Patent Description:
On average over the past decade, <NUM> new peptides have entered clinical trials every year with approval rates (from phase <NUM> to marketing) of twice that of small molecules (<NPL>; <NPL>), and more than <NUM> peptide drug candidates are currently in development (<NPL>).

The development of peptide drugs presents specific challenges, however. For example, peptide drugs are subject to proteolytic cleavage and are cleared quickly in vivo. Oral delivery remains challenging, in part due to low permeability of biological membranes. Attempts to remedy these shortcomings have been made by incorporating conformational constraints such as cyclic peptide formation (e.g., by lactamizations and click-cyclizations), hydrocarbon stapling, lipidation, and the development of polymer drug conjugates.

One example of a peptide drug is SAR425899, a dual receptor agonist that targets the glucagon and GLP1 receptors and can facilitate glycemic control and weight loss in type <NUM> diabetes mellitus patients, as well as weight loss in healthy volunteers (<NPL>). However, due to rapid in vivo clearance, a once daily dose of SAR425899 is required.

Hyaluronic acid (HA), also referred to as hyaluronan, is a naturally occurring non-sulfate linear polysaccharide composed of repeating disaccharide units of d-glucuronic acid and N-acetyl-d-glucosamine linked by β-<NUM>-<NUM> and β-<NUM>-<NUM> glycosidic bonds (<NPL>)). HA is an important structural element in the skin and participates in a number of cell surface receptor interactions. HA has immunosuppressive and antiangiogenic activity, and is present in brain tissue, hyaline cartilage, and synovial joint fluid. Due to its strong hydrophilic character and its high molecular weight in biological tissues that can absorb a large amount of water, up to <NUM> times its solid volume, HA exhibits important structural and functional roles in the body.

Because of its biocompatibility and biodegradability, HA has found numerous applications in biomedical and pharmaceutical applications. However, HA is highly soluble and often exhibits very poor mechanical properties with rapid degradation behavior in vivo. Thus, HA has been chemically modified and/or crosslinked to improve its properties, including mechanical, viscosity, solubility, degradation, and biologic. HA derivatives have been created and utilized in scaffolds for tissue engineering, in soft tissue surgery such as vocal fold augmentation, drug delivery, intracellular delivery of siRNA, wound healing, and as a device in surgical procedures.

Crosslinked hyaluronic acid (xHA) forms a multi-million Dalton hydrogel in an aqueous environment, and can be linked to a drug (e.g., a peptide drug) to stabilize the drug in vivo. However, it is difficult to both quantify the amount of drug loaded in the hydrogel, and to quantify the release rate of the drug from the hydrogel. Simple UV measurements typically applied for basic quantification of peptides in solution cannot be applied for hydrogels, as such analyses require that the drug linked hydrogel complex be fully dissolved.

Alternative methods for accelerated hydrolysis of linker and release of a peptide drug using elevated temperature or elevated pH lead to partial degradation of the peptide drug, thus producing a heterogeneous mixture that is challenging to quantify. Accordingly, analytical quantification methods are needed to quantify drug loading of prodrug compositions on crosslinked HA hydrogels.

<NPL>) developed a long-acting hyaluronic acid-interferon alpha (HA-IFNα) conjugate for the treatment of HCV infection. <NPL>) designed pH-sensitive coiled-coil peptide-cross-linked hyaluronic acid nanogels (HA-cNGs) and tested them for targeted intracellular protein delivery to CD44 overexpressing MCF-<NUM> breast cancer cells. <NPL>) developed a hyaluronic acid (HA) hydrogel networks by Michael addition between aminoethyl methacrylated HA and thiolated HA, which enabled the controlled release of human growth hormone (hGH).

The present invention is defined in the appended set of claims. To address the analytical challenges of determining drug loading on a crosslinked HA (xHA) hydrogel, e.g., to enable peptide load determination of an xHA hydrogel, novel methods were discovered that permit accurate quantification of drug load (e.g., biopolymeric peptide prodrug load) measured as weight / weight percentage. The methods described herein are highly selective for drug load determination that can be applied to any small molecule (e.g., a polypeptide, a polynucleotide or the like) which can be broken down into smaller components (e.g., by proteolytic digestion, hydrolysis or the like), and can broadly be applied for drug load determination of prodrugs, e.g., biopolymer-containing prodrugs. The methods described herein are useful, for example, for controlling dose delivery of the drug in vivo, and for quality control of drug load among prodrug batches during manufacturing. The methods described herein enable the production of drugs, e.g., peptide drugs, that have increased half-lives in vivo, e.g., drugs such as peptide drugs having half-lives that are increased from minutes to days.

The methods described herein provide a variety of advantages over previous methods known in the art, such as NMR-based methods. For example, NMR-based methods take longer and have lower throughput than the methods described herein. NMR-based methods require milligrams of material, whereas the novel methods described herein require only nanograms of material. Furthermore, the dynamic range of NMR-based methods is lower than that of the novel methods described herein. In addition, NMR-based methods are more susceptible to interference by contaminating peptides than the novel methods described herein.

Novel methods are provided herein based on the discovery that a double enzymatic digestion followed by quantification of digestion product(s) can be used to accurately quantify drug load (e.g., peptide drug load) on a hydrogel. In certain embodiments of the method, a crosslinked hyaluronic acid-linker-peptide (xHA-L-P) is incubated with an enzyme to digest the crosslinked hyaluronic acid. Subsequently, a proteolytic enzyme is applied to digest the attached peptide and produce proteolytic digest products. The proteolytic digest products, which represent the peptide, are subsequently quantified.

In certain exemplary embodiments of the method, a crosslinked hyaluronic acid linker peptide conjugate (xHA-L-P) is weighed and suspended as a hydrogel in buffer. The hydrogel is enzymatically degraded to oligomeric hyaluronic acid-linker-peptide (oHA-L-P), which is soluble. The resulting oligomers of oHA-L-P are present as a heterogeneous mixture, consisting of peptide, linker and oligomeric hyaluronic acids of different lengths. This oligomeric hyaluronic acid heterogeneity is undesirable for mass spectrometry based quantification but can be applied for a less specific UV or fluorescence based assay. Accordingly, for a mass spectrometry-based reference assay, a second endoproteolytic digest step is introduced to digest the peptide drug and produce a homogeneous peptide digest product (e.g., the <NUM>-amino acid C-terminal peptide digest product DFIEWLKAGGPSSGAPPPS-NH2 (<FIG>)). The peptide product can then be detected and quantified using assays such as, e.g., liquid chromatography / high resolution mass spectrometry (LC/MS).

