DECLARATION OF LEONARD J. CHYALL, Pu.D.
`
`I, the undersigned, Dr. Leonard J. Chyall, U.S Passport No. 432624896, with a business
`
`address of 3065 Kent Avenue, West Lafayette, in the State oflndiana, USA, having been
`
`warned that I must state the truth and that I shall be liable to the penalties prescribed by
`
`law should I fail to do so, hereby declare in writing as follows:
`
`I.
`
`INTRODUCTION
`
`1.
`
`I have been retained by Teva Pharmaceutical Industries Ltd. ("Teva") to
`
`provide opinions and analyses related to sitagliptin phosphate. This report sets forth my
`
`analyses and opinions relating to this topic, based on the work carried out by me or under
`
`my supervision or instruction, as well as on the materials I reviewed for the purpose of
`
`that work. Based on this work and the materials I have reviewed, it is my conclusion, as
`
`explained below, that upon reaction of sitagliptin free base with phosphoric acid, only
`
`sitagliptin dihydrogen phosphate (1: 1 adduct between sitagliptin base and phosphoric
`
`acid) is formed.
`
`A.
`
`2.
`
`Background And Qualifications
`
`I am a Ph.D. chemist specializing in the study of organic materials,
`
`including organic materials in the solid state and solution. I have specific qualifications
`
`in the preparation and analyses of pharmaceutical drug substances. As outlined below, I
`
`am an organic chemist by profession, with training and experience in these areas
`
`3.
`
`I am currently employed by Aptuit, Inc. ("Aptuit"). Aptuit is a research
`
`and information company that provides problem solving and analytical research to a
`
`broad range of pharmaceutical companies. Aptuit provides a complete range of services
`
`covering all aspects of pharmaceutical development. Among other things, the scientists
`
`at Aptuit perform syntheses and chemical analyses of pharmaceuticals. Aptuit also offers
`
`a broad range of analytical testing services. Aptuit performs work for both innovator and
`
`generic companies. Aptuit also has expertise in the characterization of materials in the
`
`solid state and in solution. Aptuit also performs solid form and salt selection studies for
`
`active pharmaceutical ingredients ("API"). Aptuit also provides pharmacopoeia-based
`
`- I -
`
`Merck Exhibit 2225, Page 1
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`analytical testing, stability testing of drug substances in the solid state and in solution,
`
`stabilization studies of drugs either as solids or solutions, and consulting on regulatory
`
`issues, among other services.
`
`4.
`
`My laboratory at Aptuit, which is located in West Lafayette, Indiana, is a
`
`current Good Manufacturing Practices ("cGMP") laboratory. To maintain cGMP
`
`compliance, our employees constantly monitor and comply with United States Food and
`
`Drug Administration ("FDA") guidance documents. Our facility is the subject of routine
`
`compliance audits by FDA and our clients. Our work meets the highest standards of
`
`control and reliability set by FDA and the pharmaceutical industry. As a cGMP
`
`laboratory, our studies on drug substances and drug products (formulations) are routinely
`
`submitted to FDA.
`
`5.
`
`I obtained my Bachelor of Arts degree in chemistry from Oberlin College
`
`in 1986 and my Ph.D. in chemistry from the University of Minnesota in 1991. My
`
`doctoral research involved the synthesis and characterization of novel organic molecules.
`
`My dissertation focused on understanding the reactivity of high-energy cyclopropane
`
`molecules.
`
`6.
`
`I was a postdoctoral fellow from 1992-1996 in the chemistry department
`
`at Purdue University, where I furthered my understanding of the properties and reactivity
`
`of organic molecules. These studies involved both small molecular weight molecules as
`
`well as large biological molecules.
`
`7.
`
`Following my postdoctoral fellowship, I worked as a research chemist at
`
`Great Lakes Chemical Corporation. My research involved the identification,
`
`characterization and development of new products for the company. In 2000, I became a
`
`research investigator at SSCI, Inc. and in 2003, I became a senior research investigator.
`
`In 2006, SSCI was acquired by Aptuit. SSCI now operates as an integrated division of
`
`Aptuit. I currently hold the title of Director in the SSCI division of Aptuit.
`
`8.
