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`EXHIBIT 4
`EXHIBIT 4
`
`

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`eeeESTATETTT
`
`US010888605B2
`
`a2) United States Patent
`US 10,888,605 B2
`(0) Patent No.:
`Jan. 12, 2021
`(45) Date of Patent:
`Moelleret al.
`
`(54) GLP-1 COMPOSITIONS AND USES
`THEREOF
`
`(71) Applicant: Novo Nordisk A/S, Bagsvaerd (DK)
`
`FOREIGN PATENT DOCUMENTS
`
`8/2015
`2015522573 A
`JP
`9/1990
`9010020 Al
`WO
`10/1992
`9217482 Al
`WO
`6/1998
`9824767 Al
`WO
`
`WO 0069413 Al—11/2000
`(72)
`Inventors: Eva Horn Moeller, Alleroed (DK);
`WO
`0101774 Al
`1/2001
`Michael Duelund Soerensen, Soeborg
`WO
`0102369 A2
`1/2001
`(DK); Joakim Lundqvist, Malmoe
`WO
`2005042488
`5/2005
`(SE)
`WO
`2005044294
`5/2005
`WO
`2005081711
`9/2005
`WO
`2006055603
`5/2006
`WO
`2006072065
`7/2006
`WO
`2006/097537 A2
`9/2006
`WO
`200609646 1
`9/2006
`WO
`2006099561
`9/2006
`WO
`2007075720
`7/2007
`WO
`2007094893
`8/2007
`WO
`2008019115
`2/2008
`WO
`200905 1992
`4/2009
`WO
`2011069629
`6/2011
`WO
`2012107476 Al
`8/2012
`WO
`2012151248
`11/2012
`WO
`2013072406
`5/2013
`WO
`2013151663
`10/2013
`WO
`2013151668
`10/2013
`WO
`2013190384
`12/2013
`WO
`2014060472 Al
`4/2014
`WO
`2014182950
`11/2014
`WO
`2015009616
`1/2015
`WO
`2016001862
`1/2016
`
`(73) Assignee: Novo Nordisk A/S, Bagsvaerd (DK)
`
`(*) Notice:
`
`Subject to any disclaimer, the term ofthis
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21) Appl. No.: 16/774,666
`
`(22)
`
`Filed:
`
`Jan. 28, 2020
`
`(65)
`
`Prior Publication Data
`
`US 2020/0164042 Al
`
`May 28, 2020
`
`Related U.S. Application Data
`
`application
`of
`(63) Continuation
`PCT/EP2018/072835, filed on Aug. 24, 2018.
`
`No.
`
`(30)
`
`Foreign Application Priority Data
`
`Aug. 24, 2017
`
`(EP) wceecsceeeceseecnenecetsenees 17187676
`
`(51)
`
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2017.01)
`
`Int. Cl.
`AGIK 38/26
`AGIK 9/00
`AGIK 9/08
`AGIK 47/02
`AGIK 47/10
`(52) U.S. Cl.
`CPC wee AGIK 38/26 (2013.01); AGLK 9/0019
`(2013.01); A6IK 9/08 (2013.01); A6IK 47/02
`(2013.01); A61K 47/10 (2013.01)
`(58) Field of Classification Search
`CPC ...... A61K 38/26; A61K 9/0019; A61K 47/10;
`A61K 9/08; A61K 47/02
`See application file for complete search history.
`
`(56)
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`OTHER PUBLICATIONS
`
`BASF: “BASF Chemical Emergency Medical Guidelines,” Jan. 1,
`2016, Retrieved from the Internet: URL: https://www.basf.com/
`documents/corp/en/sustainability/employees-and-society/employees/
`occupational -medicine/medical-guidelines/Phenol_B_BASF_
`medGuidelines_E104.pdf , retrieved on Nov. 20, 2017.
`Lau et al., Journal of Medicinal Chemistry, 2015, vol. 58, No. 18,
`pp. 7370-7380.
`Marbury et al., “Pharmacokinetics and Tolerability of a Single Dose
`of Semaglutide, a Once-Weekly Human GLP-1 Analogue, in Sub-
`jects With and Without Renal Impairment,” Diabetologia, 2014, vol.