The present invention provides a method for determining the amount of drug present in a crosslinked hyaluronic acid-linker-peptide (xHA-L-P) prodrug formulation is provided. The method includes the steps of contacting a sample of the xHA-L-P prodrug formulation with a hyaluronoglucosidase, e.g., a hyaluronidase (HAase) or a hyaluronate (HA) lyase, to generate oligomeric hyaluronic acid-linker-peptide drug (oHA-L-P), contacting the oHA-L-P with an endoproteinase to generate peptide digest products of the drug, and detecting the peptide digest products to determine the amount of the drug present in the xHA-L-P prodrug formulation, wherein the endoproteinase is selected from the group consisting of Glu-C, Asp-N, Lys-C, Arg-C, trypsin and chymotrypsin.

In certain exemplary embodiments, the peptide digest products are between about <NUM> amino acids and about <NUM> amino acids in length, between about <NUM> amino acids and about <NUM> amino acids in length, are between about <NUM> amino acids and about <NUM> amino acids in length, are between about <NUM> amino acids and about <NUM> amino acids in length, are between about <NUM> amino acids and about <NUM> amino acids in length, or are about <NUM> amino acids in length. In certain exemplary embodiments, the peptide digest products are about <NUM>, about <NUM> or about <NUM> amino acids in length.

In certain exemplary embodiments, the step of detecting the peptide digest products is performed by a method selected from the group consisting of one or a combination of liquid chromatography-mass spectrometry (LC-MS), liquid chromatography tandem mass spectrometry (LC-MS-MS), liquid chromatography-high resolution mass spectrometry (LC-HRMS), ultraviolet (UV) absorbance and fluorescence detection.

In certain exemplary embodiments, the hyaluronoglucosidase is an HAase selected from the group consisting of HAase <NUM>, HAase <NUM>, HAase <NUM>, HAase <NUM>, HAase <NUM> and HAase <NUM>. In other exemplary embodiments, the hyaluronoglucosidase is HAase <NUM> or HAase <NUM>. In still other exemplary embodiments, the hyaluronoglucosidase is HAase <NUM>. In certain exemplary embodiments, the hyaluronoglucosidase is HA lyase EC <NUM>.

In certain exemplary embodiments, the endoproteinase is Asp-N.

In certain exemplary embodiments, the method further comprises the use of an internal standard. In certain exemplary embodiments, the internal standard comprises one or more heavy isotopes.

In certain exemplary embodiments, the amount of drug present is determined using a calibration curve.

In certain exemplary embodiments, the xHA-L-P is contacted with the hyaluronoglucosidase in a pressure cycler. In certain exemplary embodiments, pressure in the pressure cycler is greater than atmospheric pressure. In certain exemplary embodiments, the pressure is about <NUM> KPSI, about <NUM> KPSI or about <NUM> KPSI.

In certain exemplary embodiments, the oHA-L-P is contacted with the second enzyme in a pressure cycler. In certain exemplary embodiments, pressure in the pressure cycler is greater than atmospheric pressure. In certain exemplary embodiments, the pressure is about <NUM> KPSI, about <NUM> KPSI or about <NUM> KPSI.

The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings.

The present invention is based on the development of a novel quantification method for determining the amount of drug loaded on a polymer. According to the present invention, the method comprises cleaving hyaluronic acids (HAs) present in a sample of crosslinked hyaluronic acid-linker-peptide drug (xHA-L-P) to form a heterogeneous mixture of oligomeric hyaluronic acid-linker-peptide drug (oHA-L-P). The oHA-L-P is then be proteolytically digested to yield a homogenous peptide digest product that can be detected and quantified using well-known methods in the art such as, e.g., liquid chromatography, mass spectrometry, UV absorbance, fluorescence-based assays and the like. Also disclosed but not forming part of the invention are compositions comprising xHA-L-P (e.g., the SAR425899 prodrug) in which "P" is the peptide drug SAR425899, "L" is a self-cleaving crosslinker, and "xHA" is crosslinked hyaluronic acid (<FIG>).

As used herein, the term "prodrug" is intended to refer to a compound that undergoes biotransformation before exhibiting its pharmacological effects. Prodrugs can thus be viewed as biologically active moieties containing specialized non-toxic protective groups used in a transient manner to alter or to eliminate undesirable properties in the parent molecule. Typical prodrugs may be a carrier-linked prodrug that contains a temporary linkage of a given active substance with a transient carrier group that produces improved physicochemical or pharmacokinetic properties and that can be easily removed in vivo, usually by a hydrolytic cleavage; a cascade prodrug for which the cleavage of the carrier group becomes effective only after unmasking an activating group.

To enhance physicochemical or pharmacokinetic properties of a drug such as SAR425899 in vivo, such drug can be conjugated with a carrier (e.g., xHA). If the drug is transiently bound to a carrier and/or a linker, such systems are commonly assigned as carrier-linked prodrugs. According to the definitions provided by IUPAC, a carrier-linked prodrug is a prodrug that contains a temporary linkage of a given active substance with a transient carrier group that produces improved physicochemical or pharmacokinetic properties and that can be easily removed in vivo, usually by a hydrolytic cleavage.

The linkers employed in carrier-linked prodrugs described herein can be any known cleavable linker or non-cleavable linker, e.g., enzyme labile linkers (e.g., acid-cleavable linkers, reducible linkers (e.g., disulfide linkers), β-glucuronide linkers, and the like), photo-cleavable linkers, thioether linkers, maleimidocaproyl linkers, peptide linkers (e.g., dipeptide linkers), crosslinkers and the like. Suitable linkers are commercially available from a variety of vendors such as, e.g., Sigma-Aldrich, Millipore Sigma, Creative Biolabs and the like.

The linkers employed in carrier-linked prodrugs described herein can be transient (e.g., self-cleavable), meaning that they are non-enzymatically, hydrolytically degradable (cleavable) under physiological conditions with half-lives ranging from, for example, one hour to three months.

