Methods for quantifying drug concentration in prodrug composition

Abstract

To provide analytical quantification methods in order to quantify drug loading of prodrug compositions on crosslinked HA hydrogels.SOLUTION: A method for determining the amount of drug present in a crosslinked hyaluronic acid-linker-peptide (xHA-L-P) prodrug formulation comprises: contacting a sample of the xHA-L-P prodrug formulation with a hyaluronoglucosidase to generate an oligomeric hyaluronic acid-linker-peptide drug (oHA-L-P); contacting the oHA-L-P with a second enzyme 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.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 844,579, filed May 7, 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present invention relates to a method for quantifying the amount of drug present in a prodrug composition. [Background technology]

[0003] Over the past decade, on average, 17 new peptides have entered clinical trials each year, with approval rates (from phase I to market) twice that of small molecules (Non-Patent Document 1; Non-Patent Document 2), and over 150 peptide drug candidates are currently in development (Non-Patent Document 3).

[0004] However, peptide drug development presents unique challenges. For example, peptide drugs undergo proteolytic cleavage in vivo and are rapidly cleared. Oral delivery remains difficult, in part due to poor permeability through biological membranes. Attempts to address these shortcomings have been made by incorporating structural constraints such as cyclic peptide formation (e.g., by lactamization and click cyclization), hydrocarbon stapling, lipid addition, and the development of polymer-drug conjugates.

[0005] One example of a peptide drug is SAR42589, a dual receptor agonist that targets the glucagon receptor and the GLP1 receptor, which can promote glycemic control and weight loss in type 2 diabetes patients and in healthy volunteers (Non-Patent Document 4). However, SAR425899 has rapid in vivo clearance and therefore requires once-daily administration.

[0006] Hyaluronic acid (HA), also called hyaluronan, is a natural nonsulfated linear polysaccharide composed of repeating disaccharide units of d-glucuronic acid and N-acetyl-d-glucosamine linked by β-1-3 and β-1-4 glycosidic bonds (Non-Patent Document 5). HA is an important structural element in the skin and is involved in the interaction of multiple cell surface receptors. HA has immunosuppressive and antiangiogenic activities and is present in brain tissue, hyaline cartilage and synovial fluid. HA plays important structural and functional roles in the body due to its strong hydrophilic character, which allows it to absorb large amounts of water up to 1000 times its actual volume, and its high molecular weight in biological tissues.

[0007] HA has found many uses in biomedical and pharmaceutical applications due to its biocompatibility and biodegradability. However, HA is highly soluble and often exhibits very poor mechanical properties with rapid degradation behavior in vivo. For this reason, HA has been chemically modified and / or crosslinked to improve its properties, including mechanical properties, viscosity, solubility, degradation, and biological properties. HA derivatives have been created and utilized in scaffolds for tissue engineering, in soft tissue surgery such as vocal fold augmentation, for drug delivery, intracellular delivery of siRNA, in wound healing, and as devices in surgical procedures. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Dharanipragada(2013)Future Medicinal Chemistry 5(7):831 pages [Non-Patent Document 2] Kaspar and Reichert (2013) Drug Discovery Today 18:807-817 [Non-Patent Document 3] Lau and Dunn (2018) Bioorganic & Medicinal Chemistry 26:2700-2707 [Non-Patent Document 4] Tillner et al. (2019) Diabetes Obes. Metab. 21:120, doi:10.1111 / dom.13494, Epub 2018-09-16 [Non-Patent Document 5] Khunmanee et al. (2017) J. Tissue Engineering Vol. 8 (doi:doi.org / 10.1177 / 2041731417726464) Summary of the Invention [Problem to be solved by the invention]

[0009] Cross-linked hyaluronic acid (xHA) forms hydrogels of millions of daltons in an aqueous environment and can be linked to drugs (e.g., peptide drugs) to stabilize the drugs in vivo. However, it is difficult to both quantify the amount of drug loaded into the hydrogel and to quantify the release rate of the drug from the hydrogel. UV-based measurements, which are usually applied to the basic quantification of dissolved peptides, cannot be applied to hydrogels because such analysis requires that the drug linked to the hydrogel complex is sufficiently dissolved.

[0010] Alternative methods for accelerated hydrolysis of the linker and release of the peptide drug, using high temperature or high pH, ​​result in partial degradation of the peptide drug, thus producing a heterogeneous mixture that is difficult to quantitate. Therefore, analytical quantification methods are needed to quantify the drug loading of the prodrug composition into the crosslinked HA hydrogel. [Means for solving the problem]

[0011] To address the analytical challenge of determining drug loading in crosslinked HA (xHA) hydrogels, a novel method has been discovered that allows accurate quantification of drug loading (e.g., peptide prodrug loading of biopolymers) as weight / weight percent, for example, to enable peptide loading determination of xHA hydrogels. The method described herein is highly selective for drug loading determination that can be applied to any small molecule (e.g., polypeptide or polynucleotide) that can be broken down into smaller components (e.g., by proteolytic digestion or hydrolysis), and can be broadly applied to drug loading determination of prodrugs, e.g., prodrugs containing biopolymers. The method described herein is useful, for example, to manage drug dose delivery in vivo and to quality control drug loading between batches of prodrugs during manufacturing. The method described herein allows for the production of drugs with increased half-lives in vivo, e.g., peptide drugs, e.g., peptide drugs with increased half-lives from minutes to days.

[0012] The method described herein provides various advantages over conventional methods known in the art, such as NMR-based methods.For example, NMR-based methods are more time-consuming and have lower throughput than the method described herein.NMR-based methods require milligrams of material, whereas the novel method described herein requires only nanograms of material.Furthermore, the dynamic range of NMR-based methods is narrower than that of the novel method described herein.In addition, NMR-based methods are more susceptible to interference from contaminating peptides than the novel method described herein.

[0013] The novel method uses dual enzymatic digestion followed by quantification of the digestion products to determine the degree of drug incorporation into hydrogels. Based on the discovery that it is possible to accurately quantify loading (e.g., peptide drug loading), provided herein.In a certain embodiment of this method, crosslinked hyaluronic acid-linker-peptide (xHA-LP) is incubated with enzyme to digest crosslinked hyaluronic acid.Then, proteolytic enzyme is applied to digest the peptide that is linked to it, producing proteolytic digestion products.Then, the proteolytic digestion products that correspond to peptides are quantified.

[0014] In certain exemplary embodiments of the method, cross-linked hyaluronic acid linker peptide conjugates (xHA-LP) are weighed and suspended as a hydrogel in a buffer solution. The hydrogel is enzymatically degraded to soluble, oligomeric hyaluronic acid-linker-peptide (oHA-LP). The resulting oHA-LP oligomers exist as a heterogeneous mixture, consisting of peptides, linkers and oligomeric hyaluronic acid of different lengths. This heterogeneity of oligomeric hyaluronic acid is undesirable for mass spectrometry-based quantification, but can be applied to less specific UV or fluorescence-based assays. Thus, in the mass spectrometry-based reference assay, a second enzymatic (e.g., endoproteolytic) digestion step is introduced to digest the peptide drug and produce a homogenous peptide digestion product (e.g., the 19 amino acid C-terminal peptide digestion product, DFIEWLKAGGPSSGAPPPS-NH2 (Figure 3)). The peptide products can then be detected and quantified using assays such as, for example, liquid chromatography / high resolution mass spectrometry (LC / MS).

[0015] In one embodiment, a method is provided for determining the amount of drug present in a crosslinked hyaluronic acid-linker-peptide (xHA-LP) prodrug formulation, comprising contacting a sample of the xHA-LP prodrug formulation with a hyaluronoglucosidase, such as hyaluronidase (HAase) or hyaluronic acid (HA) lyase, to produce an oligomeric hyaluronic acid-linker-peptide drug (oHA-LP), contacting the oHA-LP with the enzyme to produce a peptide digestion product of the drug, and detecting the peptide digestion product to determine the amount of drug present in the xHA-LP prodrug formulation.

[0016] In certain exemplary embodiments, the peptide digestion products are between about 2 and about 100 amino acids in length, between about 3 and about 75 amino acids in length, between about 4 and about 50 amino acids in length, between about 6 and about 30 amino acids in length, between about 15 and about 20 amino acids in length, or about 19 amino acids in length. In certain exemplary embodiments, the peptide digestion products are about 1, about 2, or about 3 amino acids in length.

[0017] In certain exemplary embodiments, detecting the peptide digestion products is carried out 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 methods.

[0018] In certain exemplary embodiments, the hyaluronoglucosidase is a HAase selected from the group consisting of HAase 1, HAase 2, HAase 3, HAase 4, HAase 5 and HAase 6. In other exemplary embodiments, the hyaluronoglucosidase is HAase 1 or HAase 2. In yet other exemplary embodiments, the hyaluronoglucosidase is HAase 2. In certain exemplary embodiments, the hyaluronoglucosidase is HA lyase EC 4.2.2.1.