`
`Through my education and work experiences, I have obtained extensive
`
`knowledge and training in chemistry with specific experience in the areas of organic
`
`- 2 -
`
`Merck Exhibit 2225, Page 2
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`chemistry and pharmaceutical sciences. I have authored or co-authored 22 publications
`
`in peer reviewed scientific journals that are listed in the attached curriculum vitae
`
`(Exhibit A). The most recent of these publications have involved scientific research that
`
`is related to the properties of pharmaceuticals. I am the inventor or co-inventor of three
`
`patented inventions granted by the United States Patent and Trademark Office. I have
`
`given numerous scientific presentations at various technical meetings.
`
`9.
`
`While at SSCI and Aptuit, I have worked on numerous projects providing
`
`research and consulting services related to the development of new pharmaceutical
`
`products. My scientific expertise has been applied to the characterization of drug
`
`substances in both the solid state and in solution. For example, I have managed research
`
`protocols involving the identification and selection of the appropriate crystalline forms of
`
`a drug substance that are suitable for further development and commercialization. These
`
`research protocols have included polymorph screening experiments, salt selection work,
`
`cocrystal screening and studies of amorphous pharmaceuticals, among other areas. I have
`
`worked with solid oral dosage forms and pharmaceuticals being developed for parenteral,
`
`topical and transdermal dosage forms.
`
`10.
`
`Based upon my education and experience, I am qualified to conduct
`
`chemistry experiments and perform analytical tests involving organic chemicals and
`
`pharmaceuticals. In particular, I am qualified to perform the experiments and analyses
`
`set out herein. All of this work was conducted by either myself, by those under my direct
`
`supervision, or by qualified outside laboratories at my request and under my instruction.
`
`11. My background and qualifications, and a complete list of my publications
`
`are more fully set out in my curriculum vitae, attached as Exhibit A.
`
`B.
`
`12.
`
`Compensation
`
`I have no financial interest in the outcome of this matter. I am not
`
`specially compensated for my work on this case, and receive my salary from my
`
`employer.
`
`- 3 -
`
`Merck Exhibit 2225, Page 3
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`II.
`
`EXPERIMENTAL BACKGROUND
`
`A.
`
`13.
`
`General Considerations
`
`I have been asked by counsel for Teva to perform laboratory experiments
`
`and analyses of samples of sitagliptin free base and sitagliptin phosphate salt. More
`
`specifically, I have been asked to perform experiments designed to probe whether
`
`sitagliptin could combine with phosphoric acid in a manner to produce a salt other than
`
`sitagliptin dihydrogen phosphate.
`
`14.
`
`I understand the chemical structure of sitagliptin base to be the structure
`
`depicted in Figure 1.
`
`F
`
`F
`
`Figure 1: Structure of sitagliptin.
`
`15.
`
`I understand phosphoric acid to have the molecular formula of H3PO4.
`
`B.
`
`16.
`
`Materials Reviewed
`
`I received from Teva two sample containers labeled Lot sal-069 and sal-
`
`008,087, which I understand to contain approximately 10 and 20 grams of sitagliptin base,
`
`respectively. Both samples were characterized by X-ray Powder Diffraction (XRPD).
`
`The XRPD patterns obtained for these samples (attached as Exhibit B) confirmed that
`
`the material is crystalline sitagliptin base, as disclosed in U.S. Patent Application Serial
`
`No. 12/740,693 (specification filed April 30, 2010) (attached as Exhibit C). The XRPD
`
`patterns for these materials were used as reference patterns in subsequent pH solubility
`
`experiments.
`
`- 4 -
`
`Merck Exhibit 2225, Page 4
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`17.
`
`In addition, the two samples of sitagliptin base were analyzed by proton
`
`NMR spectroscopy and optical microscopy. These analyses are attached as Exhibit D.
`
`The chemical shifts and the integration of the proton resonances in the NMR spectrum
`
`indicate that the material is sitagliptin base. The NMR analysis also indicated that the
`
`sample was acceptably pure to conduct the experiments described herein. The optical
`
`microscopy images provide additional confirmation that the samples are crystalline due
`
`the presence of birefringence and extinction when viewed under magnification using
`
`polarized light.
`
`18.
`
`I have also received from Teva two containers of material labeled lot no.
`
`D-1895NN-13067/3 and lot no. D1895MM14084/2. The sample container labeled lot no.