As used herein, "physiological conditions" refer to the temperature, pH and the like with respect to a particular environment of the body where a drug is intended to be released. For example, release in the plasma would occur at a pH of between <NUM> and <NUM> at a temperature of about <NUM> (e.g., between about <NUM> and about <NUM>), and release in a lysosome would occur at a pH of between about <NUM> to about <NUM> at a temperature of about <NUM>.

A drug (e.g., a peptide drug) can be transiently linked to a hydrogel carrier via a self-cleaving linker, e.g., an xHA hydrogel carrier. The terms "hydrogel prodrug" and "hydrogel-linked prodrug" refer to prodrugs of biologically active agents transiently linked to a hydrogel and are used synonymously.

The terms "drug," "peptide drug," "biologically active molecule," "biologically active moiety," "biologically active agent," "active agent" and the like, refer to any substance which can affect any physical or biochemical properties of a biological organism, including but not limited to viruses, bacteria, fungi, plants, animals, and humans. In particular, as used herein, biologically active molecules include any substance intended for diagnosis, cure, mitigation, treatment, or prevention of disease in humans or other animals, or to otherwise enhance physical or mental well-being of humans or animals. In certain exemplary embodiments, the terms "drug," "biologically active molecule," "biologically active moiety," "biologically active agent," "active agent," and the like refer to a peptide drug such as, e.g., SAR425899.

A "free form" of a drug refers to a drug (e.g., SAR425899) in its unmodified, pharmacologically active form, such as after being released from a hydrogel conjugate prodrug (e.g., SAR425899 prodrug).

As used herein, the phrase "anti-cancer therapeutic agent" or "anti-cancer agent" refers to a molecule which is detrimental to the growth and/or proliferation of neoplastic or tumor or cancer cells and may act to reduce, inhibit or destroy malignancy.

As used herein, the term "cytostatic" refers to a molecule that inhibits cell growth and multiplication.

As used herein, the phrase "cytotoxic nucleoside" refers to a nucleobase or nucleoside analogue that exerts cytotoxic effects by mimicking endogenous nucleosides.

As used herein, the phrase "tubulin binding agent" refers to a molecule that associates directly with the tubulin system.

As used herein, the term "hormone" refers to any member of a class of signaling molecules that are produced by glands in multicellular organisms and that are transported by the circulatory system to target distant organs to regulate physiology and/or behavior. A "hormone antagonist" is a specific type of receptor antagonist which acts upon hormone receptors.

As used herein, the phrase "anti-angiogenesis agent" refers to a molecule that inhibits the physiological process of angiogenesis, through which new blood vessels form from pre-existing vessels.

As used herein, the phrase "enzyme inhibitor" refers to a molecule that inhibits the function of a particular enzyme.

As used herein, the phrase "gene regulator" refers to a molecule that can positively or negatively influence the transcription of a gene.

As used herein, the phrase "cytotoxic therapeutic agent" refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term "cytotoxic agent" is intended to include chemotherapeutic agents, enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, and the various antitumor or anti-cancer agents disclosed below. In some cases, the cytotoxic agent is a taxoid, a vincas, a maytansinoid or maytansinoid analog such as DM1 or DM4, a small drug, a leptomycin derivative, an auristatin or dolastatin analog, a prodrug, topoisomerase II inhibitors, a DNA alkylating agent, an anti-tubulin agent, a CC-<NUM> or CC-<NUM> analog.

As used herein, the phrase "pharmaceutically-acceptable carrier" refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, and the like that are physiologically compatible. Examples of suitable carriers, diluents and/or excipients include one or more of water, amino acids, saline, phosphate buffered saline, buffer phosphate, acetate, citrate, succinate; amino acids and derivates such as histidine, arginine, glycine, proline, glycylglycine; inorganic salts NaCl, calcium chloride; sugars or polyalcohols such as dextrose, glycerol, ethanol, sucrose, trehalose, mannitol; surfactants such as Polysorbate <NUM>, polysorbate <NUM>, poloxamer <NUM>; and the like, as well as combination thereof. In many cases, it will be suitable to include isotonic agents, such as sugars, polyalcohols, or sodium chloride in the composition, and formulation may also contain an antioxidant such as tryptamine and a stabilizing agent such as Tween <NUM>.

As used herein, the terms "hyaluronic acid," "HA" and "hyaluronan" are used interchangeably, and refer to a non-sulfate linear polysaccharide composed of repeating disaccharide units of d-glucuronic acid and N-acetyl-d-glucosamine linked by β-<NUM>-<NUM> and β-<NUM>-<NUM> glycosidic bonds (e.g., →<NUM>)-β-d-GlcpA-(<NUM>→<NUM>)-β-d-GlcpNAc-(<NUM>→). HA occurs with different molecular weights. High molecular weight HA (HMWHA) is greater than about <NUM> × <NUM><NUM> Da, and low molecular weight HA (LMWHA) is about <NUM> to about <NUM> × <NUM><NUM> Da. Oligomeric HA is typically less than about <NUM> × <NUM><NUM> Da.

HA is commercially available from vendors such as Sigma-Aldrich (St. Louis, MO), Novozymes (Blair, NE) and Stanford Chemicals (Lake Forest, CA). FDA-approved HA includes, but is not limited to, Hyalovet (Boehringer Ingelheim Vetmedica, approved for veterinary use), Hylira (Hawthorn), Hylase (ECR), Hylartin V (Zoetis, approved for veterinary use), Hyvisc (Anika Therapeutics, approved for veterinary use), Legend (Bayer Animal Health, approved for veterinary use), NexHA (Vetoquinol, approved for veterinary use), Orthovisc (DePuy Mitek), ProVisc (Alcon), Shellgell (Cytosol Opthalmics), Solesta (Salix), Supartz (Bioventus), Synacid (Intervet, approved for veterinary use), Healon5 (Abbott Medical Optics), Healon GV (Abbott), Healon Endocoat (Abbott Medical Optics), Healon (Abbott), Euflexxa (Ferring Pharmaceuticals), Equron (Zoetis, approved for veterinary use), Coease (Abbott Medical Optics), Bionect (Cipher), Amvisc (Chiron), Synvisc (Genzyme), Gel-One (Zimmer Biomet), and Hyaglan (Fidia Pharma).