[0019] In certain exemplary embodiments, oHA-LP is contacted with an endoproteinase, such as Glu-C, Asp-N, Lys-C, Arg-C, trypsin, or chymotrypsin. In certain exemplary embodiments, the endoproteinase is Asp-N.

[0020] In certain exemplary embodiments, the method further comprises the use of an internal standard, hi certain exemplary embodiments, the internal standard comprises one or more heavy isotopes.

[0021] In certain exemplary embodiments, the amount of drug present is determined using a calibration curve.

[0022] In certain exemplary embodiments, xHA-LP is contacted with hyaluronoglucosidase in a pressure cycler. In certain exemplary embodiments, the pressure in the pressure cycler is higher than atmospheric pressure. In certain exemplary embodiments, the pressure is about 5KPSI, about 10KPSI or about 15KPSI.

[0023] In certain exemplary embodiments, oHA-LP is contacted with the second enzyme in a pressure cycler.In certain exemplary embodiments, the pressure in the pressure cycler is higher than atmospheric pressure.In certain exemplary embodiments, the pressure is about 35KPSI, about 40KPSI or about 45KPSI.

[0024] In another embodiment, a method is provided for determining the amount of drug present in a crosslinked hyaluronic acid-linker-peptide (xHA-LP) prodrug formulation, comprising contacting a sample of the xHA-LP prodrug formulation with hyaluronoglucosidase to produce an oligomeric hyaluronic acid-linker-peptide drug (oHA-LP), contacting the oHA-LP with an endoproteinase to produce a peptide digestion product of the drug, and detecting the peptide digestion product to determine the amount of drug present in the xHA-LP prodrug formulation.

[0025] In certain exemplary embodiments, the step of detecting the peptide digestion products is carried out by a method selected from the group consisting of one or a combination of LC-MS, LC-MS-MS, LC-HRMS, UV absorbance and fluorescence detection methods.

[0026] In certain exemplary embodiments, the hyaluronoglucosidase is HAase 1 or HAase 2. In certain exemplary embodiments, the hyaluronoglucosidase is HA lyase EC 4.2.2.1.

[0027] In certain exemplary embodiments, the endoproteinase is selected from the group consisting of Glu-C, Asp-N, Lys-C, Arg-C, trypsin and chymotrypsin.

[0028] In certain exemplary embodiments, the endoproteinase is Asp-N.

[0029] In certain exemplary embodiments, the method further comprises the use of an internal standard, hi certain exemplary embodiments, the internal standard comprises one or more heavy isotopes.

[0030] In certain exemplary embodiments, the amount of drug present is determined using a calibration curve.

[0031] In certain exemplary embodiments, xHA-LP is subjected to hyaluronan cycling in a pressure cycler. In certain exemplary embodiments, the pressure in the pressure cycler is greater than atmospheric pressure. In certain exemplary embodiments, the pressure is about 5 KPSI, about 10 KPSI, or about 15 KPSI.

[0032] In certain exemplary embodiments, oHA-LP is contacted with endoproteinase in a pressure cycler.In certain exemplary embodiments, the pressure in the pressure cycler is higher than atmospheric pressure.In certain exemplary embodiments, the pressure is about 35KPSI, about 40KPSI or about 45KPSI.

[0033] In another embodiment, a method is provided for determining the amount of drug present in a crosslinked hyaluronic acid-linker-peptide (xHA-LP) prodrug formulation, comprising contacting an xHA-LP prodrug with hyaluronidase 2 to produce an oligomeric hyaluronic acid-linker-peptide drug (oHA-LP), contacting the oHA-LP with Asp-N to produce a peptide digestion product of the drug, and detecting the peptide digestion product to determine the amount of drug present in the xHA-LP prodrug formulation.

[0034] In certain exemplary embodiments, the peptide digestion products are between about 2 and about 100 amino acids in length, between about 3 and about 75 amino acids in length, between about 4 and about 50 amino acids in length, between about 6 and about 30 amino acids in length, between about 15 and about 20 amino acids in length, or about 19 amino acids in length. In certain exemplary embodiments, the peptide digestion products are about 1, about 2, or about 3 amino acids in length.

[0035] In certain exemplary embodiments, the step of detecting the peptide digestion products is carried out by a method selected from the group consisting of one or a combination of LC-MS, LC-MS-MS, LC-HRMS, UV absorbance and fluorescence detection methods.

[0036] In certain exemplary embodiments, the method further comprises the use of an internal standard, hi certain exemplary embodiments, the internal standard comprises one or more heavy isotopes.

[0037] In certain exemplary embodiments, the amount of drug present is determined using a calibration curve.

[0038] In certain exemplary embodiments, xHA-LP is contacted with hyaluronidase 2 in a pressure cycler. In certain exemplary embodiments, the pressure in the pressure cycler is higher than atmospheric pressure. In certain exemplary embodiments, the pressure is about 5 KPSI, about 10 KPSI, or about 15 KPSI.

[0039] In certain exemplary embodiments, oHA-LP is contacted with Asp-N in a pressure cycler. In certain exemplary embodiments, the pressure in the pressure cycler is higher than atmospheric pressure. In certain exemplary embodiments, the pressure is about 35KPSI, about 40KPSI or about 45KPSI.

[0040] In another embodiment, a method is provided for determining the amount of drug present in a crosslinked hyaluronic acid-linker-peptide (xHA-LP) prodrug formulation, the method comprising the steps of contacting an xHA-LP prodrug with HA lyase EC 4.2.2.1 to form an oligomeric hyaluronic acid-linker-peptide drug (oHA-LP), contacting the oHA-LP with Asp-N to form a peptide digestion product of the drug, and detecting the peptide digestion product to determine the amount of drug present in the xHA-LP prodrug formulation. The method includes a step of determining.

[0041] In certain exemplary embodiments, the peptide digestion products are between about 2 and about 100 amino acids in length, between about 3 and about 75 amino acids in length, between about 4 and about 50 amino acids in length, between about 6 and about 30 amino acids in length, between about 15 and about 20 amino acids in length, or about 19 amino acids in length. In certain exemplary embodiments, the peptide digestion products are about 1, about 2, or about 3 amino acids in length.

[0042] In certain exemplary embodiments, the step of detecting the peptide digestion products is carried out by a method selected from the group consisting of one or a combination of LC-MS, LC-MS-MS, LC-HRMS, UV absorbance and fluorescence detection methods.

[0043] In certain exemplary embodiments, the method further comprises the use of an internal standard, hi certain exemplary embodiments, the internal standard comprises one or more heavy isotopes.

[0044] In certain exemplary embodiments, the amount of drug present is determined using a calibration curve.

[0045] In certain exemplary embodiments, xHA-LP is contacted with HA lyase EC 4.2.2.1 in a pressure cycler. In certain exemplary embodiments, the pressure in the pressure cycler is higher than atmospheric pressure. In certain exemplary embodiments, the pressure is about 5KPSI, about 10KPSI, or about 15KPSI.

[0046] In certain exemplary embodiments, oHA-LP is contacted with Asp-N in a pressure cycler. In certain exemplary embodiments, the pressure in the pressure cycler is higher than atmospheric pressure. In certain exemplary embodiments, the pressure is about 35KPSI, about 40KPSI or about 45KPSI.