`
`D-1895NN-13067/3 contained approximately 10 grams of material that I understood to
`
`be sitagliptin dihydrogen phosphate. This sample was characterized by proton NMR
`
`spectroscopy, which confirmed the chemical identity of this material. In addition, it was
`
`characterized by XRPD, which demonstrated that the sample is a crystalline solid. The
`
`proton NMR spectra and XRPD patterns for lot no. D-1895NN-13067/3 are attached as
`
`Exhibit E. Comparison of the XRPD pattern so obtained with XRPD data of known
`
`solid forms of sitagliptin dihydrogen phosphate as disclosed in, inter alia, US
`
`2010/0041885 Al (Exhibit F) and WO 2005/020920 (Exhibit G), confirmed that the
`
`material in this vial is sitagliptin dihydrogen phosphate. The so obtained diffraction
`
`pattern was used as a reference pattern for subsequent pH solubility experiments
`
`described herein. In addition, this sample was analyzed by optical microscopy (images
`
`attached as Exhibit H). The optical microscopy images provide additional confirmation
`
`that the samples are crystalline due the presence of birefringence and extinction when
`
`viewed under magnification using polarized light.
`
`19.
`
`The container labeled lot no. D1895MM14084/2 contains approximately
`
`10 grams of material that I understand to be sitagliptin dihydrogen phosphate. I did not
`
`use this sample in any testing conducted at Aptuit.
`
`20.
`
`Phosphoric acid and other chemical reagents were obtained from
`
`commercial suppliers and used as-received. As a cGMP-validated laboratory and in
`
`- 5 -
`
`Merck Exhibit 2225, Page 5
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`order to be in compliance with our standard operating procedures (SOPs), my laboratory
`
`at Aptuit obtains Certificates of Analyses for all chemicals and reagents purchased from
`
`its suppliers, and no expired reagents were used in the testing conducted for the work
`
`described herein.
`
`C.
`
`21.
`
`Materials Relied Upon
`
`In reaching my opinions described herein, I relied on the documents
`
`referenced herein. I also relied on my general knowledge and experience, as well as my
`
`own scientific analyses. The opinions I express in this report are based on the
`
`information and evidence currently available to me.
`
`III.
`
`SITAGLIPTIN SALT FORMATION EXPERIMENTS
`
`22.
`
`I performed twelve salt formation experiments involving chemical
`
`reactions between sitagliptin base (Figure 1) and phosphoric acid and analyzed the solid
`
`products obtained from these reactions using a variety of techniques. The following
`
`summarizes these experiments.
`
`23.
`
`The aforementioned twelve salt formation experiments were conducted by
`
`varying common parameters used in screening for potential salts of pharmaceutical AP Is.
`
`These include the composition of the solvent, the temperature during the reaction and the
`
`molar ratio of acid to base. Many of these experiments were a deliberate attempt to
`
`obtain a phosphate salt other than a 1: 1 adduct of sitagliptin and phosphoric acid.
`
`-6-
`
`Merck Exhibit 2225, Page 6
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`24.
`
`A tabular summary of the salt formation experiments conducted between
`
`sitagliptin and phosphoric acid is given in Table 1.
`
`Table 1: Salt Formation Experiments Conducted with Sitagliptin
`
`Sample
`ID
`233140
`233141
`234636
`233142
`234584
`234874
`234872
`234873
`235805
`235806
`235848
`235849
`
`Notebook
`No.
`4063-02-01
`4063-03-01
`4063-18-01
`4063-04-01
`4063-19-01
`4063-35-01
`4063-34-01
`4063-32-01
`4063-50-01
`4063-51-01
`4063-56-01
`4063-57-01
`
`Ratio of
`API:H3P04
`1.00: 1.05
`1.00: 2.10
`3.00: 1.00
`2.04: 1.00
`1.00: 5.01
`2.04: 1.00
`1.00: 1.05
`1.00: 2.10
`1.00: 2.10
`2.04: 1.00
`1.00: 2.10
`2.04: 1.00
`
`Reaction Solvent
`
`Temperature
`
`methanol
`methanol
`methanol
`methanol
`methanol
`12.5 % water in methanol
`12.5 % water in methanol
`12.5 % water in methanol
`methanol
`methanol
`methanol
`methanol
`
`ambient
`ambient
`ambient
`ambient
`ambient
`ambient
`ambient
`ambient
`0°C
`0°C
`65°C
`65 °C
`
`25.