`57, Supplement: 1, pp. S358-S359.
`
`* cited by examiner
`
`Primary Examiner — Sudhakar Katakam
`(74) Attorney, Agent, or Firm — Rosemarie R.
`Wilk-Orescan
`
`(57)
`
`ABSTRACT
`
`5,169,771 A
`9,764,003 B2
`2006/0178304 Al
`2009/0156478 AL*
`
`2012/0208755 Al
`2016/0235855 Al
`
`12/1992 Christneretal.
`9/2017 Jensen
`8/2006 Juul-Mortensenetal.
`6/2009 Lau ween A61P 3/10
`514/1.1
`
`The present invention relates to pharmaceutical composi-
`tions of the GLP-1 peptide semaglutide comprising no more
`than 0.01% (w/w) phenol, their preparation, kits comprising
`such compositions as well as uses thereof.
`
`8/2012 Leung
`8/2016 Xiong etal.
`
`14 Claims, No Drawings
`
`

`

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`US 10,888,605 B2
`
`1
`GLP-1 COMPOSITIONS AND USES
`THEREOF
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`This application is a continuation of International Appli-
`cation PCT/EP2018/072835 (WO/2019/038412), filed Aug.
`24, 2018, which claimspriority to European Patent Appli-
`cation 17187676.6, filed Aug. 24, 2017;
`the contents of
`which are incorporated herein by reference.
`The present invention relates to the field of pharmaceu-
`tical compositions comprising the GLP-1 peptide sema-
`glutide.
`
`BACKGROUND
`
`GLP-1 peptides are known to be prone to develop lack of
`stability in liquid solutions, for example lack of physical
`stability. Thus, liquid pharmaceutical compositions compris-
`ing GLP-1 peptides with even better stability are desired.
`Such improved stability may be physical stability and/or
`chemical stability.
`
`SUMMARY
`
`In some embodiments the invention relates to liquid
`pharmaceutical compositions comprising semaglutide and
`no more than 0.01% (w/w) phenol. In some embodiments
`the invention relates to kits comprising the pharmaceutical
`composition as defined herein. In some embodiments the
`invention relates to the pharmaceutical composition as
`defined herein for use in medicine.
`
`DESCRIPTION
`
`The present invention relates to liquid pharmaceutical
`compositions comprising the GLP-1 peptide semaglutide
`and no more than 0.01% (w/w) phenol. Surprisingly, the
`present
`inventors
`found that
`such compositions have
`improved chemical and/or physical
`stability.
`In some
`embodiments the composition comprises no phenol. In some
`embodiments the composition comprises 0.01-10 mg/ml
`semaglutide. In some embodiments the composition has a
`pH in the range of 6.0-10.0, such as pH 7.0-7.8.
`In some embodiments the composition of the invention is
`a liquid pharmaceutical composition comprising sema-
`glutide and no more than 0.01% (w/w)phenol, wherein said
`composition
`a. is for parenteral administration;
`b. is an aqueous solution comprising at least 60% w/w
`water; or
`c. further comprises one or more pharmaceutically accept-
`able excipients selected from the group consisting of a
`buffer or an isotonic agent.
`In some embodiments the composition of the invention is
`a liquid pharmaceutical composition comprising sema-
`glutide, no more than 0.01% (w/w) phenol, and optionally
`one or more pharmaceutically acceptable excipients,
`wherein the formulation is for parenteral administration,
`such as subcutaneous administration.
`In some embodiments the composition of the invention is
`a liquid pharmaceutical composition comprising sema-
`glutide, no more than 0.01% (w/w) phenol, at least 60% w/w
`water, and optionally one or more pharmaceutically accept-
`able excipients.
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`2
`In some embodiments the term “stability” as used herein
`refers to stability of semaglutide in a liquid pharmaceutical
`composition. In some embodiments stability is chemical
`stability of the GLP-1 peptide (e.g. determined by HPLC,
`such as Assay(I) herein), and optionally physical stability of
`the GLP-1 peptide (e.g. determined by Thioflavine T assay,
`such as Assay (II) herein).