[0047] The foregoing and other features and advantages of this invention will be better understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0048] [Figure 1] FIG. 1 depicts a polymeric carrier and a self-cleaving linker that slowly releases an active peptide drug (i.e., SAR425899, a once-daily GLP-1 / GCC receptor agonist) from an inactive prodrug form in vivo. [Diagram 2] FIG. 1 depicts the complexity and size of a once-weekly GLP-1 / GCG agonist (i.e., SAR425899) according to certain exemplary embodiments, attached to a high molecular weight cross-linked hyaluronic acid hydrogel via a cleavable linker. D-serine is the amino acid at position 2 of SAR425899 (HdSQGTFTSDLSKQK(γE-palmitate)ESKAAQDFIEWLKAGGPSSGAPPPS-NH2). [Diagram 3] FIG. 1 is a schematic representation of digestion of SAR425899 with endoproteinase Asp-N to produce digestion products, including a C-terminal peptide for use in quantification. D-Serine is the amino acid at position 2 of SAR425899 (HdSQGTFTSDLSKQK(γE-palmitate)ESKAAQDFIEWLKAGGPSSGAPPPS-NH2). A calibration curve was generated using synthetic unlabeled C-terminal peptides corresponding to the proteolytic digestion products, and a heavy isotope-labeled C-terminal peptide was applied as an internal standard. 20-40 μg of peptide was digested with 1.20-0.4 μg Asp-N (Sigma-Aldrich) (ratio 1:100) in 100 mM ammonium bicarbonate overnight at 37° C. or for 1 hour at 37° C. alternating between atmospheric and 40 KPSI with a cycle time of 1 minute using a Pressure Bio Cycler. [Figure 4]Figure 1: xHA-LP was digested using hyaluronoglucosidase (hyaluronidase (HAase)) followed by digestion with AspN and subsequent quenching. Hydrolysis was carried out at 37°C for 24 hours to release any attached intact peptides. An internal standard (IS) was added prior to LC / MS analysis. AspN completely digested SAR425899 in buffer in the presence of HA digestion products. The ratio of released intact peptides to C-terminal peptides was 0.0006. [Diagram 5] 1 shows that digestion of SAR425899 prodrug with Asp-N produces a C-terminal peptide digestion product detectable by liquid chromatography / mass spectrometry (LC / MS). SAR425899 prodrug is xHA-linker-SAR425899; SAR425899 is HdSQGTFTSDLSKQK(E-palmitate)ESKAAQDFIEWLKAGGPSSGAPPPS-NH2. The C-terminal peptide digestion product of digestion of HA-linker-SAR425899 with Asp-N was detectable by LC / MS and showed moderate yield. [Figure 6] Schematic representation of quantification of SAR425899 prodrug cleavage. Labeled peptide internal standard (IS) is a C-terminal peptide labeled with 13C15N-Lys. 200 μL of 1 mg / mL HA-linker-peptide was digested with 30 μL of 100 U / mL hyaluronate lyase EC4.2.2.1 (Sigma-Aldrich) in 100 mM ammonium bicarbonate for 24 hours at 37° C. Subsequent digestion with Asp-N was performed. [Figure 7A] HA digestion products did not produce major matrix effects. Figure 7A shows HAase 1 digested HA plus C-terminal Asp-N digestion products. [Figure 7B] HA digestion products did not produce major matrix effects. Figure 7B shows that HAase 2 digested HA was supplemented with C-terminal Asp-N digestion products. [Figure 7C] Figure 7C is a control showing intact SAR425899 digested with Asp-N in buffer, showing that the HA digestion products did not produce major matrix effects. [Figure 7D] Figure 7D shows that the HA digestion products did not produce major matrix effects. HA was digested with HAase 1, to which the intact SAR425899 peptide was added. Digestion with Asp-N was then performed. [Figure 7E] Figure 7E shows that the HA digestion products did not produce major matrix effects. HA was digested with HAase 2, to which the intact SAR425899 peptide was added. Digestion with Asp-N was then performed. [Figure 7F] HA digestion products did not produce major matrix effects. Figure 7F is a control showing intact SAR425899 digested with Asp-N in buffer. The same level of intensity of the C-terminal peptide was observed for the two HAase digests with C-terminal peptide and the control, and for the two HAase digests with SAR425899 and the control digested with Asp-N. [Figure 8A] Figure 8 shows that HAase 2 improved subsequent digestion with Asp-N compared to HAase 1. Figure 8A shows HA-linker-SAR425899 digested with HAase 1 and then digested with Asp-N. [Figure 8B] Figure 8B shows that HAase 2 improved subsequent Asp-N digestion compared to HAase 1. Figure 8B shows HA-linker-SAR425899 digested with HAase 2 and then Asp-N. The C-terminal Asp-N digestion product is indicated by the peak at 956.99 m / z. [Figure 9]9A-9B show the quantification of digestion products. Figure 9A shows that acceptable linearity was observed for the standard C-terminal peptide curve from 10 ng / mL to 10 μg / mL. Figure 9B shows very good fragmentation of the C-terminal peptides from the Asp-N digest, making it suitable for multiple reaction monitoring (MRM) methods. [Figure 10] FIG. 1 Schematic representation of the assessment of the completeness of the HAase / Asp-N digestion. C-terminal peptides were detected, indicating the yield of the process. Intact peptides were detected, indicating the peptide release by subsequent hydrolysis. Only 0.05% intact protein was detected, indicating a desired complete digestion process close to 100%. [Figure 11] 11A-11B show cross-linked hyaluronic acid (xHA) digested with HAase in matrix and xHA digested with HAase in buffer. FIG. 11A shows the raw data. FIG. 11B is a graphical representation of the curves in matrix and in buffer. Excellent linearity was observed. The curve slopes were 3.1% apart from each other. Therefore, matrix effects are not significant and the buffer curve can be applied for quantitative analysis. [Figure 12] 12A-B show that the intensities of the internal standard did not show any matrix effect, with consistent results being obtained in the buffer and matrix curves. [Figure 13] 13A-13B show the quantification of HA-linker-peptide triplicates. Good reproducibility was observed (CV was 7.4%). After applying the dilution factor, the total peptide percentage was found to be 18%. [Figure 14] Figure 1 shows peptide (P) loading of three batches of xHA-LP, where P is SAR425899. Excellent linearity was observed without matrix effects. Good reproducibility was observed for all process replicates. These results were consistent with those obtained by orthogonal NMR. [Figure 15] Figure 1 depicts results obtained using mild hydrolysis conditions, which resulted in a significant increase in the rate of enzymatic digestion. The linker-peptide intended as a control for hydrolytic release of the peptide contained high levels of synthetic impurities. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] The present invention is based on the development of a novel quantification method for determining the amount of drug loaded on the polymer. In certain embodiments, the method comprises cleaving hyaluronic acid (HA) present in a sample of crosslinked hyaluronic acid-linker-peptide drug (xHA-LP) to form a heterogeneous mixture of oligomeric hyaluronic acid-linker-peptide drug (oHA-LP). The oHA-LP can then be proteolytically digested to obtain homogeneous peptide digestion products that can be detected and quantified using methods well known in the art, such as, for example, liquid chromatography, mass spectrometry, UV absorption, and fluorescence-based assays. In a particular exemplary embodiment, a composition is provided that includes xHA-LP (e.g., SAR425899 prodrug), where "P" is the peptide drug SAR425899, "L" is a self-cleaving crosslinker, and "xHA" is crosslinked hyaluronic acid (Figure 2).

[0050] As used herein, the term "prodrug" refers to a compound that undergoes biotransformation before exerting its pharmacological effect. Thus, prodrugs can be regarded as biologically active moieties that contain specialized non-toxic protective groups that are used temporarily to change or eliminate undesirable properties in the parent molecule. Typical prodrugs can be carrier-linked prodrugs, which contain a temporary bond between a given active substance and a temporary carrier group that improves physicochemical or pharmacokinetic properties and can usually be easily removed in vivo by hydrolytic cleavage, i.e., cascade prodrugs, in which the cleavage of the carrier group only becomes effective after the activating group is exposed.

[0051] In order to enhance the physicochemical or pharmacokinetic properties of drugs such as SAR425899 in vivo, such drugs can be conjugated with carriers (e.g. xHA).When a drug is temporarily bound to a carrier and / or linker, such a system is generally designated as a carrier-linked prodrug.According to the definition provided by IUPAC, a carrier-linked prodrug is a prodrug that contains a temporary bond between a given active substance and a temporary carrier group that improves physicochemical or pharmacokinetic properties and can be easily removed in vivo, usually by hydrolytic cleavage.

[0052] The linkers used in the carrier-linked prodrugs described herein can be any known cleavable or non-cleavable linker, such as enzyme labile linkers (e.g., acid cleavable linkers, reducible linkers (e.g., disulfide linkers, and β-glucuronide linkers), photocleavable linkers, thioether linkers, maleimidocaproyl linkers, peptide linkers (e.g., dipeptide linkers), and crosslinkers. Suitable linkers are available from, for example, Sigma-Aldrich, Millipore It is commercially available from a variety of sources, such as Sigma, and Creative Biolabs.

[0053] In certain exemplary embodiments, the linkers used in the carrier-linked prodrugs described herein are transient (e.g., self-cleavable), which means that they are non-enzymatically, hydrolytically degradable (cleavable) under physiological conditions, with half-lives ranging, for example, from 1 hour to 3 months.

[0054] As used herein, "physiological conditions" refers to the temperature, pH, etc., of the particular environment of the body in which the drug is intended to be released. For example, release in plasma occurs at a pH between 7.35 and 7.45 and a temperature of about 37°C (e.g., between about 36°C and about 38°C), while release in lysosomes occurs at a pH between about 6.5 and about 4.5 and a temperature of about 37°C.

[0055] In certain exemplary embodiments, a drug (e.g., a peptide drug) is transiently linked to a hydrogel carrier, e.g., a xHA hydrogel carrier, via a self-cleaving linker. The terms "hydrogel prodrug" and "hydrogel-linked prodrug" refer to a prodrug of a biologically active agent that is transiently linked to a hydrogel and are used interchangeably.

[0056] Terms such as "drug", "peptide drug", "biologically active molecule", "biologically active moiety", "biologically active agent" and "active agent" refer to any substance that can affect any physical or biochemical property of a living 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 the diagnosis, cure, mitigation, treatment or prevention of disease in humans or other animals, or otherwise for enhancing the physical or mental health of humans or animals. In certain exemplary embodiments, terms such as "drug", "biologically active molecule", "biologically active moiety", "biologically active agent" and "active agent" refer to peptide drugs, such as, for example, SAR425899.

[0057] The "free form" of a drug refers to the unmodified, pharmacologically active form of the drug (e.g., SAR425899), such as after release from a hydrogel-conjugated prodrug (e.g., SAR425899 prodrug).

[0058] As used herein, the term "anti-cancer therapeutic agent" or "anti-cancer agent" refers to a molecule that is detrimental to the growth and / or proliferation of neoplastic or tumor or cancer cells and can act to reduce, inhibit or destroy malignancies.