`
`In some of these experiments an excess of sitagliptin base was used for the
`
`purpose of promoting the formation of a stable salt species containing either two or three
`
`molecules of sitagliptin for each molecule of phosphoric acid. As an example, for
`
`Sample ID 233142 I attempted to prepare a species corresponding to the salt with the
`formula (SG-H+)2HPO/- where "SG" corresponds to sitagliptin base (Figure 1 ). This
`stoichiometry represents a 2: 1 adduct of sitagliptin with phosphoric acid.
`
`26.
`
`In some of these experiments an excess of phosphoric acid was used for
`
`the purpose of attempting to promote formation of a stable salt species containing two or
`
`more molecules of phosphoric acid for each molecule of sitagliptin. As an example, for
`Sample ID 234873 I attempted to prepare a species corresponding to the salt with the
`formula SG-H/+(H2P04) 2. This stoichiometry represents a 1 :2 adduct of sitagliptin with
`phosphoric acid.
`
`27.
`
`For some experiments a slight excess of one of the reactants (either
`
`sitagliptin base or phosphoric acid) based on the stoichiometry of the intended chemical
`
`reaction was used. As an example, Sample ID 235805 was conducted with the intention
`
`- 7 -
`
`Merck Exhibit 2225, Page 7
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`of providing a 1 :2 adduct of sitagliptin with phosphoric acid, e.g., SG-H/+(H2P04-)2.
`This experiment involved the use of 2.1 equivalents of phosphoric acid which is a 5%
`
`molar excess based on the intended stoichiometry. The motivation for using a slight
`
`excess of one of the reactants is to drive the chemical reaction to completion.
`
`28.
`
`In the salt formation experiments, the salts were isolated from their
`
`solutions using common laboratory techniques as described below for each of the salt
`
`formation experiments performed at Aptuit.
`
`29.
`
`Each of the twelve samples prepared at Aptuit during the salt formation
`
`experiments was extensively characterized by a variety of analytical techniques to
`
`determine the composition of the salt. The chemical identity and approximate purity of
`
`the samples was evaluated using proton NMR spectroscopy. XRPD was used to
`
`determine that crystalline products were obtained for each of the experiments. XRPD
`
`was also used to determine whether the product is sitagliptin dihydrogen phosphate,
`
`based on comparison with published XRPD data. Differential scanning calorimetry
`
`(DSC) was also used to confirm crystallinity by the presence of melt endotherms in the
`
`DSC plots for each of the samples, and to demonstrate that the samples are free of
`
`substantial amounts of solvent and other volatile material.
`
`30.
`
`Each of the twelve samples prepared at Aptuit during the salt formation
`
`experiments was also analyzed under my instruction and direction for carbon, hydrogen,
`
`phosphorus and nitrogen content. The purpose of this testing was to determine the ratio
`
`of sitagliptin to phosphoric acid in these samples and to obtain an indication of the
`
`overall purity of the samples. Galbraith Laboratories ("Galbraith") was hired by Aptuit
`
`as a subcontractor laboratory for this testing. Galbraith is a contract research and services
`
`laboratory which provides a variety of chemical testing services, including elemental
`
`analysis, on a fee for service basis. Its facilities are periodically audited by the FDA to
`
`ensure compliance with GMP regulations, and are also periodically audited by the Aptuit
`
`quality assurance department to ensure cGMP compliance. Galbraith Laboratories is a
`
`longstanding provider of testing services to SSCI and Aptuit. I have only had positive
`
`- 8 -
`
`Merck Exhibit 2225, Page 8
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`experiences with Galbraith in over ten years of using them as sub-contractor laboratory,
`
`and consider the testing and data that Galbraith provides me as highly reliable.
`
`31.
`
`A detailed description of the twelve salt formation experiments is provided
`
`below. Furthermore, as shall be explained below, the various analytical data obtained
`
`clearly show that despite the different conditions used in each experiment, all twelve of
`
`the salt formation experiments resulted in sitagliptin dihydrogen phosphate, which is a
`
`salt that results upon the combination of one molecule of sitagliptin for each molecule of
`
`phosphoric acid. This salt is generated in solution by transfer of one proton (H+) from
`
`phosphoric acid to sitagliptin base.
`
`A.
`
`32.
`
`Procedures For Salt Formation Experiments
`
`Given below are the specific procedures used in each of the twelve salt
`
`formation experiments, observations made during the respective experiments, and
`
`identification of the analyses conducted on the solids resulting from the respective
`
`experiments.