`In some embodiments the term “chemical stability” in
`relation to semaglutide as used herein refers to the covalent
`bonds of the semaglutide compound being substantially
`intact. In some embodiments chemicalstability of a GLP-1
`peptide is determined by HPLC, such as Assay(I) herein. In
`some embodiments a composition possess chemicalstability
`if its covalent bonds are intact in at least 80% (w/v) ofsaid
`GLP-1 peptides after storage for 3 months at 25° C. In some
`embodiments chemical stability of semaglutide is deter-
`mined by Assay (IV) herein.
`In some embodiments the term “physical stability” in
`relation to semaglutide as used herein refers to semaglutide
`forming substantially no aggregates, e.g. in the form offibril
`formation. In some embodiments physicalstability is deter-
`mined by Thioflavine T assay, such as Assay (II) herein.
`In some embodiments the composition of the present
`invention is a stable pharmaceutical composition. The term
`“stable pharmaceutical composition” when used herein
`refers to a pharmaceutical composition, e.g. a solution or
`suspension, comprising GLP-1 peptide, and which compo-
`sition following storage comprisesat least 80% (w/v) of said
`GLP-1 peptide (e.g. after quiescent storage for 3 months at
`25° C.). Storage conditions for stability testing may be 2-8°
`C., such as 5° C., or at least 2.5 years at 5° C. Alternatively,
`storage conditions for stability testing may be at least 4
`weeks, such as 6 weeks or 3 months, optionally at 30° C. The
`conditions of storage for this stable pharmaceutical compo-
`sition may be at 5° C. for 1 or 2 years. The conditions of
`storage for this stable pharmaceutical composition may be at
`5° C. for 3 years. Alternatively, the conditionsofthis storage
`may be at 25° C. for 24 hours or 1 week. In yet another
`alternative,
`the conditions of this storage may be room
`temperature for two months, such as up to two months.
`In some embodiments, chemical stability of the GLP-1
`peptide requires at least 80% (w/v), such as at least 90%
`(w/v)or at least 95% (w/v), of said GLP-1 peptide remaining
`with its covalent bonds intact at the end of the storage
`period. In some embodiments chemical stability of the
`GLP-1 peptide requires at least 95% (w/v), such as at least
`97% (wiv) or at least 99% (w/v), of said GLP-1 peptide
`remaining with its covalent bonds intact at the end of the
`storage period.
`The composition of the invention comprises no more than
`0.01% (w/w) phenol. In some embodiments the composition
`comprises substantially no phenol.
`Pharmaceutical Compositions
`The terms “pharmaceutical composition” and “composi-
`tion” are used interchangeably herein and refer to pharma-
`ceutical compositions suitable for administration to a subject
`in need thereof.
`
`In some embodiments the composition comprises 0.01-
`100 mg/ml semaglutide. In some embodiments the compo-
`sition comprises 0.1-50 mg/ml, such as 0.5-25 mg/ml or
`1-15 mg/ml, semaglutide. In some embodiments the com-
`position comprises 0.1-10 mg/ml, such as 0.5-5 mg/ml or
`1-2 mg/ml, semaglutide. In some embodiments the compo-
`sition comprises 0.01-10 mg/ml, such as 0.01-5 mg/ml,
`semaglutide. In some embodiments the composition com-
`prises no more than 9 mg/ml, such as no more than 8 mg/ml
`or no more than 7 mg/ml, semaglutide. In some embodi-
`
`

`

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`US 10,888,605 B2
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`3
`ments the composition comprises no more than 6 mg/ml,
`such as no more than 5 mg/ml or no more than 4 mg/ml,
`semaglutide. In some embodiments the composition com-
`prises no more than 3 mg/ml, such as no more than 2 mg/ml
`or no more than 1 mg/ml, semaglutide. In some embodi-
`ments the composition comprises at least 0.01 mg/ml, such
`as at least 0.02 mg/ml or at least 0.05 mg/ml, semaglutide.
`In some embodiments the composition comprises 1.34
`mg/ml semaglutide.
`In some embodiments the composition of the invention
`has a pH in the range of 3-10, such as pH 6-10 or 6-9. In
`some embodiments the composition of the invention has a
`pH in the range of pH 6.5-8.5, such as pH 7.0-7.8.
