

In one embodiment of the invention a pharmaceutical formulation according to the present invention further comprises an isotonic agent. In one embodiment, the isotonic agent is selected from the group consisting of a salt (e.g. sodium chloride), a sugar or sugar alcohol, an amino acid (e.g. glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), an alditol (e.g. glycerol (glycerine), 1 ,2-propanediol (propyleneglycol), 1 ,3-propanediol, 1 ,3-butanediol) polyethyleneglycol (e.g. PEG400), or mixtures thereof. Any sugar such as mono-, di-, or polysaccharides, or water-soluble glucans, including for example fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch and carboxymethylcellulose-Na may be used. In one embodiment the sugar additive is sucrose. Sugar alcohol is defined as a C₄.₈-hydrocarbon having at least one -OH group and includes, for example, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. In one embodiment the sugar alcohol additive is mannitol. The sugars or sugar alcohols mentioned above may be used individually or in combination. There is no fixed limit to the amount used, as long as the sugar or sugar alcohol is soluble in the liquid preparation and does not adversely effect the stabilizing effects achieved using the methods of the invention. In one embodiment, the sugar or sugar alcohol concentration is between about 1 mg/ml and about 150 mg/ml. In one embodiment of the invention the isotonic agent is present in a concentration from 1 mg/ml to 50 mg/ml. In one embodiment of the invention the isotonic agent is present in a concentration from 1 mg/ml to 7 mg/ml. In one embodiment of the invention the isotonic agent is present in a concentration from 8 mg/ml to 24 mg/ml. In one embodiment of the invention the isotonic agent is present in a concentration from 25 mg/ml to 50 mg/ml. Each one of these specific isotonic agents constitutes an alternative embodiment of the invention. The use of an isotonic agent in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 20^th edition, 2000.

In one embodiment of the invention a pharmaceutical formulation according to the present invention further comprises a chelating agent. In one embodiment of the invention the chelating agent is selected from salts of ethylenediaminetetraacetic acid (EDTA), citric acid, and aspartic acid, and mixtures thereof. In one embodiment of the invention the chelating agent is present in a concentration from 0.1 mg/ml to 5mg/ml. In one embodiment of the invention the chelating agent is present in a concentration from 0.1 mg/ml to 2mg/ml. In one embodiment of the invention the chelating agent is present in a concentration from 2mg/ml to 5mg/ml. Each one of these specific chelating agents constitutes an alternative embodiment of the invention. The use of a chelating agent in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 20^th edition, 2000. In one embodiment of the invention a pharmaceutical formulation according to the present invention further comprises a stabilizer. The use of a stabilizer in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 20^th edition, 2000.

More particularly, compositions of the invention are stabilized liquid pharmaceutical compositions whose therapeutically active components include a polypeptide that possibly exhibits aggregate formation during storage in liquid pharmaceutical formulations. By "aggregate formation" is intended a physical interaction between the polypeptide molecules that results in formation of oligomers, which may remain soluble, or large visible aggregates that precipitate from the solution. By "during storage" is intended a liquid pharmaceutical composition or formulation once prepared, is not immediately administered to a subject. Rather, following preparation, it is packaged for storage, either in a liquid form, in a frozen state, or in a dried form for later reconstitution into a liquid form or other form suitable for administration to a subject. By "dried form" is intended the liquid pharmaceutical composition or formulation is dried either by freeze drying (i.e., lyophilization; see, for example, Williams and PoIIi (1984) J. Parenteral Sci. Technol. 38:48-59), spray drying (see Masters (1991) in Spray-Drying Handbook (5th ed; Longman Scientific and Technical, Essez, U.K.), pp. 491 - 676; Broadhead et al. (1992) Drug Devel. Ind. Pharm. 18:1169-1206; and Mumenthaler et al. (1994) Pharm. Res. 11 :12-20), or air drying (Carpenter and Crowe (1988) Cryobiology 25:459-470; and Roser (1991 ) Biopharm. 4:47-53). Aggregate formation by a polypeptide during storage of a liquid pharmaceutical composition can adversely affect biological activity of that polypeptide, resulting in loss of therapeutic efficacy of the pharmaceutical composition. Furthermore, aggregate formation may cause other problems such as blockage of tubing, membranes, or pumps when the polypeptide-containing pharmaceutical composition is administered using an infusion system.

In one embodiment of the invention a pharmaceutical formulation according to the present invention further comprises an amount of an amino acid base sufficient to decrease aggregate formation by the polypeptide during storage of the composition. By "amino acid base" is intended an amino acid or a combination of amino acids, where any given amino acid is present either in its free base form or in its salt form. Where a combination of amino acids is used, all of the amino acids may be present in their free base forms, all may be present in their salt forms, or some may be present in their free base forms while others are present in their salt forms. In one embodiment, amino acids for use in a pharmaceutical formulation according to the present invention are those carrying a charged side chain, such as arginine, lysine, aspartic acid, and glutamic acid. Any stereoisomer (i.e., L, D, or mixtures thereof) of a particular amino acid (e.g. glycine, methionine, histidine, imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine and mixtures thereof) or combinations of these stereoisomers, may be present in the pharmaceutical compositions of the invention so long as the particular amino acid is present either in its free base form or its salt form. In one embodiment the L-stereoisomer is used. Compositions of the invention may also be formulated with analogues of these amino acids. By "amino acid analogue" is intended a derivative of the naturally occurring amino acid that brings about the desired effect of decreasing aggregate formation by the polypeptide during storage of the liquid pharmaceutical compositions of the invention. Suitable arginine analogues include, for example, aminoguanidine, ornithine and N-monoethyl L-arginine, suitable methionine analogues include ethionine and buthionine and suitable cysteine analogues include S- methyl-L cysteine. As with the other amino acids, the amino acid analogues are incorporated into the compositions in either their free base form or their salt form. In one embodiment of the invention the amino acids or amino acid analogues are used in a concentration, which is sufficient to prevent or delay aggregation of the protein.