`
`33.
`
`Sitagliptin base : phosphoric acid (1.00 : 1.05) in methanol
`(Sample 4063-02-01): Sitagliptin base (503.4 mg) was weighed into a 100 mL round
`
`bottom flask. Methanol (5 mL) was added resulting in a clear, colorless solution. The
`
`flask was placed in a water bath (T=22.0 °C). Phosphoric acid stock solution (1.298 mL
`
`of l .0M phosphoric acid in methanol) was added drop-wise with stirring. White solids
`
`formed after approximately 15 minutes and were allowed to slurry for one day. The
`
`solids were collected by vacuum filtration and allowed to air-dry. The solids were
`
`analyzed by XRPD (file 398023), DSC (file 399011), TGA (file 398706), proton NMR
`
`(file 400883) and elemental analysis (LIMS 233140).
`
`34.
`
`Sitagliptin base : phosphoric acid (1.00 : 2.10) in methanol
`(Sample 4063-03-01): Sitagliptin base (502.3 mg) was weighed into a 100 mL round
`
`bottom flask. Methanol (5 mL) was added resulting in a clear, colorless solution. The
`
`flask was placed in a water bath (T=2 l .0 0 C). Phosphoric acid stock solution (2.589 mL
`
`of l.0M phosphoric acid in methanol) was added drop-wise with stirring. White solids
`
`-9-
`
`Merck Exhibit 2225, Page 9
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`formed after approximately 15 minutes and were allowed to slurry for one day. The
`
`solids were collected by vacuum filtration and allowed to air-dry. The solids were
`
`analyzed by XRPD (file 398024), DSC (file 399012), proton NMR (file 400888), and
`
`elemental analysis (LIMS 233141).
`
`35.
`
`Sitagliptin base : phosphoric acid (3.00 : 1.00) in methanol
`
`(Sample 4063-18-01): Sitagliptin base (1000.3 mg) was weighed into a 100 mL round
`
`bottom flask. Methanol (10 mL) was added resulting in a clear, colorless solution. The
`
`flask was placed in a water bath (T=23 .0 °C). Phosphoric acid stock solution (0. 818 mL
`
`of 1.0M phosphoric acid in methanol) was added drop-wise with stirring. The solution
`
`remained clear and colorless for at least 4 hours. The sample was checked after
`
`approximately 15 hours and white solids were observed. The solids were allowed to
`
`slurry for approximately one day. The solids were collected by vacuum filtration and
`
`allowed to air-dry. The solids were analyzed by XRPD (file 401165), DSC (file 401166),
`
`proton NMR (file 401158), and elemental analysis (LIMS 234636).
`
`36.
`
`Sitagliptin base : phosphoric acid (2.04 : 1.00) in methanol
`
`(Sample 4063-04-01): Sitagliptin base (501.0 mg) was weighed into a 100 mL round
`
`bottom flask. Methanol (5 mL) was added resulting in a clear, colorless solution. The
`
`flask was placed in a water bath (T=21.0 °C). Phosphoric acid stock solution (0.603 mL
`
`of l .0M phosphoric acid in methanol) was added drop-wise with stirring. White solids
`
`were observed after approximately 3 hours. The solids were allowed to slurry for
`
`approximately one day. The solids were collected by vacuum filtration and allowed to
`
`air-dry. The solids were analyzed by XRPD (file 398025), DSC (file 399013), proton
`
`NMR (file 400891), and elemental analysis (LIMS 233142).
`
`37.
`
`Sitagliptin base: phosphoric acid (1.00: 5.01) in methanol
`
`(Sample 4063-19-01): Sitagliptin base (499.9 mg) was weighed into a 100 mL round
`
`bottom flask. Methanol (5 mL) was added resulting in a clear, colorless solution. The
`
`flask was placed in a water bath (T=22.0 °C). Phosphoric acid stock solution (1.230 mL
`
`of 5.0M phosphoric acid in methanol) was added drop-wise with stirring. Slight turbidity
`
`was noted after 3 hours 40 minutes. The solids were allowed to slurry for approximately
`
`- 10 -
`
`Merck Exhibit 2225, Page 10
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`one day. The solids were collected by vacuum filtration and allowed to air-dry. The
`
`solids were analyzed by XRPD (file 401062), DSC (file 401063), proton NMR (file
`
`401064), and elemental analysis (LIMS 234584).