`In some embodiments the composition of the invention
`comprises one or more pharmaceutically acceptable excipi-
`ents.
`
`In some embodiments the composition of the invention
`comprises an isotonic agent, such as propylene glycol. In
`some embodiments the isotonic agent is propylene glycol or
`sodium chloride.
`
`In some embodiments the composition of the invention
`comprises a buffer, such as phosphate buffer, TRIS, citrate,
`or no buffer. In some embodiments the phosphate buffer is
`a sodium phosphate buffer, such as disodium hydrogen
`phosphate.
`In some embodiments the composition of the invention
`comprises no preservative.
`The composition of the inventionis in the form ofa liquid
`pharmaceutical composition. In some embodiments the liq-
`uid pharmaceutical composition is a solution or a suspen-
`sion. In some embodiments the composition of the invention
`is in the form of a solution, such as an aqueous solution. In
`some embodiments the term “aqueous solution” as used
`herein refers to a solution comprising at least 60% w/w
`water. In some embodiments the aqueoussolution comprises
`60-99% w/w water. In some embodiments the aqueous
`solution comprises at least 75% w/w water, such as at least
`80% w/w water or at
`least 85% w/w water.
`In some
`embodiments the aqueous solution comprises at least 90%
`w/w water, such as at least 92% w/w wateror at least 94%
`w/w water.
`
`4
`herein and instructions for use. In some embodiments the
`instructions for use comprise the package insert of a drug.
`In some embodiments the invention relates to a kit
`
`comprising the pharmaceutical composition as defined
`herein and an injection device. In some embodiments the
`injection device is selected from the group consisting of a
`durable pen anda prefilled pen. Examples of durable pens
`are NovoPen® 4 or NovoPen® 5 (both from Novo Nordisk
`A/S, Denmark). An example ofa prefilled pen is FlexPen®
`(Novo Nordisk A/S, Denmark).
`Indications
`In some embodiments the compositions of the invention
`are for use in medicine. In some embodiments the compo-
`sition of the invention maybeusedfor the following medical
`treatments:
`
`(i) prevention and/or treatment of all forms of diabetes,
`such as hyperglycaemia, type 2 diabetes, impaired glucose
`tolerance, type 1 diabetes, non-insulin dependent diabetes,
`MODY(maturity onset diabetes of the young), gestational
`diabetes, and/or for reduction of HbAI1c;
`(ii) delaying or preventing diabetic disease progression,
`such as progression in type 2 diabetes, delaying the pro-
`gression of impaired glucose tolerance (IGT) to insulin
`requiring type 2 diabetes, and/or delaying the progression of
`non-insulin requiring type 2 diabetes to insulin requiring
`type 2 diabetes;
`(i11) prevention and/or treatment of eating disorders, such
`as obesity, e.g. by decreasing food intake, reducing body
`weight, suppressing appetite, inducing satiety; treating or
`preventing binge eating disorder, bulimia nervosa, and/or
`obesity induced by administration of an antipsychotic or a
`steroid; reduction of gastric motility; and/or delaying gastric
`emptying.
`In some
`In some embodiments the indication is (i).
`embodiments the indication is (11). Ina still further particular
`aspect
`the indication is (111). In some embodiments the
`indication is type 2 diabetes and/or obesity.
`In some embodiments the method or use comprises pre-
`vention,
`treatment, reduction and/or induction in one or
`more diseases or conditions defined herein. In some embodi-
`
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`ments the indicationis (1) and (aii). In some embodiments the
`indication is (41) and (i11). In some embodiments the inven-
`tion comprises administration of an effective amount of a
`Semaglutide
`GLP-1 peptide. In some embodiments the invention relates
`The GLP-1 peptide semaglutide may be prepared as
`to administration of an effective amount of a GLP-1 peptide.
`described in WO2006/097537, Example 4. Semaglutide is
`also known as N°?°-{18-[N-(17-carboxyheptadecanoyl)-L-
`Generally, all subjects suffering from obesity are also
`y-glutamy1]]-10-oxo-3,6,12,15-tetraoxa-9,18-diazaoctade-
`considered to be suffering from overweight.