[0059] As used herein, the term "cell proliferation inhibitory agent" refers to a molecule that inhibits cell growth and proliferation.

[0060] As used herein, the term "cytotoxic nucleoside" refers to a nucleobase or nucleoside analog that exerts a cytotoxic effect by mimicking an endogenous nucleoside.

[0061] As used herein, the term "tubulin binding agent" refers to a molecule that binds directly to the tubulin system.

[0062] As used herein, the term "hormone" refers to any member of a class of signaling molecules produced by glands in multicellular organisms and transported by the circulatory system to distant target organs to control physiological functions and / or behavior. A "hormone antagonist" is a specific type of receptor antagonist that acts on hormone receptors.

[0063] As used herein, the term "anti-angiogenic agent" refers to a molecule that inhibits the physiological process of angiogenesis, whereby new blood vessels form from pre-existing blood vessels.

[0064] As used herein, the term "enzyme inhibitor" refers to a molecule that inhibits the function of a particular enzyme.

[0065] As used herein, the term "gene regulator" refers to a molecule that can affect the transcription of a gene, either positively or negatively.

[0066] As used herein, the term "cytotoxic therapeutic agent" refers to a substance that inhibits or prevents the function of a cell and / or causes the destruction of a cell. The term "cytotoxic agent" is intended to include chemotherapeutic agents, enzymes, antibiotics, and small molecule toxins, or toxins such as enzymatically active toxins of bacterial, fungal, plant, or animal origin and their fragments and / or variants, as well as various antitumor or anticancer agents disclosed below. In some embodiments, the cytotoxic agent is a taxoid, a vinca, a maytansinoid or maytansinoid analog such as DM1 or DM4, a small drug, a leptomycin derivative, an auristatin or dolastatin analog, a prodrug, a topoisomerase II inhibitor, a DNA alkylating agent, an antitubulin agent, CC-1065 or a CC-1065 analog.

[0067] As used herein, the term "pharmaceutical acceptable carrier" refers to any physiologically compatible solvent, dispersion medium, coating agent, antibacterial and antifungal agent, and the like. Examples of suitable carriers, diluents and / or additives include one or more of water, amino acids, saline, phosphate buffered saline, phosphate buffer, acetate buffer, citrate buffer, succinate buffer; amino acids and derivatives such as histidine, arginine, glycine, proline, glycylglycine; inorganic salts NaCl, calcium chloride; sugars or polyhydric alcohols such as dextrose, glycerin, ethanol, sucrose, trehalose, mannitol; and surfactants such as polysorbate 80, polysorbate 20, poloxamer 188; and the like, and combinations thereof. In many cases, it is suitable to include an isotonicity agent such as a sugar, polyhydric alcohol or sodium chloride in the composition, and the formulation may also include an antioxidant such as tryptamine, and a stabilizer such as Tween 20. It may also contain:

[0068] As used herein, the terms "hyaluronic acid," "HA," and "hyaluronan" are used interchangeably and refer to a nonsulfated linear polysaccharide composed of repeating disaccharide units of d-glucuronic acid and N-acetyl-d-glucosamine linked by β-1-3 and β-1-4 glycosidic bonds (e.g., →4)-β-d-GlcpA-(1→3)-β-d-GlcpNAc-(1→). HA occurs in a variety of molecular weights. High molecular weight HA (HMWHA) is approximately 1×10 6 Da, low molecular weight HA (LMWHA) is about 0.8 to about 8 × 10 5 Oligomeric hyaluronan usually has a molecular weight of about 6 × 10 3 It is less than Da.

[0069] HA is commercially available from sources 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).

[0070] To improve the mechanical properties of HA and extend its duration in vivo, hydrogels can be formed by covalently crosslinking HA polymer chains into three-dimensional networks (see, e.g., Agerup, Berg, and Akermark (2005) BioDrugs 19:23; Edsman et al. (2011) Cartilage 2:384). The mechanical and physical properties of crosslinked HA (xHA) hydrogels depend on the degree of modification and crosslinking (La Gatta, Schiraldi, Papa, and De Rosa (2011) Polymer Degradation and Stability 96:603).

[0071] As used herein, the terms "crosslinking agent" and "crosslinker" are intended to include chemical agents that can react with hyaluronic acid by 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 attraction, dipole-dipole interactions and dipole-induced interactions).

[0072] The term "crosslinked," as used herein, refers to two or more polymeric chains of hyaluronic acid that are covalently and / or non-covalently linked via a crosslinking agent. Such crosslinking is distinct from inter- or intramolecular dehydration, in which lactones, anhydrides, or esters are formed within a single polymeric chain or between two or more chains. However, It is also contemplated that intramolecular crosslinking may occur in the compositions described herein.The crosslinking agent contains at least two functional groups that create covalent and / or non-covalent bonds between two or more molecules (i.e., hyaluronic acid chains).In one embodiment of the present disclosure, the crosslinking agent contains complementary functional groups to the functional groups of hyaluronic acid so that crosslinking can proceed.

[0073] Physical cross-linking of HA can be achieved using various pH, temperature, ionic strength conditions, and physicochemical interactions, such as hydrophobic interactions, hydrogen bonding, charge interactions, or stereocomplexation. In particular, temperature-responsive hydrogels have been extensively tested for various applications. Common thermogelling polymers frequently used to modify HA to produce heat-sensitive HA hydrogels include poly(N-isopropylacrylamide) (PNIPAM), pluronic acid, methylcellulose, and polyethylene glycol (PEG).

[0074] Methods for crosslinking HA to form hydrogels include, but are not limited to, modifying -COOH groups, modifying -OH groups, modifying -NHCOCH3 groups, and chemical crosslinking methods using Schiff base crosslinking, dialdehyde hyaluronate (CHO-HA), thiol modification, Diels-Alder reaction, and enzyme-mediated ligation.

[0075] Methods for crosslinking HA hydrogels are described in Kenne et al. (2013) Carb. Polymers 91:410; and Khunmanee, supra; Hoare TR, Kohane DS, Hydrogels in drug delivery: progress. and challenges.Polymer 2008;49(8):1993~2007;Gupta D, Tator CH, Shoichet MS, Fast-gelling injectable blend of hyaluronan and methylcellulose for intrathecal,localized delivery to the injured spinal cord.Biomaterials 2006;27(11):2370-2379; Fang, JY, Chen, JP, Leu, YL, Temperature-sensitive hydrogels composed of chitosan and hyaluronic acid as injectable carriers for drug delivery.Eur J Pharm Biopharm 2008;68(3):626-636; and Ha DI, Lee SB, Chong MS et al., Preparation of thermo-responsive and injectable hydrogels based on hyaluronic acid and poly(N-isopropylacrylamide) and their drug release behaviors. Macromol. Res. 2006;14(1):87-93, each of which is incorporated herein by reference in its entirety for all purposes.

[0076] Crosslinked HA is commercially available from sources such as Sigma-Aldrich (St. Louis, MO) and Stanford Chemicals (Lake Forest, CA).

[0077] As used herein, the term "hydrogel" refers to a three-dimensional, hydrophilic or amphiphilic polymer network capable of absorbing large amounts of water. The network is composed of homopolymers or copolymers and is insoluble due to the presence of covalent chemical or physical (ionic, hydrophobic interactions, entanglements) crosslinks that provide the network structure and physical integrity. Hydrogels exhibit thermodynamic compatibility with water, which allows them to swell in aqueous media. The chains of the network are connected in such a way that pores exist, with a significant proportion of these pores being between 1 nm and 1000 nm in size. .

[0078] In certain embodiments, the methods described herein utilize one or more enzymes and / or compounds to cleave xHA, e.g., cleave xHA present in xHA-LP to form oHA-LP.

[0079] Enzymes suitable for cleaving xHA include, but are not limited to, bacterial β-endoglycosidases, bacterial β-exoglycosidases (such as β-glucuronidase and β-N-acetyl-hexosaminidase), eukaryotic β-endoglycosidases (such as endo-β-n-acetylhexosaminidase and β-endoglucuronidase), eukaryotic β-exoglycosidases (such as β-exoglucuronidase and exo-β-N-acetylglucosaminidase), hyaluronidase, and hyaluronoglucosidase.

[0080] Non-enzymatic methods of cleaving xHA include, but are not limited to, acid hydrolysis, alkaline hydrolysis, sonication, thermolysis, degradation by oxidants (e.g., superoxide anion radical, hydrogen peroxide, singlet oxygen, hydroxyl radical, nitric oxide, peroxynitrite anion, hypochlorite anion, carbonate radical anion, and dichloride radical anion, etc.), microwave irradiation, UV irradiation, γ-irradiation, and Hg lamp irradiation, etc. (See Stern et al. (2007) Biotechnol. Adv. 25:537, which is incorporated by reference in its entirety for all purposes.)