`
`38.
`
`Sitagliptin base: phosphoric acid (2.04: 1.00) in methanol and
`
`approximately 12.5% water (Sample 4063-35-01): Sitagliptin base (750.4 mg) was
`
`weighed into a 100 mL round bottom flask. Methanol (7 mL) was added resulting in a
`
`clear, colorless solution. The flask was placed in a water bath (T=21.0 °C). Phosphoric
`
`acid stock solution (1.002 mL of 0.9M phosphoric acid in water) was added drop-wise
`
`with stirring. White solids were noted after 35 minutes. The solids were allowed to
`
`slurry for approximately one day. The solids were collected by vacuum filtration and
`
`allowed to air-dry. The solids were analyzed by XRPD (file 401683), DSC (file 401686),
`
`proton NMR (file 401689), and elemental analysis (LIMS 2345874).
`
`39.
`
`Sitagliptin base: phosphoric acid (1.00: 1.05) in methanol and
`
`approximately 12.5% water (Sample 4063-34-01): Sitagliptin base (500.8 mg) was
`
`weighed into a 100 mL round bottom flask. Methanol ( 5 mL) was added resulting in a
`
`clear, colorless solution. The flask was placed in a water bath (T=21.0 °C). Phosphoric
`
`acid stock solution (0.716 mL of 1.8M phosphoric acid in water) was added drop-wise
`
`with stirring. Slight turbidity was noted after 5 minutes. The solids were allowed to
`
`slurry for approximately one day. The solids were collected by vacuum filtration and
`
`allowed to air-dry. The solids were analyzed by XRPD (file 401681), DSC (file 401684),
`
`proton NMR (file 401687), and elemental analysis (LIMS 234872).
`
`40.
`
`Sitagliptin base : phosphoric acid (1.00 : 2.10) in methanol and
`
`approximately 12.5% water (Sample 4063-32-01): Sitagliptin base (499.7 mg) was
`
`weighed into a 100 rnL round bottom flask. Methanol (5 mL) was added resulting in a
`
`clear, colorless solution. The flask was placed in a water bath (T=21.0 °C). Phosphoric
`
`acid stock solution (0.716mL of 3.6M phosphoric acid in water) was added drop-wise
`
`with stirring. White solids were noted after approximately 2 hours. The solids were
`
`allowed to slurry for approximately one day. The solids were collected by vacuum
`
`- 11 -
`
`Merck Exhibit 2225, Page 11
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`filtration and allowed to air-dry. The solids were analyzed by XRPD (file 401682), DSC
`
`(file 401685), proton NMR (file 401688), and elemental analysis (LIMS 234873).
`
`41.
`
`Sitagliptin base: phosphoric acid (1.00: 2.10) in methanol at 0 °C
`
`(Sample 4063-50-01): Sitagliptin base (1004.6 mg) was weighed into a 100 mL round
`
`bottom flask. The flask was placed in an ice-water bath (T=4.0 °C). Methanol (0 °C, 10
`
`mL) was added resulting in a clear, colorless solution. Phosphoric acid stock solution
`
`( 5 .179 mL of I .OM phosphoric acid in methanol) was added drop-wise with stirring.
`
`White solids were noted after approximately 45 minutes. The solids were slurried and
`
`the solution was allowed to come to ambient temperature over approximately 4 hours.
`
`The solids were collected by vacuum filtration and allowed to air-dry. The solids were
`
`analyzed by XRPD (file 403327), DSC (file 403329), proton NMR (file 403325), and
`
`elemental analysis (LIMS 235805).
`
`42.
`
`Sitagliptin base : phosphoric acid (2.04 : 1.00) in methanol at 0 °C
`
`(Sample 4063-51-01)): Sitagliptin base (1004.4 mg) was weighed into a 100 mL round
`
`bottom flask. The flask was placed in an ice-water bath (T=O 0 C). Methanol (0 °C, 10
`
`mL) was added resulting in a clear, colorless solution. Pre-chilled phosphoric acid stock
`
`solution ( 1.208 mL of I .OM phosphoric acid in methanol) was added drop-wise with
`
`stirring. Slight turbidity was noted after approximately 3.5 hours. The solids were
`
`slurried for an additional IO hours. The solids were collected by vacuum filtration and
`
`allowed to air-dry. The solids were analyzed by XRPD (file 403328), DSC (file 403330),
`
`proton NMR (file 403326), elemental analysis (LIMS 235806).
