Invented by Shawn DeFrees, Kyle Kinealy, Novo Nordisk AS

The market for a method for purifying polypeptide conjugates containing polyalkylene oxide by hydrophobic interaction-chromatography is experiencing significant growth due to the increasing demand for high-quality and pure polypeptide conjugates in various industries, including pharmaceuticals, biotechnology, and research.

Polypeptide conjugates are molecules that consist of a polypeptide chain attached to a polyalkylene oxide (PAO) moiety. These conjugates have gained immense importance in the field of drug delivery systems, as they offer enhanced stability, improved solubility, and prolonged circulation time in the body. However, the purification of these conjugates is a challenging task due to the presence of impurities, such as unreacted polypeptides, by-products, and aggregates.

Hydrophobic interaction-chromatography (HIC) has emerged as a powerful technique for the purification of polypeptide conjugates containing PAO. HIC utilizes the hydrophobic interactions between the PAO moiety and the stationary phase to separate the conjugates from impurities. This technique offers several advantages, including high selectivity, scalability, and compatibility with a wide range of solvents and buffer conditions.

The increasing adoption of polypeptide conjugates in drug development has fueled the demand for efficient purification methods. The pharmaceutical industry, in particular, is witnessing a surge in the development of peptide-based therapeutics, which require stringent purification processes to meet regulatory standards. The use of HIC for purifying polypeptide conjugates containing PAO ensures the removal of impurities, resulting in highly pure and bioactive conjugates.

Furthermore, the biotechnology industry is also driving the market growth for this purification method. Polypeptide conjugates are extensively used in diagnostics, imaging agents, and targeted therapies. The ability to purify these conjugates efficiently and reliably is crucial for ensuring their efficacy and safety. HIC offers a robust and reproducible purification process, making it an ideal choice for large-scale production of polypeptide conjugates.

Research institutions and academic laboratories are also contributing to the market growth. Scientists and researchers are constantly exploring new applications for polypeptide conjugates, ranging from drug delivery systems to bioimaging agents. The availability of a reliable and efficient purification method like HIC enables them to obtain pure conjugates for their studies, accelerating the pace of research and development in this field.

In terms of geographical distribution, North America and Europe are the leading markets for the purification of polypeptide conjugates containing PAO by HIC. The presence of a well-established pharmaceutical and biotechnology industry, along with significant investments in research and development, has propelled the demand for this purification method in these regions. Asia-Pacific is also witnessing substantial growth, driven by the increasing focus on biopharmaceutical development and the presence of a large patient pool.

In conclusion, the market for a method for purifying polypeptide conjugates containing polyalkylene oxide by hydrophobic interaction-chromatography is experiencing rapid growth due to the rising demand for high-quality and pure polypeptide conjugates in various industries. The ability of HIC to efficiently remove impurities and provide highly pure conjugates is driving its adoption in pharmaceuticals, biotechnology, and research. With advancements in technology and increasing investments in R&D, this market is expected to witness further expansion in the coming years.

The Novo Nordisk AS invention works as follows

The present invention provides methods for manufacturing polypeptide conjugates. The invention, in particular, provides methods for purifying polypeptide conjugates that contain at least one polymeric modification group, such as poly(alkylene dioxide) moiety. Poly(ethylene glycol), poly(propylene glycol) and poly(ethylene oxide) are exemplary poly(alkylene oxid) moieties. Hydrophobic interaction (HIC), a chromatographic technique, is used in an exemplary procedure to separate the glycoforms of glycoPEGylated Polypeptides.

Background for A method for purifying polypeptide conjugates containing polyalkylene oxide by hydrophobic interaction-chromatography

The current literature is a goldmine of information on polypeptide purification methods, which are primarily chromatographic and membrane filtration approaches. There are no effective methods to purify modified polypeptides, such as PEGylated Polypeptides. Modification of polypeptides by polymeric moieties results in a major change in their chemical and physical properties. “Methods that are effective for purifying non-modified versions of polypeptides may not be as efficient in capturing the modified versions.