In one embodiment of the invention methionine (or other sulphuric amino acids or amino acid analogous) may be added to inhibit oxidation of methionine residues to methionine sulfoxide when the polypeptide acting as the therapeutic agent is a polypeptide comprising at least one methionine residue susceptible to such oxidation. By "inhibit" is intended minimal accumulation of methionine oxidized species over time. Inhibiting methionine oxidation results in greater retention of the polypeptide in its proper molecular form. Any stereoisomer of methionine (L, D, or mixtures thereof) or combinations thereof can be used. The amount to be added should be an amount sufficient to inhibit oxidation of the methionine residues such that the amount of methionine sulfoxide is acceptable to regulatory agencies. Typically, this means that the composition contains no more than about 10% to about 30% methionine sulfoxide. Generally, this can be achieved by adding methionine such that the ratio of methionine added to methionine residues ranges from about 1 :1 to about 1000: 1 , such as 10: 1 to about 100: 1. In one embodiment of the invention a pharmaceutical formulation according to the present invention further comprises a stabilizer selected from the group of high molecular weight polymers or low molecular compounds. In one embodiment of the invention the stabilizer is selected from polyethylene glycol (e.g. PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, carboxy-/hydroxycellulose or derivates thereof (e.g. HPC, HPC-SL, HPC-L and HPMC), cyclodextrins, sulphur-containing substances as monothioglycerol, thioglycolic acid and 2-methylthioethanol, and different salts (e.g. sodium chloride). Each one of these specific stabilizers constitutes an alternative embodiment of the invention.

A pharmaceutical formulation according to the present invention may also comprise additional stabilizing agents, which further enhance stability of a therapeutically active polypeptide therein. Stabilizing agents of particular interest to the present invention include, but are not limited to, methionine and EDTA, which protect the polypeptide against methionine oxidation, and a nonionic surfactant, which protects the polypeptide against aggregation associated with freeze-thawing or mechanical shearing. In one embodiment of the invention the formulation further comprises a surfactant. In one embodiment of the invention the surfactant is selected from a detergent, ethoxylated castor oil, polyglycolyzed glycerides, acetylated monoglycerides, sorbitan fatty acid esters, polyoxypropylene-polyoxyethylene block polymers (eg. poloxamers such as Pluronic^® F68, poloxamer 188 and 407, Triton X-100 ), polyoxyethylene sorbitan fatty acid esters, polyoxyethylene and polyethylene derivatives such as alkylated and alkoxylated derivatives (tweens, e.g. Tween-20, Tween-40, Tween-80 and Brij-35), monoglycerides or ethoxylated derivatives thereof, diglycerides or polyoxyethylene derivatives thereof, alcohols, glycerol, lectins and phospholipids (eg. phosphatidyl serine, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, diphosphatidyl glycerol and sphingomyelin), derivates of phospholipids (eg. dipalmitoyl phosphatidic acid) and lysophospholipids (eg. palmitoyl lysophosphatidyl-L-serine and 1-acyl-sn-glycero-3-phosphate esters of ethanolamine, choline, serine or threonine) and alkyl, alkoxyl (alkyl ester), alkoxy (alkyl ether)- derivatives of lysophosphatidyl and phosphatidylcholines, e.g. lauroyl and myristoyl derivatives of lysophosphatidylcholine, dipalmitoylphosphatidylcholine, and modifications of the polar head group, that is cholines, ethanolamines, phosphatidic acid, serines, threonines, glycerol, inositol, and the positively charged DODAC, DOTMA, DCP, BISHOP, lysophosphatidylserine and lysophosphatidylthreonine, and glycerophospholipids (eg. cephalins), glyceroglycolipids (eg. galactopyransoide), sphingoglycolipids (eg. ceramides, gangliosides), dodecylphosphocholine, hen egg lysolecithin, fusidic acid derivatives- (e.g. sodium tauro- dihydrofusidate etc.), long-chain fatty acids and salts thereof C6-C12 (eg. oleic acid and caprylic acid), acylcarnitines and derivatives, N^α-acylated derivatives of lysine, arginine or histidine, or side-chain acylated derivatives of lysine or arginine, NT-acylated derivatives of dipeptides comprising any combination of lysine, arginine or histidine and a neutral or acidic amino acid, NT-acylated derivative of a tripeptide comprising any combination of a neutral amino acid and two charged amino acids, DSS (docusate sodium, CAS registry no [577-1 1- 7]), docusate calcium, CAS registry no [128-49-4]), docusate potassium, CAS registry no [7491 -09-0]), SDS (sodium dodecyl sulphate or sodium lauryl sulphate), sodium caprylate, cholic acid or derivatives thereof, bile acids and salts thereof and glycine or taurine conjugates, ursodeoxycholic acid, sodium cholate, sodium deoxycholate, sodium taurocholate, sodium glycocholate, N-Hexadecyl-N,N-dimethyl-3-ammonio-1 - propanesulfonate, anionic (alkyl-aryl-sulphonates) monovalent surfactants, zwitterionic surfactants (e.g. N-alkyl-N,N-dimethylammonio-1 -propanesulfonates, 3-cholamido-1 - propyldimethylammonio-1 -propanesulfonate, cationic surfactants (quaternary ammonium bases) (e.g. cetyl-trimethylammonium bromide, cetylpyridinium chloride), non-ionic surfactants (eg. Dodecyl β-D-glucopyranoside), poloxamines (eg. Tetronic's), which are tetrafunctional block copolymers derived from sequential addition of propylene oxide and ethylene oxide to ethylenediamine, or the surfactant may be selected from the group of imidazoline derivatives, or mixtures thereof. Each one of these specific surfactants constitutes an alternative embodiment of the invention. The use of a surfactant in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 20^th edition, 2000.

It is possible that other ingredients may be present in a pharmaceutical formulation according to the present invention. Such additional ingredients may include wetting agents, emulsifiers, antioxidants, bulking agents, tonicity modifiers, chelating agents, metal ions, oleaginous vehicles, proteins (e.g., human serum albumin, gelatine or proteins) and a zwitterion (e.g., an amino acid such as betaine, taurine, arginine, glycine, lysine and histidine). Such additional ingredients, of course, should not adversely affect the overall stability of the pharmaceutical formulation of the present invention.

The ingredients for a pharmaceutical formulation according to the present invention may advantageously be part of the buffer used in step (b) of a method according to the present invention, or may be added to the eluate of step (b) of a method according to the present invention. A pharmaceutical formulation according to the present invention may be administered to a patient in need of such treatment at several sites, for example, at topical sites, for example, skin and mucosal sites, at sites which bypass absorption, for example, administration in an artery, in a vein, in the heart, and at sites which involve absorption, for example, administration in the skin, under the skin, in a muscle or in the abdomen. Administration of pharmaceutical compositions according to the invention may be through several routes of administration, for example, lingual, sublingual, buccal, in the mouth, oral, in the stomach and intestine, nasal, pulmonary, for example, through the bronchioles and alveoli or a combination thereof, epidermal, dermal, transdermal, vaginal, rectal, ocular, for examples through the conjunctiva, uretal, and parenteral to patients in need of such a treatment. As it is clearly evident for a person skilled in the art, the route of administration is dependent on what makes sense for any given therapeutic peptide.