To improve the mechanical properties and prolong the duration of HA in vivo, a hydrogel can be formed by covalently crosslinking HA polymer chains into a three-dimensional network (see e.g. <NPL>; <NPL>). Mechanical and physical properties of crosslinked HA (xHA) hydrogels are dependent on the degrees of modification and crosslinking (<NPL>;).

As used herein, the terms "crosslinking agent" and "crosslinker" are intended to cover a chemical agent that could react with hyaluronic acid through at least one of covalent and/or non-covalent bonds. Non-limiting examples of non-covalent bonds include ionic bonds, hydrophobic interactions, hydrogen bonds and van der Waals forces (dispersion attractions, dipole-dipole and dipole-induced interactions).

The term "crosslinked" as used herein is intended to refer to two or more polymer chains of hyaluronic acid which have been covalently and/or non-covalently bonded via a crosslinking agent. Such crosslinking is differentiated from intermolecular or intramolecular dehydration which results in lactone, anhydride, or ester formation within a single polymer chain or between two or more chains. Although, it is contemplated that intramolecular crosslinking may also occur in the compositions as described herein. Crosslinking agents contain at least two functional groups that create covalent and/or non-covalent bonds between two or more molecules (i.e. hyaluronic acid chains). The crosslinking agents may comprise complimentary functional groups to that of hyaluronic acid such that the crosslinking can proceed.

Physical crosslinking of HA can be accomplished using a variety of pH, temperature, ionic strength conditions, and physicochemical interactions, for example, hydrophobic interactions, hydrogen bonding, charge interaction, or stereocomplexation. In particular, temperature-responsive hydrogels have been extensively examined for various applications. Common thermo-gelling polymers that are frequently used to modify HA to prepare thermally sensitive HA hydrogels include poly(N-isopropylacrylamide) (PNIPAM), pluronic acid, methylcellulose, and polyethylene glycol (PEG).

Methods of crosslinking HA to form hydrogels include, but are not limited to, methods that modify -COOH groups, methods that modify -OH groups, methods that modify -NHCOCH<NUM> groups, and chemical crosslinking methods using Schiff-base crosslinking, dialdehyde hyaluronic acid (CHO-HA), thiol modifications, Diels-Alder reactions, and enzyme-mediated linking.

Methods of crosslinking HA hydrogels are described in <NPL>; and Khunmanee, Supra; <NPL>; <NPL>; <NPL>; and <NPL>.

Crosslinked HA is commercially available from vendors such as Sigma-Aldrich (St. Louis, MO) and Stanford Chemicals (Lake Forest, CA).

As used herein the term "hydrogel" is intended to refer to a three-dimensional, hydrophilic or amphiphilic polymeric network capable of taking up large quantities of water. The networks are composed of homopolymers or copolymers, are insoluble due to the presence of covalent chemical or physical (ionic, hydrophobic interactions, entanglements) crosslinks. The crosslinks provide the network structure and physical integrity. Hydrogels exhibit a thermodynamic compatibility with water which allows them to swell in aqueous media. The chains of the network are connected in such a fashion that pores exist and that a substantial fraction of these pores are of dimensions between <NUM> and <NUM>.

The methods described herein utilize one or more hyaluronoglucosidases, e.g., hyaluronidases (HAases) to cleave xHA, e.g., to cleave xHA present in xHA-L-P to form oHA-L-P. In certain exemplary embodiments, a combination of two, three, four, five or more HAases are used to cleave xHA. In other embodiments, a single HAase is used to cleave xHA.

As used herein, "hyaluronidase" refers to a hyaluronoglucosidase that cleaves the (<NUM>-><NUM>)-linkages (EC <NUM>. <NUM>) or (<NUM>-><NUM>)-linkages (EC <NUM>. <NUM>) between N-acetylglucosamine and glucuronate in order to catalyze the degradation of HA.

There are three general classes of hyaluronidases: <NUM>. Mammalian-type hyaluronidases, (EC <NUM>. <NUM>) which are endo-beta-N-acetylhexosaminidases with tetrasaccharides and hexasaccharides as the major end products. They have both hydrolytic and trans glycosidase activities, and can degrade hyaluronan and chondroitin sulfates (CS), specifically C4-S and C6-S; <NUM>. Bacterial hyaluronidases (EC <NUM>. <NUM>) degrade hyaluronan and, and to various extents, CS and DS. They are endo-beta-N-acetylhexosaminidases that operate by a beta elimination reaction that yields primarily disaccharide end products; and <NUM>. Hyaluronidases (EC <NUM>. <NUM>) from leeches, other parasites, and crustaceans are endo-beta-glucuronidases that generate tetrasaccharide and hexasaccharide end products through hydrolysis of the beta <NUM>-<NUM> linkage.

Mammalian hyaluronidases can be further divided into two groups: neutral active and acid active enzymes. There are six hyaluronidase-like genes in the human genome, HYAL1, HYAL2, HYAL3 HYAL4, HYALP1 and PH20/SPAM1. HYALP1 is a pseudogene, and HYAL3 has not been shown to possess enzyme activity toward any known substrates. HYAL4 is a chondroitinase and lacks activity towards hyaluronan. HYAL1 (also known as LUCA1, MPS9 and NAT6) is the prototypical acid-active enzyme and PH20 is the prototypical neutral-active enzyme. Acid active hyaluronidases, such as HYAL1 and HYAL2 lack catalytic activity at neutral pH. For example, HYAL1 has no catalytic activity in vitro over pH <NUM> (<NPL>). HYAL2 is an acid active enzyme with a very low specific activity in vitro.

HYAL5 is an HAase originally discovered in mice that is located on the plasma and acrosomal membranes of acrosome-intact sperm that is released during the acrosome reaction. HYAL6 is an HAase that was also discovered in mice.

Chemical grade HAases are commercially available from vendors such as Sigma-Aldrich (St. Louis, MO), Millipore Sigma (Burlington, MA), and Calzyme Laboratories (San Luis Obispo, CA). FDA-approved HAases include, but are not limited to, Amphadase (bovine hyaluronidase; new drug application (NDA) No. <NUM>; Amphastar Pharmaceuticals), Hydase (bovine hyaluronidase; NDA No. <NUM>; Akorn Inc. ), Hylenex (recombinant human hyaluronidase; NDA No. <NUM>; Halozyme); Vitrase (ovine hyaluronidase; NDA No. <NUM>; Bausch and Lomb), and Wydase (bovine hyaluronidase; NDA No. <NUM>; Baxter Healthcare).