`
`43.
`
`Sitagliptin base: phosphoric acid (1.00: 2.10) in methanol under
`
`reflux (Sample 4063-56-01): Sitagliptin base (1006.9 mg) was weighed into a 100 mL
`
`round bottom flask. Methanol (9 mL) was added resulting in a clear, colorless solution.
`
`The solution was heated to reflux (65 °C) in an oil bath with stirring. Phosphoric acid
`
`stock solution (5.1912 mL of I.OM phosphoric acid in methanol) was added drop-wise
`
`with stirring. Immediate precipitation was observed. The solution was allowed to come
`
`to room temperature at a rate of approximately 5 °C/hour prior to harvesting solids. The
`
`solids were collected by vacuum filtration and allowed to air-dry. The solids were
`
`- 12 -
`
`Merck Exhibit 2225, Page 12
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`analyzed by XRPD (file 403551), DSC (file 403548), proton NMR (file 403553), and
`
`elemental analysis (LIMS 235848).
`
`44.
`
`Sitagliptin base : phosphoric acid (2.04 : 1.00) in methanol under
`
`reflux (Sample 4063-57-01): Sitagliptin base (1004.8 mg) was weighed into a 100 mL
`
`round bottom flask. Methanol (9 mL) was added resulting in a clear, colorless solution.
`
`The solution was heated to reflux (65-70 °C) in an oil bath with stirring. Phosphoric acid
`
`stock solution (1.2088 mL of I.OM phosphoric acid in methanol) was added drop-wise
`
`with stirring. Turbidity was noted after 15 minutes; white solids after a total of 30
`
`minutes. The solution was allowed to come to room temperature at a rate of
`
`approximately 5 °C/hour prior to harvesting solids. The solids were collected by vacuum
`
`filtration and allowed to air-dry. The solids were analyzed by XRPD (file 403552), DSC
`
`(file 403550), proton NMR (file 403554), and elemental analysis (LIMS 235849).
`
`45.
`
`For each of the twelve salt formation experiments described above, the
`
`XRPD patterns are provided in Exhibit I, the NMR spectra are provided in Exhibit J,
`and the DSC plots are provided in Exhibit K. 1 A summary of the elemental analyses for
`the solids resulting from the salt formation experiments is provided in Exhibit L.
`
`B.
`
`46.
`
`Results of the Salt Formation Experiments
`
`Based on the analytical data obtained in the salt formation experiments
`
`(XRPD, proton NMR, DSC and elemental analysis), it is my conclusion that all twelve
`
`salt formation experiments resulted in sitagliptin dihydrogen phosphate. I note that
`
`elemental analysis and proton NMR provide information about the chemical content and
`
`structure of the compound. Furthermore, XRPD and DSC data provide information about
`
`the physical properties of the compound ( crystalline form and melting temperature,
`
`respectively). Based on all of the analytical data I conclude that the samples are
`
`sitagliptin dihydrogen phosphate. The above conclusion is supported by the comparison
`
`of the observed physical properties to the characteristic physical properties of sitagliptin
`
`dihydrogen phosphate published in the literature.
`
`The TGA plot for Sample ID 233140 is also provided in Exhibit K.
`
`- 13 -
`
`Merck Exhibit 2225, Page 13
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`47.
`
`Upon analysis of the XRPD patterns obtained on the twelve experimental
`
`samples, it is my conclusion that all salt formation experiments resulted in known
`
`crystalline forms or mixtures of known crystalline forms of sitagliptin dihydrogen
`
`phosphate disclosed in US 2010/0041885 Al and WO 2005/020920. Additional support
`
`for the identification of these samples as crystalline solids was obtained from the DSC
`
`analyses of the materials, which display a melt endotherm for the materials. The
`
`chemical shift positions of the resonances and their corresponding integrated intensities
`
`in the proton NMR spectra demonstrate that the salt formation experiment samples
`
`prepared at Aptuit are sitagliptin dihydrogen phosphate. In addition, the NMR spectra for
`
`these twelve samples match the spectrum obtained for the sitagliptin dihydrogen
`
`phosphate sample obtained from Teva.
`
`- 14 -
`
`Merck Exhibit 2225, Page 14
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`48.