A pharmaceutical formulation according to the present invention may be administered in several dosage forms, for example, as solutions, suspensions, emulsions, microemulsions, multiple emulsion, foams, salves, pastes, plasters, ointments, tablets, coated tablets, rinses, capsules, for example, hard gelatine capsules and soft gelatine capsules, suppositories, rectal capsules, drops, gels, sprays, powder, aerosols, inhalants, eye drops, ophthalmic ointments, ophthalmic rinses, vaginal pessaries, vaginal rings, vaginal ointments, injection solution, in situ transforming solutions, for example in situ gelling, in situ setting, in situ precipitating, in situ crystallization, infusion solution, and implants.

A pharmaceutical formulation according to the present invention may further be compounded in, or attached to, for example through covalent, hydrophobic and electrostatic interactions, a drug carrier, drug delivery system and advanced drug delivery system in order to further enhance stability of the peptide of the present invention, increase bioavailability, increase solubility, decrease adverse effects, achieve chronotherapy well known to those skilled in the art, and increase patient compliance or any combination thereof. Examples of carriers, drug delivery systems and advanced drug delivery systems include, but are not limited to, polymers, for example cellulose and derivatives, polysaccharides, for example dextran and derivatives, starch and derivatives, polyvinyl alcohol), acrylate and methacrylate polymers, polylactic and polyglycolic acid and block co-polymers thereof, polyethylene glycols, carrier proteins, for example albumin, gels, for example, thermogelling systems, for example block co-polymeric systems well known to those skilled in the art, micelles, liposomes, microspheres, nanoparticulates, liquid crystals and dispersions thereof, L2 phase and dispersions there of, well known to those skilled in the art of phase behaviour in lipid- water systems, polymeric micelles, multiple emulsions, self-emulsifying, self- microemulsifying, cyclodextrins and derivatives thereof, and dendrimers.

A pharmaceutical formulation according to the present invention may be used in the formulation of controlled, sustained, protracting, retarded, and slow release drug delivery systems. Without limiting the scope of the invention, examples of useful controlled release system and compositions are hydrogels, oleaginous gels, liquid crystals, polymeric micelles, microspheres, and nanoparticles.

The following is a non-limiting list of embodiments of the present invention. Embodiment 1 : A method for purifying a peptide having a pi below 6.5, which peptide is recombinantly expressed in a host cell, wherein the recombinant peptide is produced in inclusion bodies, which purification method comprises the steps of:

(i) recovering the peptide in a biologically active form from inclusion bodies produced in the recombinant expression,

(ii) if necessary, adjusting the conductivity of the solution comprising the recovered recombinant peptide to between 0 and 4 mS/cm, (a) subjecting the solution from step ii) to a first chromatographic separation with an anionic exchanger and collecting an eluate comprising the recombinant peptide; and

(b) performing a second chromatographic separation on the eluate from step (a) with a multimodal exchanger and collecting an eluate comprising the recombinant peptide. Embodiment 2: A method for preparing a pharmaceutical formulation of a peptide having a pi below 6.5, which peptide is recombinantly expressed in a host cell, wherein the recombinant peptide is produced in inclusion bodies, which method comprises the steps of: (i) recovering the peptide in a biologically active form from inclusion bodies produced in the recombinant expression, (ii) if necessary, adjusting the conductivity of the solution comprising the recovered recombinant peptide to between 0 and 4 mS/cm,

Embodiment 3: A method according to embodiment 2, wherein the pharmaceutical formulation is a lyophilized pharmaceutical formulation, which is made by lyophilizing the eluate from step (b).

Embodiment 4: A method according to embodiment 2, wherein the pharmaceutical formulation is an aqueous pharmaceutical formulation, which is made by reconstitution of a lyophilized pharmaceutical formulation, which is made by lyophilizing the eluate from step (b).

Embodiment 5: A method according to embodiment 3 or embodiment 4, wherein one or more additional components of the pharmaceutical formulation are added to the eluate of step (b) before lyophilization thereof.

Embodiment 6: A method according to embodiment 2, wherein the eluate from step (b) is used as the pharmaceutical formulation.

Embodiment 7: A method according to embodiment 6, wherein additional components of the pharmaceutical formulation is added to the eluate of step (b) for finalizing the preparation of the pharmaceutical preparation.

Embodiment 8: A method according to any of embodiments 1 to 7, wherein step (i) comprises the steps of:

(ia) solubilising the insoluble aggregate; and (ib) refolding the recombinant peptide to a biologically active conformation. Embodiment 9: A method according to embodiment 8, wherein step (ia) comprises addition of a solubilisation agent selected from a guanidinium salt, urea, a detergent, or other organic solvent.

Embodiment 10: A method according to embodiment 8 or embodiment 9, wherein step (ib) comprises the use of a refolding buffer comprising arginine salts and/or Tris.

Embodiment 11 : A method according to any of embodiments 8 to 10, which additionally comprises a washing step before step (ia).

Embodiment 12: A method according to any of embodiments 1 to 1 1 , wherein said host cell is a prokaryotic cell. Embodiment 13: A method according to embodiment 12, wherein said prokaryotic cell is Escherichia coli.

Embodiment 14: A method according to any of embodiments 1 to 13, wherein the level of endotoxins in the eluate in step (b) is acceptable for pharmaceutic use.

Embodiment 15: A method for purifying a peptide having a pi below 6.5, which peptide is recombinantly expressed in a host cell, wherein the recombinant peptide is produced in soluble form, which purification method comprises the steps of: (i) obtaining a solution comprising the peptide from the recombinant expression,

(b) performing a second chromatographic separation on the eluate from step (a) with a multimodal exchanger and collecting an eluate comprising the recombinant peptide. Embodiment 16: A method for preparing a pharmaceutical formulation of a peptide having a pi below 6.5, which peptide is recombinantly expressed in a host cell, wherein the recombinant peptide is produced in soluble form, which method comprises the steps of: (i) obtaining a solution comprising the peptide from the recombinant expression,

(a) subjecting the solution from step ii) to a first chromatographic separation with an anionic exchanger and collecting an eluate comprising the recombinant peptide; (b) performing a second chromatographic separation on the eluate from step (a) with a multimodal exchanger and collecting an eluate comprising the recombinant peptide. Embodiment 17: A method according to embodiment 16, wherein the pharmaceutical formulation is a lyophilized pharmaceutical formulation, which is made by lyophilizing the eluate from step (b). Embodiment 18: A method according to embodiment 16, wherein the pharmaceutical formulation is an aqueous pharmaceutical formulation, which is made by reconstitution of a lyophilized pharmaceutical formulation, which is made by lyophilizing the eluate from step (b). Embodiment 19: A method according to embodiment 17 or embodiment 18, wherein one or more additional components of the pharmaceutical formulation are added to the eluate of step (b) before lyophilization thereof.