Additional suitable HAases for use herein are described in: worldwide web site: brenda-enzymes. org/enzyme. php?ecno=<NUM>. <NUM>;<NPL>); <NPL>; <NPL>; <NPL>; <NPL>; <NPL>; <NPL>".

"Hyaluronidase lyase," "hyaluronate lyase," "HA lyase," "EC <NUM>. <NUM>" or "HA lyase <NUM>. <NUM>" refers to a hyaluronoglucosidase (i.e., a bacterial carbon-oxygen lyase) that cleaves hyaluronan chains at a beta-D-GalNAc-(<NUM>-><NUM>)-beta-D-GlcA bond, ultimately digesting the polysaccharide to <NUM>-(<NUM>-deoxy-beta-D-gluc-<NUM>-enuronosyl)-N-acetyl-D-glucosamine. Hyaluronate lyases can be isolated from bacteria and Streptomyces, and differ from hyaluronidases from other sources by their mode of action, as they catalyze an elimination reaction, rather than hydrolysis, of the beta <NUM>,<NUM>-glycosidic linkage between N-acetyl-beta-D-glucosamine and D-glucuronic acid residues.

The method of the invention utilizes one or more endoproteinases selected from the group consisting of Glu-C, Asp-N, Lys-C, Arg-C, trypsin and chymotrypsin to cleave a peptide drug present as a free drug or a prodrug, e.g., as free SAR425899 or as SAR425899 prodrug, in order to generate a peptide digest product of the drug or prodrug. Criteria used to choose an appropriate endoproteinase was as follows: <NUM>) to generate as few highly-specific fragments as possible; <NUM>) to generate a C-terminal fragment that does not contain glutamate-palmitate linked to lysine; and <NUM>) to use common, commercially available enzymes.

In certain exemplary embodiments, endoproteinase Asp-N is used in the methods described herein. Endoproteinases are commercially available from companies such as Sigma-Aldrich (St. Louis, MO), New England Biolabs (Ipswich, MA), Thermo Scientific (Lenexa, KS), and Promega (Fitchburg, WI).

In certain embodiments, a pressure cycler may be used to improve (e.g., decrease) xHA and/or oHA digest time. In certain embodiments, xHA and/or oHA digestion may be performed in a pressure cycler under cycling pressure ranging between about <NUM> KPSI and about <NUM> KPSI, e.g., at about <NUM> KPSI, about <NUM> KPSI, about <NUM> KPSI, about <NUM> KPSI, about <NUM> KPSI, about <NUM> KPSI, about <NUM> KPSI, about <NUM> KPSI, about <NUM> KPSI, about <NUM> KPSI, about <NUM> KPSI, about <NUM> KPSI, about <NUM> KPSI, about <NUM> KPSI, about <NUM> KPSI or about <NUM> KPSI.

In certain embodiments, a pressure cycler is used with one or more hyaluronoglucosidases (e.g., HAases) to digest xHA at a pressure below about <NUM> KPSI, below about <NUM> KPSI, below about <NUM> KPSI, or below about <NUM> KPSI. In certain embodiments, a pressure cycler is used with one or more hyaluronoglucosidases (e.g., HAases) to digest xHA at a pressure of about <NUM> KPSI, about <NUM> KPSI or about <NUM> KPSI. In certain embodiments, a pressure cycler is used with one or more hyaluronoglucosidases (e.g., HAases) to digest xHA at a pressure of about <NUM> KPSI.

In certain embodiments, a pressure cycler is used with one or more enzymes (e.g., Glu-C, Asp-N, Lys-C, Arg-C, trypsin, or chymotrypsin) to digest peptide at a pressure below about <NUM> KPSI, below about <NUM> KPSI, below about <NUM> KPSI, or below about <NUM> KPSI. In certain embodiments, a pressure cycler is used with one or more enzymes (e.g., Glu-C, Asp-N, Lys-C, Arg-C, trypsin, or chymotrypsin) to digest peptide at a pressure of about <NUM> KPSI, about <NUM> KPSI or about <NUM> KPSI. In certain embodiments, a pressure cycler is used with one or more enzymes (e.g., Glu-C, Asp-N, Lys-C, Arg-C, trypsin, or chymotrypsin) to digest peptide at a pressure of about <NUM> KPSI.

In certain embodiments, a pressure cycler reduces peptide, xHA, and/or oHA digest time by at least about one hour, about two hours, about three hours, about four hours, about five hours, about six hours, about seven hours, about eight hours, about nine hours, about ten hours, about eleven hours, about twelve hours, about thirteen hours, about fourteen hours, about fifteen hours, about sixteen hours, about seventeen hours, about eighteen hours, about nineteen hours, about twenty hours, about twenty-one hours, about twenty-two hours, about twenty-three hours, about twenty-four hours or more as compared to digestion under non-pressurized conditions.

As used herein, a "peptide digest product" refers to any part or portion of a peptide drug (e.g., a free drug or a prodrug) that is generated by an enzyme and is smaller than the intact peptide drug. In certain exemplary embodiments, a peptide digest product is less than <NUM> amino acids long, e.g., between about <NUM> and about <NUM> amino acids in length, between about <NUM> and about <NUM> amino acids in length, between about <NUM> and about <NUM> amino acids in length, between about <NUM> and about <NUM> amino acids in length, between about <NUM> and about <NUM> amino acids in length, between about <NUM> and about <NUM> amino acids in length, or between about <NUM> and about <NUM> amino acids in length or any values or sub-ranges within these ranges. In certain exemplary embodiments, a peptide digest product is about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or about <NUM> amino acids in length.

In particular embodiments, a peptide digest product is about <NUM> amino acids in length. In other particular embodiments, a peptide digest product is about <NUM>, about <NUM> or about <NUM> amino acids in length. In yet other particular embodiments, a peptide digest product is between about <NUM> and about <NUM> amino acids in length. In still other particular embodiments, a peptide digest product is between about <NUM> and about <NUM> amino acids in length.