[0081] In other embodiments, the methods described herein utilize one or more hyaluronoglucosidases, such as hyaluronidases (HAases), to cleave xHA. For example, xHA present in xHA-LP is cleaved to form oHA-LP. In certain exemplary embodiments, a combination of two, three, four, or five or more HAases is used to cleave xHA. In other embodiments, a single HAase is used to cleave xHA.

[0082] As used herein, "hyaluronidase" refers to hyaluronoglucosidase, which catalyzes the degradation of HA by cleaving the (1->4)-linkage (EC 3.2.1.35) or (1->3)-linkage (EC 3.2.1.36) between N-acetylglucosamine and glucuronate.

[0083] There are three general classes of hyaluronidases: 1. mammalian-type hyaluronidases (EC 3.2.1.35), which are endo-beta-N-acetylhexosaminidases with tetrasaccharides and hexasaccharides as the major end products. They have both hydrolytic and transglycosidase activity and can degrade hyaluronan and chondroitin sulfate (CS), especially C4-S and C6-S; 2. bacterial hyaluronidases (EC 4.2.99.1), which degrade hyaluronan and, to a lesser extent, CS and DS. They are endo-beta-N-acetylhexosaminidases that function primarily by beta-elimination reactions producing disaccharide end products; and 3. hyaluronidases (EC 3.2.1.36). It is an endo-beta-glucuronidase derived from leeches, other parasites, and crustaceans that hydrolyzes beta 1-3 bonds to produce tetra- and hexasaccharide end products.

[0084] 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 no enzymatic activity towards any of the known substrates. None of the enzymes have been shown to have catalytic activity against hyaluronan. HYAL4 is a chondroitinase and lacks activity against hyaluronan. HYAL1 (also known as LUCA1, MPS9, and NAT6) is a typical acid-active enzyme, and PH20 is a typical neutral-active enzyme. Acid-active hyaluronidases such as HYAL1 and HYAL2 lack catalytic activity at neutral pH. For example, HYAL1 has no catalytic activity above pH 4.5 in vitro (Frost et al. (1997) Anal. Biochemistry). HYAL2 is an acid-active enzyme with very low specific activity in vitro.

[0085] HYAL5 is an HAase originally discovered in mice that is located in plasma and in the acrosomal membrane of acrosome-intact spermatozoa and is released during the acrosome reaction.HYAL6 is an HAase also discovered in mice.

[0086] Chemical grade HAases are commercially available from sources 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. 021665; Amphastar Pharmaceuticals), Hydase (bovine hyaluronidase; NDA No. 021716; Akorn Inc.), Hylenex (recombinant human hyaluronidase; NDA No. 021859; Halozyme), Vitrase (ovine hyaluronidase; NDA No. 021640; Bausch and Lomb), and Wydase (bovine hyaluronidase; NDA No. 006343; Baxter Healthcare).

[0087] Additional HAases suitable for use herein are described in: World Wide Web site: brenda-enzymes.org / enzyme.php?ecno=3.2.1.35; Karl Meyer and Maurice M. Rapport, Chapter "Hyaluronidases", Advances in Enzymology-and Related Areas of Molecular Biology, Vol. 13, pp. 199-236 (doi.org / 10.1002 / 9780470122587.ch6); Stern and Jedrzejas, Chem. Rev. 2006, 106, pp. 818-839, Hyaluronidases: Their Genomics, Structures, and Mechanisms of Action; Stern and Jedrzejas, Chem. Rev. 2008, 108, pp. 5061-5085; Yoshida et al. (2013) "KIAA1199, A deafness A gene of unknown function, is a new hyaluronan binding protein involved in hyaluronan depolymerization. Proc. Natl. Acad. Sci. USA 110, pp. 5612-5617; Nagaoka et al., 2015 Regulation of Hyaluronan (HA) Metabolism Mediated by HYBID, doi:10.1074 / jbc.M115.673566, first published online October 30, 2015; Yoshino et al., 2018 Biochemical and Biophysical Research Communications Vol. 505, No. 1, October 20, 2018; Yamaguchi et al., 2019 (Matrix Biol. (2019) pp. 78-79, pp. 139-146, TMEM2: A missing link in hyaluronan catabolism identified?; each of which is incorporated herein by reference in its entirety for all purposes.

[0088] "Hyaluronidase lyase," "Hyaluronic acid lyase," "HA lyase," "EC4. "2.2.1" or "HA lyase 4.2.2.1" refers to a hyaluronoglucosidase (i.e., a bacterial carbon-oxygen lyase) that cleaves hyaluronan chains at beta-D-GalNAc-(1->4)-beta-D-GlcA bonds, ultimately digesting the polysaccharide to 3-(4-deoxy-beta-D-gluc-4-enuronosyl)-N-acetyl-D-glucosamine. Hyaluronate lyase can be isolated from bacteria and Streptomyces and differs in its mode of action from hyaluronidases from other sources, as it catalyzes the elimination, rather than hydrolysis, of the beta-1,4-glycosidic bond between N-acetyl-beta-D-glucosamine and D-glucuronic acid residues.

[0089] In other embodiments, the methods described herein utilize one or more proteases to cleave peptides, for example enzymes that catalyze proteolysis by hydrolytically cleaving peptide bonds. For example, cleaving P present in xHA-LP to form oHA-LP. Suitable proteases include, but are not limited to, serine proteases, cysteine ​​proteases, threonine proteases, aspartic acid proteases, glutamic acid proteases, metalloproteases, and asparagine peptide lyases. Suitable proteases, classified according to the optimum pH at which the proteases are active, include, but are not limited to, acidic proteases, neutral proteases, and basic proteases. In certain exemplary embodiments, a combination of two, three, four, or five or more proteases is used to cleave peptides. In other embodiments, a single protease is used to cleave peptides.

[0090] In certain embodiments, the methods described herein utilize one or more enzymes and / or compounds to cleave a peptide drug present as a free drug or a prodrug, e.g., present as free SAR425899 or as a SAR425899 prodrug, to generate peptide digestion products of the drug or prodrug.

[0091] In certain embodiments, the methods described herein utilize one or more endoproteinases to cleave a peptide drug present as a free drug or a prodrug, e.g., as free SAR425899 or as a SAR425899 prodrug, to generate peptide digestion products of the drug or prodrug. The criteria used to select a suitable endoproteinase were: 1) generation of as few highly specific fragments as possible; 2) generation of C-terminal fragments that do not contain lysine-linked glutamate-palmitate; and 3) use of common commercially available enzymes.

[0092] Suitable endoproteinases include, but are not limited to, Glu-C, Asp-N, Lys-C, Arg-C, trypsin and chymotrypsin. In certain exemplary embodiments, the endoproteinase Asp-N is used in the methods described herein. Various suitable 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).

[0093] In certain embodiments, a pressure cycler can be used to improve (e.g., shorten) the digestion time of xHA and / or oHA. In certain embodiments, digestion of xHA and / or oHA can be carried out in a pressure cycler under a cycle pressure ranging between about 5KPSI and about 80KPSI, for example, about 5KPSI, about 10KPSI, about 15KPSI, about 20KPSI, about 25KPSI, about 30KPSI, about 35KPSI, about 40KPSI, about 45KPSI, about 50KPSI, about 55KPSI, about 60KPSI, about 65KPSI, about 70KPSI, about 75KPSI, or about 80KPSI.

[0094] In certain embodiments, a pressure cycler is used with one or more hyaluronoglucosidases (e.g., HAases) to digest xHA at a pressure of less than about 50 KPSI, less than about 40 KPSI, less than about 30 KPSI, or less than about 20 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 5 KPSI, about 10 KPSI, or about 15 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 10 KPSI.

[0095] In certain embodiments, a pressure cycler is used with one or more enzymes (e.g., endoproteinase, protease, Glu-C, Asp-N, Lys-C, Arg-C, trypsin, or chymotrypsin, etc.) to digest peptides at a pressure of less than about 80 KPSI, less than about 70 KPSI, less than about 60 KPSI, or less than about 500 KPSI. In certain embodiments, a pressure cycler is used with one or more enzymes (e.g., endoproteinase, protease, Glu-C, Asp-N, Lys-C, Arg-C, trypsin, or chymotrypsin, etc.) to digest peptides at a pressure of about 35 KPSI, about 40 KPSI, or about 45 KPSI. In certain embodiments, a pressure cycler is used with one or more enzymes (e.g., endoproteinase, proteolytic enzyme, Glu-C, Asp-N, Lys-C, Arg-C, trypsin, or chymotrypsin, etc.) to digest peptides at a pressure of about 40 KPSI.

[0096] In certain embodiments, the pressure cycler reduces digestion time of the peptide, xHA, and / or oHA by at least about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours or more compared to digestion under non-pressurized conditions.

[0097] As used herein, "peptide digestion product" refers to any portion or part 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, the peptide digestion product is less than 50 amino acids in length, e.g., between about 2 and about 100 amino acids in length, between about 3 and about 75 amino acids in length, between about 2 and about 50 amino acids in length, between about 4 and about 50 amino acids in length, between about 5 and about 40 amino acids in length, between about 6 and about 30 amino acids in length, or between about 15 and about 20 amino acids in length, or any value or subrange within these ranges. In certain exemplary embodiments, the peptide digestion products are about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 61, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, or about 100 amino acids in length.