Embodiment 20: A method according to embodiment 16, wherein the eluate from step (b) is used as the pharmaceutical formulation. Embodiment 21 : A method according to embodiment 20, wherein additional components of the pharmaceutical formulation is added to the eluate of step (b) for finalizing the preparation of the pharmaceutical preparation.

Embodiment 22: A method according to any of embodiments 15 to 21 , wherein said host cell is a eukaryotic cell. Embodiment 23: A method according to embodiment 22, wherein said host cell is a yeast cell.

Embodiment 24: A method according to embodiment 22, wherein said host cell is a human cell.

Embodiment 25: A method according to any of embodiments 1 to 24, wherein the peptide is a hormone compound.

Embodiment 26: A method according to any of embodiments 1 to 24, wherein the peptide is an antibody compound.

Embodiment 27: A method according to any of embodiments 1 to 24, wherein the peptide is a cytokine compound. Embodiment 28: A method according to any of embodiments 1 to 27, wherein the peptide is a prolactin compound, a growth hormone compound or a placental lactogen compound.

Embodiment 29: A method according to any of embodiments 1 to 24, wherein the peptide is a chemokine compound. Embodiment 30: A method according to any of embodiments 1 to 29, wherein the anionic exchanger used in step (a) is a strong anionic exchanger.

Embodiment 31 : A method according to any of embodiments 1 to 30, wherein the anionic exchanger used in step (a) binds to negative charged sugar residues on endotoxins or lipopolysaccarides. Embodiment 32: A method according to any of embodiments 1 to 31 , wherein the anionic exchanger used in step (a) is selected from: Q-Sepharose Fast Flow; Q-Sepharose High Performance; Source 3OQ; Toyopearl SuperQ; and UNOsphere Q.

Embodiment 33: A method according to any of embodiments 1 to 32, wherein the anionic exchanger comprises a quaternary amine resin.

Embodiment 34: A method according to any of embodiments 1 to 33, wherein the anionic exchanger used in step (a) is Q SEPHAROSE FAST FLOW™ anionic exchanger.

Embodiment 35: A method according to any of embodiments 1 to 34, which comprises the use of a buffer in step (a). Embodiment 36: A method according to embodiment 35, wherein said buffer includes triethanolamine, Tris (tris(hydroxymethyl)methylamine), TAPS (3- {[tris(hydroxymethyl)methyl]amino}propanesulfonic acid), Bicine (N,N-bis(2- hydroxyethyl)glycine), Tricine (N-tris(hydroxymethyl)methylglycine), HEPES (4-2- hydroxyethyl-1 -piperazineethanesulfonic acid), TES (2-{[tris(hydroxymethyl)- methyl]amino}ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), Cacodylate (dimethylarsinic acid), MES (2-(N- morpholino)ethanesulfonic acid) or acetate.

Embodiment 37: A method according to embodiment 35, wherein said buffer includes triethanolamine, Tris (tris(hydroxymethyl)methylamine), TAPS (3- {[tris(hydroxymethyl)methyl]amino}propanesulfonic acid), Bicine (N,N-bis(2- hydroxyethyl)glycine), Tricine (N-tris(hydroxymethyl)methylglycine), HEPES (4-2- hydroxyethyl-1 -piperazineethanesulfonic acid), TES (2-{[tris(hydroxymethyl)- methyl]amino}ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), Cacodylate (dimethylarsinic acid), or acetate. Embodiment 38: A method according to embodiment 36 or embodiment 37, wherein the buffer used in step (a) comprises triethanolamine buffer.

Embodiment 39: A method according to any of embodiments 35 to 38, wherein the buffer used in step (a) has a pH, which is +/-1 of the pi of the recombinant peptide.

Embodiment 40: A method according to any of embodiments 35 to 39, wherein chromatographic separation in step (a) is achieved by increasing the ionic strength of the buffer.

Embodiment 41 : A method according to any of embodiments 35 to 40, wherein the buffer comprises a salt selected from ammonium bicarbonate, KCI or NaCI. Embodiment 42: A method according to embodiment 41 , wherein the buffer comprises from 0 mM to 50OmM NaCI.

Embodiment 43: A method according to any of embodiments 1 to 42, wherein the multimodal exchanger used in step (b) is a negatively charged multimodal exchanger. Embodiment 44: A method according to any of embodiments 1 to 43, wherein the multimodal exchanger used in step (b) is a CAPTO MMC™ multimodal exchanger.

Embodiment 45: A method according to any of embodiments 1 to 44, which comprises the use of a buffer in step (b).

Embodiment 46: A method according to embodiment 45, wherein the buffer in step (b) is suitable for lyophilization.

Embodiment 47: A method according to embodiment 45 or embodiment 46, wherein the buffer in step (b) constituents are capable of undergoing sublimation during lyophilization.

Embodiment 48: A method according to any of embodiments 45 to 47, wherein the buffer in step (b) is an ammonium buffer. Embodiment 49: A method according to any of embodiments 45 to 48, wherein the buffer in step (b) is ammonium bicarbonate.

Embodiment 50: A method according to any of embodiments 45 to 49, wherein the buffer in step (b) is a pharmaceutically acceptable buffer.

Embodiment 51 : A method according to any of embodiments 45 to 50, wherein the buffer in step (b) further comprises phosphate buffered saline or histidine, or further comprises histidine and a sugar.

Embodiment 52: A method according to any of embodiments 1 to 48, wherein chromatographic separation in step (b) is achieved by increasing the pH of the buffer.

Embodiment 53: A method according to any of embodiments 1 to 52 , wherein the pH of the solution comprising the recovered recombinant peptide is adjusted between step (i) and step (a) to a pH above the isoelectric point (pi) of the peptide.