In certain exemplary embodiments, a peptide digest product is detected using a peptide identification method, such as liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MS-MS), liquid chromatography-high resolution mass spectrometry (LC-HRMS), nano-LC-MS-MS, high performance liquid chromatography-tandem MS (HPLC-MS-MS), nanoHPLC-MS-MS, ultra-performance-tandem MS (UPLC-MS-MS), nanoUPLC-MS-MS, ultrahigh-performance-tandem MS (UHPLC-MS-MS), nanoUHPLC-MS-MS, ultraviolet (UV) spectrometry, fluorescence spectrometry or the like.

In certain exemplary embodiments, the methods described herein utilize a separation process such as a chromatography method, e.g., liquid chromatography. According to an embodiment, detecting peptide digest products is performed by: (i) high performance liquid chromatography ("HPLC"), (ii) anion exchange, (iii) anion exchange chromatography; (iv) cation exchange; (v) cation exchange chromatography; (vi) ion pair reversed-phase chromatography; (vii) chromatography; (viii) single dimensional electrophoresis; (ix) multi-dimensional electrophoresis; (x) size exclusion; (xi) affinity; (xii) reverse phase chromatography; (xiii) capillary electrophoresis chromatography ("CEC"); (xiv) electrophoresis; (xv) ion mobility separation; (xvi) field asymmetric ion mobility separation or spectrometry ("FAIMS"); (xvii) capillary electrophoresis; and (xviii) supercritical fluid chromatography.

The amount of peptide digest product can be determined by measurement of multiple reaction monitoring (MRM) transitions consisting of the peptide precursor ion, one or more fragment ions and a retention time. This measurement is performed, for example, on a triple quadrupole instrument. The signature can also be obtained by a combination of retention time and accurate high-resolution mass spectrometric analysis of the intact peptide. These quantitation methods typically require a labeled internal standard and an external synthetic peptide calibration curve.

In certain exemplary embodiments, internal standards are used that include a labeled peptide corresponding to a peptide drug, e.g., a labeled C-terminal peptide corresponding to the C-terminal Asp-N digest product of SAR425899. The internal standards typically have a known peptide sequence and are provided in a known quantity. In some embodiments, the standards are labeled, such with one or more heavy isotopes, e.g., <NUM>C or <NUM>N.

The peptide digest product profiling methods described herein are useful for measuring the amount of drug present in a hydrogel prodrug formulation. The methods described herein are also useful for performing batch-to-batch reproducibility assessments.

As used herein, a "sample" refers to any composition containing a peptide drug (e.g., in a prodrug form). Exemplary samples include, but are not limited to, pharmaceutical compositions, dissolution or release media and the like. In certain embodiments, a sample is a hydrogel. In other embodiments, a sample is aqueous.

A sample for use in the quantification methods described herein can be a therapeutic composition, such as a liquid formulation for administration orally, sublingually, mucosally, intradermally, subcutaneously, intravenously, intramuscularly, parenterally or by inhalation.

In other embodiments, the sample will include a substrate, such as a nanoparticle, a capsule, a film or tablet, or a gel, such as a hydrogel (e.g., an xHA hydrogel). The quantification methods described herein are useful to quantify the amount of peptide drug in the substrate, such as, e.g., in a nanoparticle or capsule, or in a film or a hydrogel (e.g., an xHA hydrogel). The release can be from the interior of the substrate, e.g., an xHA hydrogel, or from the exterior (e.g., a surface) of a substrate. In one embodiment, a release profile is assayed by performing a complete release of peptide drug and then assaying for a controlled release, such as over a period of time or in different culture or solution conditions (e.g., at different temperatures, pH or the like). The amount of peptide drug released in the controlled release assay is typically reported as a fraction or percentage as compared to the amount of peptide drug released under the complete release conditions.

As used herein, "dissolution medium," "dissolution media," "release medium" and "release media" refer to a composition that is used to provide in vitro drug release information. Dissolution or release media is useful, for example, for quality control testing of a sample for determining the release and/or stability of peptide drug in a sample. In choosing a suitable dissolution or release medium, it is useful to determine the analytical target profile of the peptide drug (e.g., delayed release, constant release, extended release and the like) and/or the peptide drug solubility profile. For a review of dissolution media selection, see <NPL>.

As used herein, "release rate" refers to the rate that a peptide drug flows from a hydrogel prodrug formulation and into a surrounding medium in an in vitro release test. In one exemplary embodiment, the composition is first prepared for release testing by suspending the composition into the appropriate in vitro release medium. This is generally performed by exchanging the buffer after centrifugation to pellet the substrate (e.g., a hydrogel), and reconstituting the substrate using mild conditions. In certain embodiments, the assay is started by suspending the sample at <NUM>° C in an appropriate temperature-controlled apparatus. A sample is typically removed at various time points.

Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only.

SAR425899 is a dual receptor agonist targeting the glucagon and GLP1 receptors. Due to rapid clearance, a once daily dose is required. To reduce frequency of dosing, the once daily SAR425899 peptide drug candidate (<FIG>) was bound to a polymeric carrier (crosslinked hyaluronic acid (xHA)) via a self-cleaving linker to provide slow release of an active peptide drug from the inactive prodrug (<FIG>). The SAR425899 prodrug comprises crosslinked hyaluronic acid (xHA) bound to the SAR425899 lipopeptide (P) via a self-cleavable linker (L).

Determination of peptide load of a hydrogel is required for dosing and quality control. Crosslinked hyaluronic acid forms a multimillion Dalton hydrogel in an aqueous environment. Simple UV measurements typically applied for basic quantification of peptides in solution cannot be applied for hydrogels as this analysis require the hydrogel-linker-peptide to be fully dissolved. Whereas the linker is self-cleaving, complete cleavage under physiological conditions is by design very slow and therefore not suitable for analytical quantification methods which require certainty that a complete release has been accomplished.

To address this analytical challenge and enable peptide load determination, a novel method was developed that allows accurate quantification of peptide drug load in a biopolymeric prodrug measured as weight/weight percentage. The biopolymer-linker-peptide drug can, in principle, be applied to any small molecule (e.g., polypeptide, polynucleotide or the like) drug that can by cleaved or hydrolyzed, and the method can broadly be applied for drug load determination of biopolymer-containing prodrugs.