`
`The elemental analyses of all twelve salt formation experiment samples
`
`are conclusive for the generation of sitagliptin dihydrogen phosphate in these samples. It
`
`is not possible for any of the samples to contain other than a 1: 1 adduct of sitagliptin and
`
`phosphoric acid, which is sitagliptin dihydrogen phosphate. This is because the relative
`
`amounts of nitrogen and phosphorus in the ratio of 13.86/6.13 would be substantially
`
`different for other stoichiometries of sitagliptin and phosphoric acid. Table 2, below,
`
`provides theoretical values, the elemental analysis results, and the deviation between
`
`measured and theoretical values.
`
`Table 2: Elemental Analyses of Salt Formation Experiment Samples
`
`Notebook
`No.
`
`API:acid ratio;
`solvent; temperature
`
`C
`
`H
`
`N
`
`p
`
`Theoretical
`
`n/a
`
`38.03
`
`3.59
`
`13.86
`
`6.13
`
`C
`
`-
`
`H
`
`-
`
`N
`
`-
`
`p
`
`-
`
`Measured Values
`
`Deviation from Theory
`
`4063-19-01
`
`1.00:5.01; methanol; ambient
`
`4063-18-01
`
`3.00:1.00; methanol; ambient
`
`4063-02-01
`
`1.00:1.05; methanol; ambient
`
`4063-03-01
`
`1.00:2.1 0; methanol; ambient
`
`37.72
`
`37.89
`
`37.92
`
`37.76
`
`3.67
`
`13.64
`
`6.03
`
`-0.31
`
`0.08
`
`-0.22
`
`-0.11
`
`3.64
`
`3.43
`
`3.47
`
`3.41
`
`13.71
`
`6.12
`
`-0.14
`
`0.05
`
`-0.15
`
`-0.01
`
`13.54
`
`13.60
`
`13.62
`
`6.15
`
`5.97
`
`5.91
`
`-0.11
`
`-0.27
`
`-0.25
`
`-0.16
`
`-0.32
`
`0.02
`
`-0.12
`
`-0.26
`
`-0.16
`
`-0.18
`
`-0.24
`
`-0.22
`
`13.56
`
`6.31
`
`-1.53
`
`0.03
`
`-0.30
`
`0.18
`
`14.08
`
`13.98
`
`13.98
`
`13.96
`
`13.97
`
`5.78
`
`5.92
`
`5.85
`
`6.22
`
`6.29
`
`6.12
`
`-0.08
`
`-0.39
`
`-0.16
`
`-0.21
`
`-0.28
`
`-0.21
`
`0.00
`
`0.22
`
`-0.35
`
`-0.03
`
`0.12
`
`-0.22
`
`-0.01
`
`0.12
`
`-0.29
`
`0.04
`
`0.10
`
`-0.01
`
`0.11
`
`0.09
`
`0.16
`
`0.03
`
`0.05
`
`-0.01
`
`4063-04-01
`
`2.04: 1.00; methanol; ambient
`
`4063-50-01
`
`1.00:2.10; methanol; 0 °C
`
`4063-51-01
`
`2.04:1.00; methanol; 0 °C
`
`4063-56-01
`
`1.00:2.10; methanol; 65 °C
`
`4063-57-01
`
`2.04:1.00; methanol; 65 °C
`
`37.78
`
`36.51
`
`37.96
`
`37.64
`
`37.87
`
`4063-34-01
`
`1.00: 1.05; methanol-water; ambient
`
`37.82
`
`4063-32-01
`
`1.00:2.1 0; methanol-water; ambient
`
`37.75
`
`3.62
`
`3.60
`
`3.56
`
`3.58
`
`3.63
`
`3.58
`
`4063-35-01
`
`2.04: 1.00; methanol-water; ambient
`
`37.82
`
`3.62
`
`13.91
`
`49.
`
`As can be readily seen from Table 2, above, all but one of the elemental
`
`analysis values fall within ±0.4% of the theoretical values for anhydrous sitagliptin
`
`dihydrogen phosphate. A deviation within ±0.4% from the theoretical value is generally
`
`considered to be an acceptable demonstration of compound purity by peer-reviewed
`
`- 15 -
`
`Merck Exhibit 2225, Page 15
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`scientific journals.2 The agreement between the values obtained for the twelve samples is
`
`remarkable when one