[0098] In certain embodiments, the peptide digest is about 19 amino acids in length. In certain embodiments, the peptide digestion products are about 1 amino acid long, about 2 amino acids long, or about 3 amino acids long. In certain other embodiments, the peptide digestion products are between about 4 and about 50 amino acids long. In certain other embodiments, the peptide digestion products are between about 15 and about 20 amino acids long.

[0099] In certain exemplary embodiments, the peptide digestion products are detected using peptide identification methods 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), nano-HPLC-MS-MS, ultra-performance liquid chromatography-tandem MS (UPLC-MS-MS), nano-UPLC-MS-MS, ultra-performance liquid chromatography-tandem MS (UHPLC-MS-MS), nano-UHPLC-MS-MS, ultraviolet (UV) spectroscopy or fluorescence spectroscopy.

[0100] In certain exemplary embodiments, the methods described herein utilize separation processes such as chromatographic methods, e.g., liquid chromatography. According to one embodiment, detection of the peptide digestion products is performed using techniques such as: (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) one-dimensional electrophoresis; (ix) multidimensional electrophoresis; (x) size exclusion; (xi) affinity; (xii) reversed phase chromatography; (xiii) capillary electrophoresis chromatography ("CEC"); (xiv) electrophoresis; (xv) ion mobility separation; (xvi) field asymmetric ion mobility separation or spectroscopy. or spectrometry "FAIMS"); (xvii) capillary electrophoresis; and (xviii) supercritical fluid chromatography.

[0101] The amount of peptide digestion products can be determined by measuring multiple reaction monitoring (MRM) transitions consisting of peptide precursor ions, one or more fragment ions and retention times. This measurement is performed, for example, on a triple quadrupole instrument. Signatures can also be obtained by combining retention times with accurate high-resolution mass analysis of intact peptides. These quantification methods usually require a calibration curve of labeled internal standards and external synthetic peptides.

[0102] In certain exemplary embodiments, an internal standard is used that includes a labeled peptide corresponding to a peptide drug, for example, a labeled C-terminal peptide corresponding to the digestion product of the C-terminus of SAR425899 with Asp-N. The internal standard typically has a known peptide sequence and is provided in known amounts. In some embodiments, the standard is doped with one or more heavy isotopes, e.g., 13 C or 15 Labelled with N.

[0103] The peptide digestion product profiling methods described herein are useful for determining the amount of drug present in a hydrogel prodrug formulation. The methods described herein are also useful for performing batch-to-batch reproducibility assessments.

[0104] As used herein, "sample" refers to any composition that contains a peptide drug (e.g., in prodrug form). Exemplary samples include, but are not limited to, pharmaceutical compositions, and dissolution or release media. In certain embodiments, the sample is a hydrogel. In other embodiments, the sample is aqueous.

[0105] The samples used in the quantification methods described herein can be therapeutic compositions such as liquid formulations for oral, sublingual, mucosal, intradermal, subcutaneous, intravenous, intramuscular, parenteral or inhalation administration.

[0106] In other embodiments, the sample comprises a substrate such as nanoparticles, capsules, films or tablets, or gels such as hydrogels (e.g., xHA hydrogels). The quantification methods described herein are useful for quantifying the amount of peptide drug in a substrate such as, for example, nanoparticles or capsules, or films or hydrogels (e.g., xHA hydrogels). Release can be from the interior of the substrate, e.g., xHA hydrogels, or from the exterior (e.g., surface) of the substrate. In one embodiment, the release profile is assayed by performing a complete release of the peptide drug, and then assaying a controlled release over a period of time, or under different culture or solution conditions (e.g., various temperatures or pHs, etc.). The amount of peptide drug released in a controlled release assay is usually reported as a fraction or percentage when compared to the amount of peptide drug released under complete release conditions.

[0107] As used herein, "dissolution medium", "dissolution media", "release medium" and "release media" refer to compositions used to obtain in vitro drug release information. Dissolution or release media are useful, for example, for quality control testing of samples to determine the release and / or stability of peptide drugs in the samples. 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, sustained release, etc.) and / or the solubility profile of the peptide drug. For a review of dissolution medium selection, see Martin and Gray (Summer 2011) Journal of Validation Technology.

[0108] As used herein, "release rate" refers to the rate at which peptide drugs flow from hydrogel prodrug formulations into the surrounding medium in in vitro release testing. In an exemplary embodiment, a composition is first prepared for release testing by suspending the composition in a suitable in vitro release medium. This is generally performed by centrifuging and precipitating the substrate (e.g., hydrogel), followed by buffer exchange and reconstitution of the substrate using mild conditions. In certain embodiments, the assay is initiated by suspending the sample at 37°C in a suitable temperature control device. Samples are typically removed at various time points.

[0109] As will be readily understood by those skilled in the art, other suitable modifications and adaptations of the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. Although certain embodiments have been described in detail herein, the same will be more clearly understood by reference to the following examples. The following examples are included for illustrative purposes only and are not intended to be limiting.

[0110] [Example I] Quantification of peptide drugs present in prodrug formulations SAR425899 is a dual receptor agonist that targets the glucagon and GLP1 receptors. Its rapid clearance necessitates once-daily dosing. To reduce the frequency of dosing, the once-daily SAR425899 peptide drug candidate (Figure 1) was conjugated to a polymeric carrier (crosslinked hyaluronic acid (xHA)) via a self-cleavable linker to allow slow release of the active peptide drug from an inactive prodrug (Figure 2). The SAR425899 prodrug contains crosslinked hyaluronic acid (xHA) linked to the SAR425899 lipopeptide (P) via a self-cleavable linker (L).

[0111] Determination of peptide loading of hydrogels is necessary for dosing and quality control. Cross-linked hyaluronic acid forms hydrogels of several million daltons in an aqueous environment. UV-based measurements, which are usually applied for basic quantification of dissolved peptides, cannot be applied to hydrogels because this analysis requires the hydrogel-linker-peptide to be fully dissolved. Although the linker is self-cleaving, complete cleavage under physiological conditions is intentionally very slow, and therefore not suitable for analytical quantification methods that require certainty that complete release has been achieved.

[0112] To address this analytical challenge and enable peptide loading determination, a novel method was developed that allows accurate quantification of peptide drug loading in biopolymer prodrugs as weight / weight percent. The biopolymer-linker-peptide drug method can in principle be applied to any small molecule (e.g., polypeptide or polynucleotide) drug that can be cleaved or hydrolyzed, and this method can be broadly applied to drug loading determination of biopolymer-containing prodrugs.

[0113] The method is based on double enzymatic digestion followed by quantification of digestion products corresponding to peptides. First, cross-linked hyaluronic acid-linker-peptide (xHA-LP) is digested with an enzyme that digests cross-linked hyaluronic acid. Then, a proteolytic enzyme is applied to digest the linked peptides, producing proteolytic digestion products. The proteolytic digestion products (corresponding to peptides) are then quantified.

[0114] xHA-LP is weighed and partially dissolved as a hydrogel in buffer. The hydrogel is enzymatically degraded to multiple soluble oligomeric hyaluronic acid-linker-peptide (oHA-LP). The resulting oHA-LP hyaluronic acid oligomers are a heterogeneous mixture, consisting of peptides, linkers, and oligomeric hyaluronic acid of different lengths. This heterogeneity of oligomeric hyaluronic acid is undesirable for quantification, and for this reason a second enzymatic (e.g., proteolytic) digestion step is introduced to digest a portion of the peptide drug to produce a homogenous 19 amino acid C-terminal peptide digestion product, DFIE....PPPS-NH2 (Figure 3). This C-terminal peptide is detected and quantified using LC / HRMS.

[0115] Digestion of the prodrug with Asp-N without prior hyaluronoglucosidase treatment gave low yields of C-terminal peptides (Figure 5), likely due to steric hindrance of Asp-N activity by cross-linked hyaluronan. Linker hydrolysis was deemed impractical as a method of peptide release for quantification.

[0116] A method for digesting HA was developed (Figure 6). Suitable digestion conditions for subsequent digestion with endoproteinase (e.g., Asp-N) were used. Multiple enzymes capable of digesting HA (e.g., HAase) were tested.

[0117] Digestion of cross-linked hyaluronan did not interfere with the digestion of free SAR425899 with Asp-N (Figures 7A-F), and was shown to significantly increase the yield of the C-terminal peptide digestion product following digestion with Asp-N (Figures 8A-B). Indeed, it was found that the Asp-N protease provided nearly 100% digestion efficiency for the SAR425899 peptide.

[0118] Asp-N was shown to completely digest SAR425899 in buffer and in the presence of HA digestion products (Figure 4). Digestion was performed in triplicate on the HA-linker-peptide with HAase followed by digestion with Asp-N and quenching. Subsequent hydrolysis was carried out at 37°C for 24 h to release any bound intact peptides, and an internal standard (IS) was added prior to LC / MS analysis. The ratio of released intact peptides to C-terminal peptides was 0.0006.