When a polypeptide, whether glycosylated (or non-glycosylated), is subjected a chemical reaction for modification, it may produce side products in addition to that desired modified polypeptide. To isolate the desired product from a mixture of reaction products, it is important that the process can remove unwanted side-products as well as chemical reagents. It is particularly important when a polypeptide will be used to treat a disease. The use of polypeptide modification techniques that rely on enzyme specificity can result in reaction products with improved homogeneity compared to chemical methods. Expression of a recombinant peptide by a cell, such as a mammalian, insect or bacterial cell, results in polypeptides that are characterized, at least to some extent, by different glycans. The polypeptide is then modified, for example, by using those glycans. This results in a heterogeneous product. While remodeling glycans prior to chemical or enzyme modification of polypeptides can improve product quality, there is still a degree of heterogeneity. There is a need for production processes that can isolate the desired polypeptide from a reaction mix, which may include chemical reagents derived from unreacted modification groups, catalytic enzymes and/or polypeptide by-products. This invention addresses both of these needs.

The present invention provides methods for isolating (e.g. large-scale purification of) polypeptide conjugates. Polypeptide conjugates according to the present invention comprise a modified polypeptide, such as polymers. Water-soluble polymers are exemplary polymers. The invention is particularly useful in the isolation of polypeptides conjugates containing poly(alkylene dioxide)-based polymers such as poly(ethylene and propylene glycol). Although reverse-phase (RP), chromatography is a technique that can be used to purify the polypeptides after they have been derivatized by highly polar and water-soluble polymers such as poly(ethylene glycol) or poly(propylene glycol), it’s not recommended because the method requires water-soluble solvents such as acetonitrile. Organic solvents are associated with both environmental issues and can affect the chemical stability in the polypeptide conjugate. Process steps that use aqueous solution are therefore generally preferred. In one embodiment, this invention offers methods for separating polypeptide conjugates without using organic solvents.

An example method of the invention comprises at least one chromatographic process that is effective for separating polypeptides conjugates with at least one poly (alkylene oxide ) moiety from components of a mix. These methods can be used to separate polypeptide conjugates from any mixture. In one example, a mixture can be a reaction mix (e.g. the product of an ezymatically-catalyzed reaction such as glycoPEGylation) and optionally contain other polypeptides conjugates. Hydrophobic interaction media (HICs) are used in preferred methods. In one embodiment, HIC media are used with at least one additional step of chromatography selected from anion-exchange chromatography (ACE), mixed-mode chromatography (MCC), cation exchanged chromatography (CEC) and hydroxyapatite/fluoroapatite/fluoroapatite/fluoroapatite/hydroxyapatite/fluoroapatite/fluoroapatite/fluoroapatit In another embodiment, HIC can be used with at least one anion exchange, mixed-mode, or cation exchanging chromatography. The inventors discovered that HIC combined with cation-chromatography is an effective method for the separation of polypeptides conjugates containing at least one poly (alkylene oxide moiety). It was found that HIC followed by cation exchanging can separate EPO PEG2 from EPO PEG3 species. HIC, in combination with cation-exchange, provided a composition consisting of purified [PEG(10kDa),]3 that had a very small residual concentration of EPO[PEG (10kDa),]2.

FIG. “An example of a method that incorporates anion exchange, cation-exchange chromatography and HIC in addition to HIC” 1. In one embodiment, methods of the invention can be used to separate different glycoforms from a polypeptide-conjugate, especially those glycoforms that differ by the number poly(alkylene dioxide) moieties linked to the polypeptide. Under the same conditions that are used to create the polypeptide conjugate, unwanted glycoforms can be produced as by-products.

The invention, therefore, provides, in its first aspect, a method for making a composition containing a polypeptide-alkylene-oxide conjugate. This first polypeptide-alkyleneoxide conjugate has a number of poly(alkyleneoxide) moieties that are covalently attached to the polypeptide. The method comprises: (a), contacting a mix containing the polypeptide with a hydrophobic interactions chromatography medium (HIC); and (b), eluting a first polypeptide from the HIC media. In a first example, the mixture contains a second conjugate of polypeptides, which has a different number of poly(alkylene oxygen) moieties that are covalently attached to the second peptide. The first polypeptide has 3 poly(alkylene oxygen) moieties. However, the second polypeptide can have 0, 1, 2, or 4 poly (alkylene dioxide) moieties. “In one example, poly(alkylene glycol) is used as the poly(alkylene).

The invention also provides a method for isolating from a polypeptide-polypeptide conjugate containing a poly(alkyleneoxide) moiety covalently attached to a polypeptide a polypeptide-polypeptide conjugate containing a poly(alkyleneoxide) moiety covalently coupled to a polypeptide. In this second aspect, the first and second numbers are different. The method comprises: (a), contacting a mix containing the second polypeptide and the first conjugate with an hydrophobic interactions chromatography (HIC), and (b), eluting said first conjugate. In one example, according to this aspect the first polypeptide includes 3 poly(alkylene dioxide) moieties while the second polypeptide includes 0, 1, 2, 5, 6, or 7 poly (alkylene oxygen) moieties.