The method is based on double enzymatic digestion followed by quantification of a digestion product representing the peptide. Initially, crosslinked hyaluronic acid-linker-peptide (xHA-L-P) is digested with an enzyme that digests the crosslinked hyaluronic acid. Subsequently, a proteolytic enzyme is applied to digest the attached peptide and produce proteolytic digest products. These proteolytic digest products, representing the peptide, are subsequently quantified.

xHA-L-P is weighed and partially dissolved as a hydrogel in buffer. The hydrogel is enzymatically degraded into a plurality of oligomeric hyaluronic acid-linker-peptides (oHA-L-Ps) which are soluble. The resulting hyaluronic acid oligomers of oHA-L-P is a heterogeneous mixture, consisting of peptide, linker and oligomers of hyaluronic acid of different lengths. The oligomeric hyaluronic acid heterogeneity is undesirable for quantification, and for this reason a second enzymatic (e.g., proteolytic) digest step is introduced where the peptide drug moiety is digested to produce a homogeneous <NUM> amino acid C-terminal peptide digest product DFIE. PPPS-NH<NUM> (<FIG>). This C-terminal peptide is detected and quantified using LC / HRMS.

Digestion of the prodrug using Asp-N without prior hyaluronoglucosidase treatment produced a low yield of C-terminal peptide, presumably due to steric hindrance of Asp-N activity by the crosslinked hyaluronic acid (<FIG>). Hydrolysis of linker was determined to be impractical as a peptide release method for quantification.

An HA digestion method was developed. ) Digestion conditions that were suitable for subsequent endoproteinase (e.g., Asp-N) digestion were used. Several enzymes (e.g., HAases) capable of digesting HA were tested.

Digestion of crosslinked hyaluronic acid did not interfere with Asp-N digestion of free SAR425899 (<FIG> - <FIG>), and was demonstrated to significantly increase the subsequent yield of the Asp-N digestion C-terminal peptide digest product (<FIG> - <FIG>). Indeed, it was determined that the Asp-N proteolytic enzyme gave near <NUM>% digestion efficiency for the SAR425899 peptide.

Asp-N was demonstrated to digest SAR <NUM> completely in buffer and in presence of HA digest products (<FIG>). Digestion was performed in triplicate for HA-linker-peptide by HAase, followed by digestion with Asp-N, and quenching. Subsequent hydrolysis was performed at <NUM> for <NUM> hours to release any bound intact peptide, and an internal standard (IS) was added prior to LC/MS analysis. The ratio of released intact peptide to C-terminal peptide was <NUM>.

A double digest was performed in triplicate on HA-linker-peptide with HAase for <NUM> × <NUM> hours, followed by digestion with Asp-N, and quenching. Subsequent hydrolysis at <NUM> for was performed for <NUM> hours to release any bound intact peptide, and an IS was added prior to LC/MS analysis. No intact peptide was observed.

Digestion was performed in triplicate on HA-linker-peptide with HAase for <NUM> days, followed by digestion with Asp-N. No increase in C-terminal peptide was observed as compared to digest for <NUM> day, but C-terminal peptide isomer was observed eluting slightly earlier than the regular C-terminal peptide.

LC/MS of reaction products was performed as follows. The sample and standards were prepared in <NUM>% acetonitrile (ACN), <NUM>% formic acid (FA) to prevent observed adsorption issues. Separation was performed on an Accela <NUM> LC system equipped with an Luna Omega C18 column <NUM> × <NUM>, <NUM> A (part no. OOD-<NUM>-AN) (S/N H16-<NUM>) operated at <NUM>.

Mobile phase A: <NUM>% formic acid aq. Mo; Mobile phase B: ACN <NUM> % formic acid; flow rate was <NUM>µL/min. Gradient <NUM>% to <NUM>% B in <NUM>. <NUM>% B hold for <NUM> sec. equilibrate at <NUM>% B for <NUM>. A gradient initiating at <NUM>% B had also been applied and was feasible.

The eluate was introduced into an Orbitrap ELITE using a standard HESI ESI source operated in positive ion mode. The Orbitrap resolution of <NUM> @ m/z <NUM> was applied, m/z range <NUM>-<NUM>.

It was determined that all peptide was converted into digest products with no intact peptide remaining.

High C-terminal peptide yield following the sequential double digest was demonstrated in several ways. ) Double digestion was allowed to proceed, followed by quenching of all enzymatic activities by boiling. Subsequently, release of any intact peptide that might remain attached via a linker to hyaluronic acid was performed by allowing hydrolysis to proceed at <NUM>. No or very small amounts of intact peptide were observed, indicating that the double digest resulted in near complete digestion of all peptide drug molecules. In a separate experiment, release by hydrolysis over <NUM> weeks followed by Asp-N digestion did not produce a higher amount of C-terminal peptide than the double digest procedure. As a part of method qualification, it was analytically shown that the xHA-L-P batch obtained had a peptide load of <NUM>%, consistent with the results obtained for several lots showing around <NUM>% load (without intending to be bound by scientific theory, the lower amount of <NUM>% was attributed to water uptake of the lyophilized material). When lyophilized hydrogel takes up water, the resulting amount weighted contains a lower percentage of peptide due to water addition. This can bring the percentage of peptide from <NUM>% to <NUM>%. The remedy is to be cautious that the weighted material is not exposed to humid air conditions.

Crosslinked HA that did not contain peptide was digested and used as a matrix. The digest product (matrix) was spiked with synthetic non-labeled C-terminal peptide corresponding to the C-terminal Asp-N peptide digest product of SAR <NUM>, and a dilution series was generated. A heavy isotope labeled C-terminal peptide was applied as internal standard. The curve was compared to the equivalent curve prepared in <NUM>% ACN and <NUM>% formic acid buffer.

The curves were analyzed by LC/MS analysis to determine if the slopes were within <NUM>% of each other, which is the minimum criteria for accepting the buffer calibration curve.

Peptide quantification using the methods described herein was reproducible. Three aliquots of HA-linker peptide were weighted and digested with HAase and Asp-N. The digest products were adjusted to <NUM>% ACN and then further diluted <NUM>-fold, followed by an additional <NUM>-fold dilution by the addition of heavy internal standard (HIS). A <NUM>-point standard curve was prepared from <NUM>µg/mL to <NUM> ng/mL with <NUM> -old dilution steps. The curve was further diluted <NUM>-fold by <NUM>:<NUM> vol:vol addition of H-IS.