Embodiment 54: A method according to embodiment 53, wherein the pH of the solution comprising the recovered recombinant peptide is adjusted between step (i) and step (a) to a pH at least 1.0. units above the (pi) of the peptide. Embodiment 55: A method according to any of embodiments 1 to 54, wherein the pH of the eluate comprising the recombinant peptide from step (a) is adjusted to a pH below the isoelectric point of the peptide prior to step (b). Embodiment 56: A method according to any of embodiments 1 to 55, wherein the conductivity of the eluate comprising the recombinant peptide is adjusted to between 0 to 8 mS/cm prior to step (b).

Embodiment 57: A method according to any of embodiments 1 to 56, which does not comprise any additional upconcentration steps.

Embodiment 58: A method according to any of embodiments 1 to 57, which does not comprise any additional buffer changing steps.

Embodiment 59: A method for purifying a prolactin compound having a pi below 6.5, which prolactin compound is recombinantly expressed in a host cell, wherein the prolactin compound is produced in inclusion bodies, which purification method comprises the steps of: (i) recovering the prolactin compound in a biologically active form from inclusion bodies produced in the recombinant expression, (ii) if necessary, adjusting the conductivity of the solution comprising the recovered prolactin compound to between 0 and 4 mS/cm, (a) subjecting the solution from step ii) to a first chromatographic separation with an anionic exchanger and collecting an eluate comprising the prolactin compound; and (b) performing a second chromatographic separation on the eluate from step (a) with a multimodal exchanger and collecting an eluate comprising the prolactin compound.

Embodiment 60: A method for preparing a pharmaceutical formulation of a prolactin compound, which prolactin compound is recombinantly expressed in a host cell, wherein the prolactin compound is produced in inclusion bodies, which method comprises the steps of: (i) recovering the prolactin compound in a biologically active form from inclusion bodies produced in the recombinant expression,

(b) performing a second chromatographic separation on the eluate from step (a) with a multimodal exchanger and collecting an eluate comprising the recombinant peptide. Embodiment 61 : A method according to embodiment 60, wherein the pharmaceutical formulation is a lyophilized pharmaceutical formulation, which is made by lyophilizing the eluate from step (b).

Embodiment 62: A method according to embodiment 60, wherein the pharmaceutical formulation is an aqueous pharmaceutical formulation, which is made by reconstitution of a lyophilized pharmaceutical formulation, which is made by lyophilizing the eluate from step (b).

Embodiment 63: A method according to embodiment 61 or embodiment 62, wherein one or more additional components of the pharmaceutical formulation are added to the eluate of step (b) before lyophilization thereof.

Embodiment 64: A method according to embodiment 60, wherein the eluate from step (b) is used as the pharmaceutical formulation.

Embodiment 65: A method according to embodiment 64, wherein additional components of the pharmaceutical formulation is added to the eluate of step (b) for finalizing the preparation of the pharmaceutical preparation.

Embodiment 66: A method according to any of embodiments 59 to 65, wherein step (i) comprises the steps of: (ia) solubilising the insoluble aggregate; and

(ib) refolding the prolactin compound to a biologically active conformation. Embodiment 67: A method according to embodiment 66, wherein step (ia) comprises addition of a solubilisation agent selected from a guanidinium salt, urea, a detergent, or other organic solvent.

Embodiment 68: A method according to embodiment 66 or embodiment 67, wherein step (ib) comprises the use of a refolding buffer comprising arginine salts and/or Tris. Embodiment 69: A method according to any of embodiments 66 to 68, which additionally comprises a washing step before step (ia).

Embodiment 70: A method according to any of embodiments 59 to 69, wherein said host cell is a prokaryotic cell.

Embodiment 71 : A method according to embodiment 70, wherein said prokaryotic cell is Escherichia coli.

Embodiment 72: A method for purifying a prolactin compound having a pi below 6.5, which prolactin compound are recombinantly expressed in a host cell, wherein the prolactin compound is produced in soluble form, which purification method comprises the steps of: (i) obtaining a solution comprising the prolactin compound from the recombinant expression,

(a) subjecting the solution from step ii) to a first chromatographic separation with an anionic exchanger and collecting an eluate comprising the prolactin compound; and (b) performing a second chromatographic separation on the eluate from step (a) with a multimodal exchanger and collecting an eluate comprising the prolactin compound.

Embodiment 73: A method for preparing a pharmaceutical formulation of a prolactin compound having a pi below 6.5, which prolactin compound is recombinantly expressed in a host cell, wherein the recombinant peptide is produced in soluble form, which method comprises the steps of: (i) obtaining a solution comprising the prolactin compound from the recombinant expression,

(ii) if necessary, adjusting the conductivity of said solution to between 0 and 4 mS/cm, (a) subjecting the solution from step ii) to a first chromatographic separation with an anionic exchanger and collecting an eluate comprising the prolactin compound; (b) performing a second chromatographic separation on the eluate from step (a) with a multimodal exchanger and collecting an eluate comprising the prolactin compound.

Embodiment 74: A method according to embodiment 73, wherein the pharmaceutical formulation is a lyophilized pharmaceutical formulation, which is made by lyophilizing the eluate from step (b).

Embodiment 75: A method according to embodiment 73, wherein the pharmaceutical formulation is an aqueous pharmaceutical formulation, which is made by reconstitution of a lyophilized pharmaceutical formulation, which is made by lyophilizing the eluate from step (b).

Embodiment 76: A method according to embodiment 74 or embodiment 75, wherein one or more additional components of the pharmaceutical formulation are added to the eluate of step (b) before lyophilization thereof.

Embodiment 77: A method according to embodiment 73, wherein the eluate from step (b) is used as the pharmaceutical formulation.

Embodiment 78: A method according to embodiment 77, wherein additional components of the pharmaceutical formulation is added to the eluate of step (b) for finalizing the preparation of the pharmaceutical preparation.

Embodiment 79: A method according to any of embodiments 72 to 78, wherein said host cell is a eukaryotic cell.

Embodiment 80: A method according to embodiment 79, wherein said host cell is a yeast cell.

Embodiment 81 : A method according to embodiment 79, wherein said host cell is a human cell. Embodiment 82: A method according to any of embodiments 59 to 81 , wherein the anionic exchanger used in step (a) is a strong anionic exchanger.

Embodiment 83: A method according to any of embodiments 59 to 82, wherein the anionic exchanger used in step binds to negative charged sugar residues on endotoxins or lipopolysaccarides.

Embodiment 84: A method according to any of embodiments 59 to 83, wherein the anionic exchanger used in step (a) is selected from: Q-Sepharose Fast Flow; Q-Sepharose High Performance; Source 3OQ; Toyopearl SuperQ; and UNOsphere Q.