`In some
`embodiments the invention relates to a method for treatment
`canoyl}-[8-(2-amino-2-propanoic_acid),34-L-arginine]hu-
`man glucagon-like peptide 1(7-37),
`see WHO Drug
`or prevention of obesity. In some embodiments the invention
`Information Vol. 24, No. 1, 2010. In some embodiments
`relates to use of the composition for treatment or prevention
`semaglutide maybe present in the composition in its fully or
`of obesity. In some embodiments the subject suffering from
`partly ionised form; for example one or more carboxylic
`obesity is human, such as an adult human ora paediatric
`acid groups (—COOH) may be deprotonated into the car-
`human (including infants, children, and adolescents). Body
`boxylate group (—COO_)and/or one or more amino groups
`mass index (BMI) is a measure of body fat based on height
`(—NH.,) maybe protonated into the —NH,+ group. In some
`and weight. The formula for calculation is BMJ=weight in
`kilograms/height in meters”. A human subjectsuffering from
`embodiments semaglutide is added to the composition in the
`form ofa salt.
`obesity may have a BMIof 230; this subject may also be
`Administration and Kits
`referred to as obese.
`In some embodiments the human
`The composition of the invention is for parenteral admin-
`subject suffering from obesity may have a BMI of 235 or a
`istration.
`In some embodiments the composition is for
`BMIin the range of 230 to <40. In some embodiments the
`subcutaneous administration.
`obesity is severe obesity or morbid obesity, wherein the
`human subject may have a BMIof 240.
`In some embodiments the invention relates to a method
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`In some embodiments the composition of the invention is
`for administration once weekly. In some embodiments the
`composition of the invention is for administration once
`daily, once every second or once every third day.
`In some embodiments the invention relates to a kit
`
`comprising the pharmaceutical composition as defined
`
`65
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`for treatment or prevention of overweight, optionally in the
`presence of at least one weight-related comorbidity. In some
`embodiments the invention relates to use of the composition
`for treatment or prevention of overweight, optionally in the
`
`

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`
`5
`presenceof at least one weight-related comorbidity. In some
`embodiments the subject suffering from overweight
`is
`human, such as an adult human or a paediatric human
`(including infants, children, and adolescents).
`In some
`embodiments a human subject suffering from overweight
`may have a BMIof 225, such as a BMIof 227. In some
`embodiments a human subject suffering from overweight
`has a BMIin the range of 25 to <30 or in the range of 27 to
`<30. In some embodiments the weight-related comorbidity
`is selected from the group consisting of hypertension, dia-
`betes (such as type 2 diabetes), dyslipidaemia, high choles-
`terol, and obstructive sleep apnoea.
`In some embodiments the invention relates to a method
`
`for reduction of body weight. In some embodiments the
`invention relates to use of the composition for reduction of
`body weight. A humanto be subjected to reduction of body
`weight according to the present invention may have a BMI
`of 225, such as a BMI of 227 or a BMI of 230. In some
`embodiments the human to be subjected to reduction of
`body weight according to the present invention may have a
`BMIof 235 or a BMIof 240. The term “reduction of body
`weight” may include treatment or prevention of obesity
`and/or overweight.
`In some embodiments, as used herein, specific values
`given in relation to numbers or intervals may be understood
`as the specific value or as about the specific value(e.g. plus
`or minus 10 percent of the specific value).
`
`Embodiments of the Invention
`
`The following are non-limiting embodiments of the
`invention:
`
`1. A liquid pharmaceutical composition comprising sema-
`glutide and no more than 0.01% (w/w)phenol.
`2. A liquid pharmaceutical composition comprising sema-
`glutide and substantially no phenol.
`3. The composition according to claim 1 or 2, wherein said
`composition does not comprise phenol.
`4. The composition according to any one of the preceding
`claims, wherein said composition is an aqueous solution
`comprising at least 60% w/w water, such as at least 70%
`w/w water or at least 80% w/w water.
`
`5. The composition according to any one of the preceding
`claims, wherein the concentration of semaglutide is 0.5-
`10 mg/ml of said composition.
`6. The composition according to any one of the preceding
`claims, wherein said semaglutide is in the form of a
`pharmaceutically acceptable salt.