The method robustly tolerated the matrix. (<FIG> - <FIG>. ) C-terminal peptide calibration curves in buffer showed the same slope as calibration curves in extracted matrix generated by digest of crosslinked hyaluronic acid (xHA). (<FIG>, <FIG>, <FIG>, <FIG>. ) It was also determined that the matrix generated by digestion of xHA did not interfere with Asp-N digestion efficiency.

Several commercial hyaluronidases were tested, and HAase <NUM> was determined to be superior. It was furthermore demonstrated that the C-terminal peptide produced a near complete fragment series when subjected to gas phase collision induced fragmentation (CID), and that these fragments showed intensities that are suitable for development of a multi-reaction monitoring quantification method MRM (LC/MS MRM) as an alternative to the high-resolution mass spectrometry based method applied in the initial work (quantification by LC/HRMS).

A prerequisite for UV or fluorescence detection is that the analyte is soluble. xHA-L-P is not soluble. oHA-L-P, on the other hand, is water soluble and therefore accessible for UV or florescence analysis. The required digestion to produce oHA-L-P from xHA-L-P does however introduce a hyaluronoglucosidase, e.g., a hyaluronidase or HA lyase, into the mixture. The hyaluronoglucosidase, e.g., hyaluronidase or HA lyase, is a protein that itself absorbs UV. Accordingly, the hyaluronoglucosidase will be removed after the first digestion step followed by direct UV detection. This method would be even more suitable for a regulated analytical development / manufacturing environment.

Peptide loading of three separate batches of xHA-L-P wherein P is SAR425899 was determined (<FIG>). Excellent linearity was obtained with no matrix effects. Good reproducibility of full process replicates was achieved, and the results were consistent with the results obtained using orthogonal NMR. The methods described herein are widely applicable and are being applied to aid formulation optimization. The methods described herein are amenable to both cleavable and non-cleavable linker scenarios using a wide range of polymers and peptide drugs.

An LC/MS method was developed for analysis of HA-Linker-SAR425899, SAR425899, and fragments / digestion products. Complete cleavage of SAR425899 with Asp-N to release the C-terminal peptide fragment for quantification was demonstrated. Initial cleavage by Asp-N of HA-Linker-SAR425899 was demonstrated, but the digest was likely incomplete based on a modest LC/MS response.

Several HAases were tested, and the HA lyase (Sigma-Aldrich) EC <NUM>. <NUM> demonstrated significantly increased yields of the Asp-N digest product. Isotope-labeled C-terminal peptides were introduced, and the linearity range demonstrated after adhesion issue was resolved. Experiments evaluating digestion efficiency were carried out. Matrix curve examination showed no matrix effect, and a good matrix curve versus buffer curve alignment with only <NUM>% difference in slope. Triplicate weighing, processing and analysis showed good reproducibility with <NUM>% CV.

The total percentage peptide in a sample lot was found to be <NUM>% (the water content of the sample was unknown in particular after multiple freeze-thaw (room temperature) cycles).

The amount of drug loaded in the SAR425899 prodrug was determined using a novel pressure cycler process via hydration and denaturation of the prodrug. Digestion reactions tested SAR425899 digestion with AspN (<NUM>% reaction was achieved in one hour). Conditions were identified allowing reduction of xHA digest time from <NUM> hours to <NUM> hours using a pressure cycler.

The HAase appeared to be sensitive to very high pressure. Accordingly, a maximum pressure of <NUM> KPSI was applied and a higher enzyme concentration was used. Under the optimized conditions higher oHA-L-P product concentration was observed for the <NUM>-hour pressure cycler-aided digest.

<NUM> samples were simultaneously assayed (<NUM>-<NUM>µL/sample). The rate of pressure increase/decrease was controlled. The shape of the pressure profile (sine / square wave, etc.) was observed. Sample temperatures were controlled using a built-in electric heater. The maximum pressure used was <NUM> KPSI. A typical assay went from atmospheric pressure to <NUM>-<NUM> KPSI applied with <NUM> cycle, for a one-hour duration of time.

AspN digestion of oHA-L-P under a <NUM> kPSI pressure cycle of once per minute at <NUM> was determined to be as effective as a <NUM>-hour AspN digest of oHA-L-P at <NUM> at atmospheric pressure. All experiments were carried out using a Pressure Biosciences Barocycler (Model 2320EXT, South Easton, MA).

It was determined that AspN digestion using cycling between <NUM> KPSI pressure and atmospheric pressure with <NUM>-minute intervals for <NUM> hour at <NUM> gave the same digestion efficiency as <NUM> hours at <NUM> under atmospheric pressure. Similarly, the HAase (E. <NUM>) was digested using cycling between <NUM> KPSI pressure with a <NUM>-minute cycle time for <NUM> hours yielded the results, based on oligomers observed, as the <NUM>-hour digestion at <NUM> under atmospheric pressure.

In general, each new enzyme is optimized in terms of temperature and pressure, pressure profile, frequency and duration of the pressure applied.

Sample purity and determination of major impurities were analyzed (<FIG>). Release of intact peptide under mild hydrolysis conditions was ascertained (<NUM>, <NUM> days or <NUM>, <NUM> hours). Both release methods produced sufficient material for impurity analysis by LC/MS, down to <NUM>% or lower. Release was demonstrated to not produce impurities.

A linker-peptide control was analyzed at t=<NUM> and at full release time. A high level of impurities was observed for linker-peptide at t=<NUM>. Impurity analyses were performed by assaying LC/MS of released peptides.

LC/MS method qualification was successfully completed and implemented. Digestion conditions to produce complete AspN digestion were demonstrated. Matrix was shown not to be an issue. Good reproducibility and linearity of the method were demonstrated.

Claim 1:
A method for determining the amount of drug present in a crosslinked hyaluronic acid-linker-peptide (xHA-L-P) prodrug formulation comprising:
contacting a sample of the xHA-L-P prodrug formulation with a hyaluronoglucosidase to generate oligomeric hyaluronic acid-linker-peptide drug (oHA-L-P);
contacting the oHA-L-P with an endoproteinase to generate peptide digest products of the drug; and
detecting the peptide digest products to determine the amount of the drug present in the xHA-L-P prodrug formulation,
wherein the endoproteinase is selected from the group consisting of Glu-C, Asp-N, Lys-C, Arg-C, trypsin and chymotrypsin.