Embodiment 85: A method according to any of embodiments 59 to 84, wherein the anionic exchanger comprises a quaternary amine resin.

Embodiment 86: A method according to any of embodiments 59 to 85, wherein the anionic exchanger used in step (a) is Q SEPHAROSE FAST FLOW™ anionic exchanger.

Embodiment 87: A method according to any of embodiments 59 to 86, which comprises the use of a buffer in step (a). Embodiment 88: A method according to embodiment 87, wherein said buffer includes triethanolamine, Tris (tris(hydroxymethyl)methylamine), TAPS (3- {[tris(hydroxymethyl)methyl]amino}propanesulfonic acid), Bicine (N,N-bis(2- hydroxyethyl)glycine), Tricine (N-tris(hydroxymethyl)methylglycine), HEPES (4-2- hydroxyethyl-1 -piperazineethanesulfonic acid), TES (2-{[tris(hydroxymethyl)- methyl]amino}ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), Cacodylate (dimethylarsinic acid), MES (2-(N- morpholino)ethanesulfonic acid) or acetate.

Embodiment 89: A method according to embodiment 87, wherein said buffer includes triethanolamine, Tris (tris(hydroxymethyl)methylamine), TAPS (3- {[tris(hydroxymethyl)methyl]amino}propanesulfonic acid), Bicine (N,N-bis(2- hydroxyethyl)glycine), Tricine (N-tris(hydroxymethyl)methylglycine), HEPES (4-2- hydroxyethyl-1 -piperazineethanesulfonic acid), TES (2-{[tris(hydroxymethyl)- methyl]amino}ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), Cacodylate (dimethylarsinic acid) or acetate. Embodiment 90: A method according to embodiment 88 or embodiment 89, wherein the buffer used in step (a) comprises triethanolamine buffer.

Embodiment 91 : A method according to any of embodiments 87 to 90, wherein the first separation in step (a) is performed by first eluating with 25% 20 mM triethanolamine and then collecting the prolactin compound by eluating with 60% 20 mM triethanolamine. Embodiment 92: A method according to any of embodiments 87 to 91 , wherein the buffer used in step (a) has a pH, which is between 5.3 and 7.3.

Embodiment 93: A method according to any of embodiments 87 to 92, wherein chromatographic separation in step (a) is achieved by increasing the ionic strength of the buffer.

Embodiment 94: A method according to any of embodiments 87 to 93, wherein the buffer comprises a salt selected from ammonium bicarbonate, KCI or NaCI.

Embodiment 95: A method according to embodiment 94, wherein the buffer comprises from 0 mM to 500 mM NaCI. Embodiment 96: A method according to any of embodiments 59 to 95, wherein the multimodal exchanger used in step (b) is a negatively charged multimodal exchanger.

Embodiment 97: A method according to any of embodiments 59 to 96, wherein the multimodal exchanger used in step (b) is a CAPTO MMC™ multimodal exchanger.

Embodiment 98: A method according to any of embodiments 59 to 97, which comprises the use of a buffer in step (b).

Embodiment 99: A method according to embodiment 98, wherein the buffer in step (b) is suitable for lyophilization.

Embodiment 100: A method according to embodiment 98 or embodiment 99, wherein the buffer in step (b) constituents are capable of undergoing sublimation during lyophilization.

Embodiment 101 : A method according to any of embodiments 98 to 100, wherein the buffer in step (b) is an ammonium buffer.

Embodiment 102: A method according to any of embodiments 98 to 101 , wherein the buffer in step (b) is ammonium bicarbonate. Embodiment 103: A method according to any of embodiments 98 to 102, wherein the buffer in step (b) further comprises ingredients, which would make the solution pharmaceutically acceptable.

Embodiment 104: A method according to any of embodiments 98 to 103, wherein the buffer in step (b) further comprises phosphate buffered saline or histidine, or further comprises histidine and a sugar.

Embodiment 105: A method according to any of embodiments 59 to 101 , wherein chromatographic separation in step (b) is achieved by increasing the pH of the buffer.

Embodiment 106: A method according to embodiment 105, wherein the pH is increased from 6.5 to 8.5. Embodiment 107: A method according to any of embodiment 59 to 106, wherein the prolactin compound is human prolactin.

Embodiment 108: A method according to any of embodiment 59 to 106, wherein the prolactin compound is an antagonist of the human prolactin receptor. Embodiment 109: A method according to any of embodiments 59 to 108, wherein the prolactin compound is Ser-PRL S33A Q73L G129R K190R.

Embodiment 110: A method according to 109, wherein the buffer in step (a) is triethanolamine, and the first separation in step (a) is performed by first eluating with 25% 20 mM triethanolamine and then collecting the prolactin compound by eluating with 60% 20 mM triethanolamine.

Embodiment 11 1 : A method according to any of embodiments 59 to 1 10 , wherein the pH of the solution comprising the recovered prolactin compound is adjusted between step (i) and step (a) to a pH above the isoelectric point (pi) of the peptide.

Embodiment 112: A method according to embodiment 11 1 , wherein the pH of the solution comprising the recovered prolactin compound is adjusted between step (i) and step (a) to a pH at least 1.0. units above the (pi) of the peptide.

Embodiment 113 A method according to embodiment 107, wherein the pH of the solution comprising the recovered prolactin compound is adjusted between step (i) and step (a) to a pH at least 7.3 above the (pi) of the prolactin. Embodiment 114: A method according to any of embodiments 59 to 1 13, wherein the pH of the eluate comprising the prolactin compound from step (a) is adjusted to a pH below the isoelectric point of the prolactin compound prior to step (b).

Embodiment 115: A method according to embodiment 107, wherein the pH is adjusted to between about 5.3 and about 7.3 following step (a) and prior to step (b). Embodiment 116: A method according to any of embodiments 59 to 1 15, wherein the conductivity of the eluate comprising the prolactin compound is adjusted to between 0 to 8 mS/cm prior to step (b).

Embodiment 117: A method according to any of embodiments 1 to 116, wherein the level of endotoxin in the eluate from step (b) is acceptable for pharmaceutical use. Embodiment 118: A method according to any of embodiments 1 to 116, wherein the level of endotoxin in the eluate from step (b) is less than 5 EU/mg.

Embodiment 119: A method according to embodiment 118, wherein the level of endotoxin in the eluate from step (b) is less than 1 EU/mg. Embodiment 120: A recombinant peptide purification kit, which comprises an anionic exchange resin, a multimodal exchange resin and instructions to use said kit in accordance with a method according to any of embodiments 1 to 1 19.