[0119] Double digestion was performed in triplicate on the HA-linker-peptide with HAase for 2×24 h, followed by digestion with Asp-N and quenching. Subsequent hydrolysis at 37° C. was carried out for 24 h to release any bound intact peptide, and IS was added prior to LC / MS analysis. No intact peptide was observed.

[0120] Digestion was performed in triplicate on the HA-linker-peptide with HAase for 14 days followed by digestion with Asp-N. No increase in C-terminal peptides was observed compared to the 1-day digestion, but the C-terminal peptide isomer was observed eluting slightly earlier than the normal C-terminal peptide.

[0121] LC / MS of the reaction products was performed as follows: Samples and standards were prepared in 25% acetonitrile (ACN), 0.1% formic acid (FA) to prevent observed adsorption problems. Separation was performed on an Accela 1250 LC system equipped with a Luna Omega C18 column 100 x 2.1 mm, 100A (product number OOD-4742-AN) (S / N H16-168886) operated at 50 °C.

[0122] Mobile phase A: 0.1% formic acid in water; mobile phase B: ACN 0.1% formic acid; flow rate was 400 μL / min. The gradient was 5% to 50% B in 7 min, held at 99% B for 30 s, and equilibrated at 5% B for 2.5 min. A gradient starting at 20% B was also applied and was viable.

[0123] The eluent was introduced into the Orbitrap ELITE using a standard HESI ESI source operated in positive ion mode. An Orbitrap resolution of 60000@m / z 400 was applied. m / z range 350-1500.

[0124] It was found that no intact peptides remained and all peptides were converted into digestion products.

[0125] [Table 1] Table 1. xHA digested with HAase-1, added C-terminal peptide (corresponding to the Asp-N digest), analyzed by LC / MS (Experiment #1); xHA digested with HAase-1, added full-length peptide, digested with Asp-N, analyzed by LC / MS (Experiment #2); xHA digested with HAase-2, added C-terminal peptide corresponding to the Asp-N digest, analyzed by LC / MS (Experiment #3); xHA digested with HAase-2, added full-length peptide, digested with Asp-N, analyzed by LC / MS (Experiment #4); control-added full-length peptide, digested with Asp-N, analyzed by LC / MS (Experiment #5); xHA-linker-peptide digested with HAase-1, then digested with Asp-N, analyzed by LC / MS (Experiment #6); xHA-linker-peptide The tides were digested with HAase-2 and then with Asp-N and analyzed by LC / MS (experiment #7).

[0126] High yields of C-terminal peptides after sequential double digestion were demonstrated by several methods (Figure 10). The double digestion was allowed to proceed, after which all enzyme activity was quenched by boiling. Release of any intact peptides that may have remained attached to hyaluronic acid via linkers was then performed by allowing hydrolysis to proceed at 37°C. No or very little intact peptides were observed, indicating that the double digestion resulted in near complete digestion of all peptide drug molecules. In another experiment, digestion with Asp-N after hydrolytic release over a 2-week period did not produce more C-terminal peptides than the double digestion procedure. As part of the method qualification, it was analytically shown that the resulting batch of xHA-LP had a peptide loading of 18%, which was consistent with results obtained showing loadings around 20% for some lots (without being bound by chemical theory, the lower amount of 20% was due to water absorption of the lyophilized material). If the lyophilized hydrogel absorbs water, the weighed amount will consequently contain a lower percentage of peptide due to the addition of water. This can result in a peptide percentage between 20% and 18%. As a countermeasure, care should be taken to ensure that the material being weighed is not exposed to humid air conditions.

[0127] Cross-linked HA that did not contain peptide was digested and used as a matrix. A synthetic unlabeled C-terminal peptide corresponding to the peptide digestion product of SAR425899 with C-terminus Asp-N was added to this digestion product (matrix) to generate a dilution series. A heavy isotope-labeled C-terminal peptide was applied as an internal standard. The curves were compared with equivalent curves made in a buffer of 25% ACN and 0.1% formic acid.

[0128] Both curves were analyzed by LC / MS analysis to determine if the slopes were within 15% of each other, which is the minimum criterion for an acceptable buffer calibration curve.

[0129] Quantification of peptides using the method described herein was reproducible. Three aliquots of HA-linker peptide were weighed and digested with HAase and Asp-N. The digestion product was adjusted to 25% ACN and then further diluted 100-fold, followed by a further 2-fold dilution by adding heavy isotope-labeled internal standard (H-IS). A 10-point standard curve was generated from 10 μg / mL to 20 ng / mL in 2-fold dilution steps. The curve was further diluted 2-fold by adding H-IS 1:1 vol:vol.

[0130] The method was robustly adapted to the matrix (Figures 7A-7F). The calibration curves of the C-terminal peptides in buffer showed similar slopes to those in the extraction matrix produced by digestion of cross-linked hyaluronic acid (xHA) (Figures 9A, 9B, 11A, 11B, 12A, 12B, 13A, and 13B). It was also confirmed that the matrix produced by digestion of xHA did not interfere with the digestion efficiency of Asp-N.

[0131] Several commercially available hyaluronidases were tested and HAase 2 was determined to be predominant. Furthermore, the C-terminal peptide was found to be highly resistant to gas-phase collision-induced fragmentation. It was demonstrated that, when subjected to chemically inducible fragmentation (CID), a nearly complete series of fragments was generated, and that these fragments exhibited intensities suitable for the development of a multiple reaction monitoring quantification method, MRM (LC / MSMRM), as an alternative to the high-resolution mass spectrometry-based method (quantification by LC / HRMS) applied in the initial studies.

[0132] A prerequisite for UV or fluorescence detection methods is that the analyte is soluble. P is not soluble. On the other hand, oHA-LP is water soluble and therefore available for UV or fluorescent analysis. However, for the digestion required to produce oHA-LP from xHA-LP, hyaluronoglucosidase, e.g., hyaluronidase or HA lyase, is introduced into the mixture. Hyaluronoglucosidase, e.g., hyaluronidase or HA lyase, is itself a UV absorbing protein. Thus, hyaluronoglucosidase is removed after the first digestion step, followed by direct UV detection. This method would be much more suitable for a development / production environment for controlled analysis.

[0133] The peptide loading of three separate batches of xHA-LP, where P is SAR425899, was determined (FIG. 14). Excellent linearity was observed without matrix effects. Good reproducibility was achieved for all process repeats, and the results were consistent with those obtained using orthogonal NMR. The methods described herein are broadly applicable and have been applied to aid in formulation optimization. The methods described herein can be applied to both cleavable and non-cleavable linker cases, using a wide range of polymer and peptide drugs.

[0134] summary An LC / MS method was developed for analysis of HA-linker-SAR425899, SAR425899, and fragment / digestion products. SAR425899 was demonstrated to be completely cleaved by Asp-N, releasing a C-terminal peptide fragment for quantification. HA-linker-SAR425899 demonstrated initial cleavage by Asp-N, but the digestion was likely incomplete based on the modest LC / MS response.

[0135] Multiple HAases were tested. HA lyase (Sigma-Aldrich) EC4.2.2.1 was shown to significantly increase the yield of Asp-N digestion products. An isotopically labeled C-terminal peptide was introduced and its range of linearity was shown after the attachment issue was resolved. Experiments were performed to evaluate the digestion efficiency. Examination of the matrix curves showed no matrix effects and good matrix vs. buffer curve alignment with slopes differing by only 3%. Weighing, processing and analysis of triplicates showed good reproducibility with 7.5% CV.

[0136] The total percentage of peptides in one sample lot was found to be 18% (the water content of the sample was unknown, especially after multiple freeze-thaw (room temperature) cycles).

[0137] [Example II] A pressure cycler for quantification of peptide drugs present in prodrug formulations The amount of drug loaded into the SAR425899 prodrug was determined using a novel pressure cycler process by hydrating and denaturing the prodrug. The digestion reaction was tested for digestion of SAR425899 with AspN (100% reaction was achieved in 1 hour). Conditions were identified that allowed for a reduction in xHA digestion time from 24 hours to 2 hours using the pressure cycler.

[0138] HAase appeared to be sensitive to extremely high pressure. Therefore, a maximum pressure of 10 KPSI was applied and higher enzyme concentrations were used. Under optimal conditions, higher oHA-LP digestion concentrations were observed after 2 h of pressure cycler-assisted digestion.

[0139] 16 samples were assayed simultaneously (50-150 μL / sample). The rate of pressure increase / decrease was controlled. The shape of the pressure profile (sine wave / square wave, etc.) was observed. The temperature of the samples was controlled using a built-in electric heater. The maximum pressure used was 40 KPSI. A typical assay was performed with pressures ranging from atmospheric to 10-40 KPSI applied at 1 Hz cycles and duration of 1 hour.

[0140] AspN digestion of oHA-LP under 40 kPSI pressure cycles once per minute at 37° C. was determined to be as effective as AspN digestion of oHA-LP for 12 hours at atmospheric pressure at 37° C. All experiments were performed using a Pressure Biosciences Barocycler (Model 2320EXT, South Easton, Mass.).