In one example, according to the embodiments above, both the first and second polypeptides have the same sequence of amino acids. In another example, according to any above embodiments both the first polypeptide and the second are EPO.

The method includes: (a) contacting a mixture containing the first EPO-conjugate with an anion exchange medium; (b) eluting the first EPO-conjugate from the anion exchange medium, forming a first Eluate including the first EPO-conjugate; and, (c) contacting the first eluate with a hydrophobic interaction chromatography media. The method comprises: (a), contacting a composition containing a first EPO-conjugate with an anion-exchange medium; (b), eluting a first EPO-conjugate from the anion-exchange medium to form a first Eluate; (c), contacting the resulting first eluate against a hydrophobic interactions chromatography (HIC); and (d), eluting a first EPO conjucate from the HIC medium. After step d, the method can include: “(e) eluting first EPO conjugate using a cation-exchange chromatography medium.

In one embodiment of the method, it further comprises forming the polypeptide-conjugate either chemically or by enzymatically catalyzed glymodification (e.g. glycoPEGylation with a glycosyltransferase using an appropriate glycosyl donation molecule such as a sugar nucleotide modified). GlycoPEGylation is well-known in the art; for example, see WO 03/031464 by DeFrees and others. Or WO 04/99231, whose disclosures are incorporated by reference herein in their entirety.

The invention also provides compositions that are made using the methods of invention, as well as pharmaceutical formulas including the composition. The invention also provides treatment methods using the compositions.

The detailed description of the invention that follows will reveal other objects and benefits of the invention to those with the necessary skills.


FIG. The figure 1 shows an overview of a polypeptide conjugate separation process according to the invention. The diafiltration/ultrafiltration step following hydrophobic interaction chromatography (HIC) is optional.


FIG. The elution peak of FIG. 2A. The fractions (E),(F) and G) are indicated by the letters. (F), for example, indicates that EPO-SA-PEG-10kDa3 was eluted.

FIG. 3A shows a scheme for an exemplary EPO conjugate having a glycosylation pattern specific to insects. It includes three monoantennary N-linked glycans that are covalently attached to the amino acid residues of N24, N38, and N83. Each glycan is covalently attached to a PEG 10 kDa moiety via the terminal galactose moiety. FIG. 3A includes an example reaction scheme that can be used for the synthesis of EPO conjugate. The substrate of the enzyme-catalyzed transformations is an EPO peptide that contains at least one trimannosyl glycan. In the first step, a N-acetylglucosamine Transferase (GnT-1), which adds only one terminal mannose moiety with a GlcNAc, is used. In a second step, the galactosyl-transferase (GalT-1), which forms a terminal-GlcNAc -Gal molecule, links a Gal moiety to the newly-added GlcNAc molecule. First and second steps can be performed in the same vessel. The third step involves linking a modified sialic moiety with a PEG to the terminal gal moiety by using a ST3Gal3 sialyltransferase.

FIG. The figure 3B shows an example composition of the invention, which includes different glycoforms for an exemplary polypeptide (e.g. EPO conjugate). Each glycoform can be distinguished from the others by the number or structure of PEG moiety covalently attached to the polypeptide. The percentage values shown are examples.

FIG. The chromatogram 4B shows a reverse-phase (RP)HPLC of an exemplary invention composition containing EPO-(SAPEG-10 kDa), the main component. This composition was obtained by a method according to the invention. The numbered peaks represent: (1) tri-PEG-EPO=EPO-(SA-PEG-10 kDa)3 and (2) di-PEG-EPO-EPO-(SA-PEG-10 kDa)2.

FIG. 5 is a schematic representation of exemplary glycopegylated EPO isoforms isolated from Chinese Hamster Ovary cells. A. An exemplary 40 kilodaton O-linked pegylated glycoform. B: One of several 30 kilodalton N-linked pegylated glycoforms. The modified sialic acid moiety comprising the PEG molecule may occur on any one or more of any of the branches of the N-linked glycosyl residue. Furthermore the illustration is exemplary in that any glycosylated EPO molecule may comprise any mixture of mono-, bi- tri-, or tetra-antennary N-linked glycosyl residues and any one or more of the branches may further comprise a modified sialic acid moiety.

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