Embodiment 121 : A kit according to embodiment 120, wherein the recombinant peptide to be purified is a hormone compound.

Embodiment 122: A kit according to embodiment 120, wherein the recombinant peptide to be purified is a cytokine compound.

Embodiment 123: A kit according to any of embodiments 120 to 122, wherein the recombinant peptide to be purified is prolactin, hGH etc (check pi). Embodiment 124: A kit according to embodiment 120, wherein the recombinant peptide to be purified is an antibody compound.

Embodiment 125: A kit according to embodiment 120, wherein the recombinant peptide to be purified is a chemokine compound.

Embodiment 126: A kit according to any of embodiments 120 to 125, wherein the anionic exchange resin comprises a strong anionic exchanger.

Embodiment 127: A method according to any of embodiments 120 to 126, wherein the anionic exchanger used in step binds to negative charged sugar residues on endotoxins or lipopolysaccarides.

Embodiment 128: A kit according to any of embodiments 120 to 127, wherein the anionic exchange resin comprises an anionic exchanger selected from: Q-Sepharose Fast Flow; Q-Sepharose High Performance; Source 3OQ; Toyopearl SuperQ; and UNOsphere Q.

Embodiment 129: A kit according to any of embodiments 120 to 128, wherein the anionic exchange resin comprises a quaternary amine resin.

Embodiment 130: A kit according to any of embodiments 120 to 129, wherein the anionic exchanger is Q SEPHAROSE FAST FLOW™ anionic exchanger.

Embodiment 131 : A kit according to any of embodiments 120 to 130, which comprises a buffer for use in step (a) of a method according to any of embodiments 1 to 119.

Embodiment 132: A kit according to embodiment 131 , wherein said buffer includes triethanolamine, Tris (tris(hydroxymethyl)methylamine), TAPS (3-{[tris(hydroxymethyl)- methyl]amino}propanesulfonic acid), Bicine (N,N-bis(2-hydroxyethyl)glycine), Tricine (N- tris(hydroxymethyl)methylglycine), HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid), TES (2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid), MOPS (3-(N- morpholino)propanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), Cacodylate (dimethylarsinic acid), MES (2-(N-morpholino)ethanesulfonic acid) or acetate. Embodiment 133: A kit according to embodiment 131 , wherein said buffer includes triethanolamine, Tris (tris(hydroxymethyl)methylamine), TAPS (3-{[tris(hydroxymethyl)- methyl]amino}propanesulfonic acid), Bicine (N,N-bis(2-hydroxyethyl)glycine), Tricine (N- tris(hydroxymethyl)methylglycine), HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid), TES (2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid), MOPS (3-(N- morpholino)propanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), Cacodylate (dimethylarsinic acid), or acetate.

Embodiment 134: A kit according to embodiment 132 or embodiment 133, wherein the buffer used in step (a) comprises triethanolamine buffer. Embodiment 135: A kit according to any of embodiments 131 to 134, wherein the buffer used in step (a) has a pH, which is +/-1 of the pi of the recombinant peptide.

Embodiment 136: A kit according to any of embodiments 120 to 135, wherein the multimodal exchange resin comprises a negatively charged multimodal exchanger.

Embodiment 137: A kit according to any of embodiments 120 to 136, wherein the multimodal exchanger is a CAPTO MMC™ multimodal exchanger.

Embodiment 138: A kit according to any of embodiments 120 to 137, which comprises a buffer for use in step (b) of a method according to any of embodiments 1 to 119.

Embodiment 139: A kit according to embodiment 138, wherein said buffer is suitable for lyophilization. Embodiment 140: A kit according to embodiment 138 or embodiment 139, wherein said buffer comprises constituents, which are capable of undergoing sublimation during lyophilization.

Embodiment 141 : A kit according to any of embodiments 138 to 140, wherein said buffer is an ammonium buffer. Embodiment 142: A kit according to any of embodiments 138 to 141 , wherein said buffer is ammonium bicarbonate.

Embodiment 143: A kit according to any of embodiments 138 to 142, wherein said buffer further comprises ingredients, which would make the solution pharmaceutically acceptable. Embodiment 144: A kit according to any of embodiments 138 to 143, wherein said buffer further comprises phosphate buffered saline or histidine, or further comprises histidine and a sugar.

Embodiment 145: A recombinant peptide obtained by a method defined in any of embodiments 1 to 1 19. Embodiment 146: A recombinant peptide obtainable by a method defined in any of embodiments 1 to 1 19.

Embodiment 147: A pharmaceutical formulation obtained by a method defined in any of embodiments 1 to 119. Embodiment 148: A pharmaceutical formulation obtainable by a method defined in any of embodiments 1 to 119.

All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.

The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law.

EXAMPLES

The invention will be further defined by reference to the following examples. It will be apparent to those skilled in the art that many modifications, both to the materials and methods may be practiced without departing from the scope of the invention.

Example 1

Washing prolactin inclusion bodies

The prolactin analogue Ser-PRL S33A Q73L G129R K190R (as described in WO2008/028684) was expressed in inclusion bodies (Lan et al. 46(2), 285-93 (2006)) which were then suspended in buffer (10 mM Na₂HPO₄, 0.05% Tween 20, 1 mM EDTA, 5 mM DTT, pH 9.0) and mixed roughly in order to wash them effectively. Mixing was carried out with an Ultra-Turax™ homogenator. The inclusion bodies were centrifuged at 4°C at 9000 rpm for 20 minutes. The washing in buffer and centrifugation were then repeated.

The washed inclusion bodies were then either frozen or used directly in Example 2. Example 2

Refolding prolactin Solubilisation:

The inclusion bodies were dissolved in 30 ml of buffer (8 M Urea, 0.1 M Tris, 20 mM DTT, pH 8.5) per gram of inclusion body. The solution was shaken carefully or stirred overnight at 4°C. After mixing, the sample was centrifuged at 4°C at 10000 rpm for 20 minutes followed by filtration with a 0.7-1.0 μm deep filter. Refolding:

5 μg PMSF was added to the solubilised prolactin per gram of inclusion body. The solution was diluted by a ratio of 4:1 with buffer (0.4 M Arginine, pH 8.5), which was added very slowly over approximately 15 minutes. After dilution, the sample was stirred for a minimum of 4 hours at 15°C.

The progression of refolding was checked using HPLC. Once folding was completed, the sample was filtered through a 0.7 to 1.0 μm deep filter. The filtered sample was further diluted by a ratio of 7:1 with buffer (20 mM Tris,

0.05% Tween 20, pH 8.0), which was added very slowly over approximately 30 minutes. The sample was then refiltered through a 0.7 to 1.0 μm deep filter.