[0141] It was determined that digestion with AspN using cycling between 40 KPSI pressure and atmospheric pressure at 1 minute intervals for 1 hour at 37° C. resulted in similar digestion efficiency as 12 hours at atmospheric pressure at 37° C. Similarly, HAase (EC 4.2.2.1) was digested using cycling between 10 KPSI pressure and atmospheric pressure at 1 minute cycle times for 2 hours, which, based on the oligomers observed, gave results similar to 24 hours of digestion at 37° C. and atmospheric pressure.

[0142] In general, each new enzyme was optimized with respect to temperature and pressure, pressure profile, frequency and duration of applied pressure.

[0143] Sample purity and determination of major impurities were analyzed (Figure 15). Release of intact peptide under mild hydrolysis conditions was confirmed (4°C for 14 days or 37°C for 24 hours). Both release methods produced sufficient material for impurity analysis by LC / MS, below 0.01%. Release was demonstrated to produce no impurities.

[0144] Linker-peptide controls were analyzed at t=0 and throughout the release time. High levels of impurities were observed in the linker-peptide at t=0. Impurity analysis was performed by assaying the LC / MS of the released peptide.

[0145] Overall conclusion Quantification by LC / MS method was successfully completed and performed. Digestion conditions that yielded complete AspN digestion were demonstrated. Matrix was found to be not an issue. Good reproducibility and linearity of the method was demonstrated.

[0146] The results obtained by LC / MS were consistent with those obtained by the orthogonal NMR method. The method using a second generation pressure cycler significantly reduced the total analysis time. The method is applied both as a reference method and to assist formulation optimization studies. Methods for the release and analysis of impurities were established. For example, release conditions were established and impurities were detected and identified.

[0147] Equivalent The present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. The above-described embodiments should therefore be considered in all respects as illustrative rather than limiting of the present disclosure. The scope of the present disclosure is therefore indicated by the appended claims rather than the above description, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

1. 1. A method for determining the amount of drug present in a crosslinked hyaluronic acid-linker-peptide (xHA-LP) prodrug formulation, comprising: A sample of the xHA-LP prodrug formulation; contacting with hyaluronoglucosidase to produce an oligomeric hyaluronic acid-linker-peptide drug (oHA-LP); contacting the oHA-LP with a second enzyme to produce a peptide digestion product of the drug; and Detecting the peptide digestion products to determine the amount of drug present in the xHA-LP prodrug formulation. The method comprising:

2. 2. The method of claim 1, wherein the peptide digestion products are between about 2 amino acids in length and about 100 amino acids in length.

3. 2. The method of claim 1, wherein the peptide digestion products are between about 3 amino acids in length and about 75 amino acids in length.

4. 2. The method of claim 1, wherein the peptide digestion products are between about 4 amino acids in length and about 50 amino acids in length.

5. 2. The method of claim 1, wherein the peptide digestion products are between about 6 amino acids in length and about 30 amino acids in length.

6. 2. The method of claim 1, wherein the peptide digestion products are between about 15 and about 20 amino acids in length.

7. 2. The method of claim 1, wherein the peptide digestion products are about 1 amino acid in length, about 2 amino acids in length, about 3 amino acids in length, about 19 amino acids in length, or any combination thereof.

8. 2. The method of claim 1, wherein the step of detecting the peptide digestion products is carried out 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 methods.

9. The method of claim 1 , wherein the hyaluronoglucosidase is a hyaluronidase (HAase).

10. 10. The method of claim 9, wherein the HAase is selected from the group consisting of HAase 1, HAase 2, HAase 3, HAase 4, HAase 5 and HAase 6.

11. The method of claim 10, wherein the HAase is HAase 1 or HAase 2.

12. The method of claim 11 , wherein the HAase is HAase 2.

13. 2. The method of claim 1, wherein the hyaluronoglucosidase is a hyaluronic acid (HA) lyase EC 4.2.2.

1.

14. The method of claim 1, wherein the oHA-LP is contacted with an endoproteinase.

15. 15. The method of claim 14, wherein the endoproteinase is selected from the group consisting of Glu-C, Asp-N, Lys-C, Arg-C, trypsin and chymotrypsin.

16. The method of claim 15, wherein the endoproteinase is Asp-N.

17. The method of claim 1 further comprising the use of an internal standard.

18. 20. The method of claim 17, wherein the internal standard comprises one or more heavy isotopes.

19. The method of claim 1 , wherein the amount of drug present is determined using a calibration curve.

20. The method of claim 1, wherein the xHA-L-P is contacted with the hyaluronoglucosidase in a pressure cycler.

21. 21. The method of claim 20, wherein the pressure in the pressure cycler is greater than atmospheric pressure.

22. 22. The method of claim 21, wherein the pressure is about 5 KPSI, about 10 KPSI, or about 15 KPSI.

23. The method of claim 1, wherein the oHA-L-P is contacted with the second enzyme in a pressure cycler.

24. 24. The method of claim 23, wherein the pressure in the pressure cycler is greater than atmospheric pressure.

25. 25. The method of claim 24, wherein the pressure is about 35 KPSI, about 40 KPSI or about 45 KPSI.

26. 1. A method for determining the amount of drug present in a crosslinked hyaluronic acid-linker-peptide (xHA-LP) prodrug formulation, comprising: contacting a sample of the xHA-LP prodrug formulation with hyaluronoglucosidase to produce an oligomeric hyaluronic acid-linker-peptide drug (oHA-LP); contacting the oHA-LP with an endoproteinase to produce peptide digestion products of the drug; and Detecting the peptide digestion products to determine the amount of drug present in the xHA-LP prodrug formulation. The method comprising:

27. 27. The method of claim 26, wherein the step of detecting the peptide digestion products is carried out 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 methods.

28. 27. The method of claim 26, wherein the hyaluronoglucosidase is HAase 1 or HAase 2.

29. The hyaluronoglucosidase of claim 26 is HA lyase EC 4.2.2.

1. The method described.

30. 27. The method of claim 26, wherein the endoproteinase is selected from the group consisting of Glu-C, Asp-N, Lys-C, Arg-C, trypsin and chymotrypsin.

31. The method of claim 30, wherein the endoproteinase is Asp-N.

32. 27. The method of claim 26, further comprising the use of an internal standard.

33. 33. The method of claim 32, wherein the internal standard comprises one or more heavy isotopes.

34. 27. The method of claim 26, wherein the amount of drug present is determined using a calibration curve.

35. 27. The method of claim 26, wherein the xHA-L-P is contacted with hyaluronoglucosidase in a pressure cycler.

36. 36. The method of claim 35, wherein the pressure in the pressure cycler is greater than atmospheric pressure.

37. 37. The method of claim 36, wherein the pressure is about 5 KPSI, about 10 KPSI, or about 15 KPSI.

38. The method of claim 26, wherein the oHA-L-P is contacted with the endoproteinase in a pressure cycler.

39. 40. The method of claim 38, wherein the pressure in the pressure cycler is greater than atmospheric pressure.

40. 40. The method of claim 39, wherein the pressure is about 35 KPSI, about 40 KPSI, or about 45 KPSI.

41. 1. A method for determining the amount of drug present in a crosslinked hyaluronic acid-linker-peptide (xHA-LP) prodrug formulation, comprising: contacting a sample of the xHA-LP prodrug formulation with HA lyase EC 4.2.2.1 to produce an oligomeric hyaluronic acid-linker-peptide drug (oHA-LP); contacting the oHA-LP with Asp-N to produce a peptide digestion product of the drug; and Detecting the peptide digestion products to determine the amount of drug present in the xHA-LP prodrug formulation. The method comprising:

42. 42. The method of claim 41 , wherein the peptide digestion products are between about 2 and about 100 amino acids in length, between about 3 and about 75 amino acids in length, between about 4 and about 50 amino acids in length, between about 6 and about 30 amino acids in length, between about 15 and about 20 amino acids in length, or are about 1, about 2, about 3, or about 19 amino acids in length.

43. The step of detecting the peptide digestion products is carried out by a method selected from the group consisting 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 methods, or a combination thereof.

42. The method of claim 41 .

44. 42. The method of claim 41, further comprising the use of an internal standard.

45. 45. The method of claim 44, wherein the internal standard comprises one or more heavy isotopes.

46. 42. The method of claim 41, wherein the amount of drug present is determined using a calibration curve.

47. 42. The method of claim 41, wherein xHA-L-P is contacted with HA lyase EC 4.2.2.1 in a pressure cycler.

48. 48. The method of claim 47, wherein the pressure in the pressure cycler is greater than atmospheric pressure.

49. 49. The method of claim 48, wherein the pressure is about 5 KPSI, about 10 KPSI, or about 15 KPSI.

50. The method of claim 41, wherein oHA-L-P is contacted with Asp-N in a pressure cycler.

51. 51. The method of claim 50, wherein the pressure in the pressure cycler is greater than atmospheric pressure.

52. 52. The method of claim 51, wherein the pressure is about 35 KPSI, about 40 KPSI or about 45 KPSI.

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