Example 3

Anionic Ion Exchanger Anionic ion exchange was carried out using a Q Sepharose Fast Flow™. The flow rate was maintained between 30 to 60 cv/h. The column was equilibrated with 5 CV of 20 mM triethanolamine pH 8.5.

The pH of the sample prepared in Example 2 was adjusted to 8.5, and the conductivity was measured and adjusted to below 3 mS/cm. The sample was filtered through a 0.4 μm filter and applied to the column with a general load of 10 mg protein pr. ml resin.

The column was then washed with 10 CV of 20 mM triethanolamine pH 8.5 (buffer A). Some bound protein was eluted with 10 CV of a mix of 75% Buffer A and 25% 20 mM triethanolamine, 0.5 M NaCI (Buffer B), pH 8.5, and more bound protein was then eluted with

10 CV of a mix of 40% buffer A and 60% buffer B. The column was regenerated using 10 CV of 100% buffer B, followed by 5 CV of 1

M NaCI.

The chromatography system, tubing and column is washed for at least one hour in

10 CV of 1 M NaOH to remove any residual endotoxin in the system before and after use. Example 4

Multimodal Ion Exchanger

The multimodal ion exchange was carried out using a Capto MMC™. The column was equilibrated with 10 CV of 50 mM ammonium acetate, pH 6.5.

The pH in the sample obtained from the elution in Example 3 using the mix of 40% buffer A and 60% buffer B was adjusted to 6.5 by addition of 20 mM citrate from a 1 M sodium citrate pH 5.0, followed by the careful addition of dilute acetic acid (0.5 M). This sample was diluted with H₂O until the conductivity was below 6 mS/cm. The sample was filtered through a 0.4 to 1.0 μm filter (if necessary) and applied to the column.

Next, the column was washed with 10 CV of 50 mM ammonium acetate pH 6.5. Bound protein was eluted with 15 CV of 50 mM ammonium bicarbonate, pH 8.5. The column was regenerated with 15 CV of 50 mM ammonium bicarbonate, pH 8.5, and 1 M NaCI.

The fractions containing purified protein were then pooled, sterile filtered through a 0.2 μm filter, and then dispensed into freeze-drying vials for freeze-drying.

Example 5

Endotoxin level

Endotoxin levels of the eluates purified as described in the examples above were measured by use of the Endosafe-IPT test produced by Charles River Laboratories, Inc. Endotoxin level of the protein containing eluates from the described columns was measured and is presented below in Table 1.

Table 1

Column A is an 8 ml Q Sepharose FF™ packed in Tricorn 10/100 column

Column B is an 8 ml Capto Q™ packed in Tricorn 10/100 column Column C is a Capto MMC™ packed in a XK 50 column with a he a volume of 510 ml. Results are shown for two separate runs.

Claims

1. A method for purifying a peptide having a pi below 6.5, which peptide is recombinantly expressed in a host cell, wherein the recombinant peptide is produced in inclusion bodies, which purification method comprises the steps of: (i) recovering the peptide in a biologically active form from inclusion bodies produced in the recombinant expression,

(ii) if necessary, adjusting the conductivity of the solution comprising the recovered recombinant peptide to between 0 and 4 mS/cm,

2. A method for purifying a peptide having a pi below 6.5, which peptide is recombinantly expressed in a host cell, wherein the recombinant peptide is produced in soluble form, which purification method comprises the steps of:

(i) obtaining a solution comprising the peptide from the recombinant expression,

3. A method for preparing a pharmaceutical formulation of a peptide having a pi below 6.5, which peptide is recombinantly expressed in a host cell, wherein the recombinant peptide is produced in inclusion bodies, which method comprises the steps of:

4. A method for preparing a pharmaceutical formulation of a peptide having a pl below 6.5, which peptide is recombinantly expressed in a host cell, wherein the recombinant peptide is produced in soluble form, which method comprises the steps of: (i) obtaining a solution comprising the peptide from the recombinant expression,

5. A method according to claim 3 or claim 4, wherein the pharmaceutical formulation is a lyophilized pharmaceutical formulation, which is made by lyophilizing the eluate from step (b).

6. A method according to claim 3 or claim 4, wherein the pharmaceutical formulation is an aqueous pharmaceutical formulation, which is made by reconstitution of a lyophilized pharmaceutical formulation, which is made by lyophilizing the eluate from step (b).

7. A method according to claim 5 or claim 6, wherein one or more additional components of the pharmaceutical formulation are added to the eluate of step (b) before lyophilization thereof.

8. A method according to claim 3 or claim 4, wherein the eluate from step (b) is used as the pharmaceutical formulation.

9. A method according to claim 8, wherein additional components of the pharmaceutical formulation is added to the eluate of step (b) for finalizing the preparation of the pharmaceutical preparation.

10. A method according to any of claims 1 to 9, which comprises the use of a buffer in step (a).

11. A method according to claim 10, wherein the buffer used in step (a) has a pH, which is +/-1 of the pi of the recombinant peptide.

12. A method according to any of claims 1 to 1 1 , which comprises the use of a buffer in step (b).

13. A method according to claim 12, wherein the buffer in step (b) is a pharmaceutically acceptable buffer.

14. A method according to any of claims 1 to 13 , wherein the pH of the solution comprising the recovered recombinant peptide is adjusted between step (i) and step (a) to a pH above the isoelectric point (pi) of the peptide.

15. A method according to any of claims 1 to 14, wherein the pH of the eluate comprising the recombinant peptide from step (a) is adjusted to a pH below the isoelectric point of the peptide prior to step (b).

16. A method according to any of claims 1 to 15, wherein the conductivity of the eluate comprising the recombinant peptide is adjusted to between 0 to 8 mS/cm prior to step (b).

17. A method according to any of claims 1 to 16, which does not comprise any additional upconcentration steps.

18. A method according to any of claims 1 to 17, which does not comprise any additional buffer changing steps.

19. A method according to any of claims 1 to 18, wherein the level of endotoxin in the eluate from step (b) is acceptable for pharmaceutical use.

20. A recombinant peptide purification kit, which comprises an anionic exchange resin, a multimodal exchange resin and instructions to use said kit in accordance with a method according to any of claims 1 to 19.

21. A recombinant peptide obtainable by a method defined in any of claims 1 to 19.

22. A pharmaceutical formulation obtainable by a method defined in any of claims 1 to 19.