`7. The composition according to any one of the preceding
`claims, wherein said composition comprises one or more
`pharmaceutically acceptable excipients.
`8. The composition according to any one of the preceding
`claims, wherein said composition comprises one or more
`agents for adjusting pH, such as HCl, NaOH,oracetate.
`9. The composition according to any one of the preceding
`claims, wherein said composition comprises a buffer
`and/or an isotonic agent.
`10. The composition according to any one of the preceding
`claims, wherein said buffer is present in a concentration of
`0.01-50 mM ofsaid composition.
`11. The composition according to any oneof the preceding
`claims, wherein said buffer is a phosphate buffer.
`12. The composition according to any one of the preceding
`claims, wherein said phosphate buffer is selected from the
`group consisting of sodium dihydrogen phosphate, diso-
`dium hydrogen phosphate, and sodium phosphate.
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`13. The composition according to any one of the preceding
`claims, wherein said isotonic agent is present in a con-
`centration from 8 mg/ml to 50 mg/ml, such as 14 mg/ml
`to 30 mg/ml, of said composition.
`14. The composition according to any one of the preceding
`claims, wherein said isotonic is propylene glycol.
`15. The composition according to any one of the preceding
`claims, wherein said composition comprises no preserva-
`tive.
`16. The composition according to any one of the preceding
`claims, wherein said composition has a pH in the range of
`6.0-10.0.
`
`17. The composition according to any one of the preceding
`claims, wherein said composition is for parenteral admin-
`istration.
`
`18. The composition according to any one of the preceding
`claims, wherein said composition is for subcutaneous
`administration.
`19. A kit comprising the pharmaceutical composition as
`defined in any one of the preceding claims and instruc-
`tions for use.
`
`20. A kit comprising the pharmaceutical composition as
`defined in any one of the preceding claims and an injec-
`tion device for administration of said composition to a
`subject, wherein said injection device is selected from the
`group consisting of a durable pen anda prefilled pen.
`21. A pharmaceutical composition as defined in any one of
`the preceding claims for use in medicine.
`22. The pharmaceutical composition for use as defined in
`any one of the preceding claims for use in the treatment
`of diabetes or obesity.
`23. A method for the prevention or treatment of diabetes or
`obesity, wherein the pharmaceutical composition as
`defined in any one of the preceding claims is administered
`to a subject in need thereof.
`
`EXAMPLES
`
`General Methods and Characterisation
`Preparation of Semaglutide Compositions:
`Unless otherwise noted, compositions of semaglutide
`were prepared by dissolving buffer (e.g. disodiumhydrogen-
`phosphate dihydrate), isotonic agent (e.g. propylene glycol)
`and optionally preservative (phenol) in water. Semaglutide
`was dissolved therein, pH was adjusted to 7.4 using sodium
`hydroxide and/or hydrochloric acid, and the composition
`wasfinally sterilised by filtration through a 0.22 um sterile
`filter.
`Preparation of Liraglutide Compositions:
`Unless otherwise noted, compositions of liraglutide were
`prepared from Solution 1 and Solution 2: Solution 1 was
`prepared by dissolving buffer (disodiumhydrogenphosphate
`dihydrate), isotonic agent (mannitol), and optionally preser-
`vative (phenol) in water. Solution 2 was prepared by dis-
`solving liraglutide while stirring slowly. Solution 1 and
`Solution 2 were mixed, pH was adjusted to 8.15 using
`sodium hydroxide and/or hydrochloric acid, and the com-
`position wasfinally sterilised by filtration through a 0.22 um
`sterile filter.
`
`Assay(I): Determination of High Molecular Weight Proteins
`(HMWP) Content of Semaglutide Compositions
`Determination of HMWPcontent was performed using
`size exclusion chromatography (SE-HPLC) using a Waters
`Insulin HMWP column with a mobile phase of sodium
`chloride, sodium phosphate, phosphoric acid and isopropa-
`nol, isocratic elution and detection at 280 nm. Content of
`HMWPis given in % as the combined area of chromato-
`
`

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`7
`graphic peaks eluting earlier than the semaglutide monomer
`peak (i.e. HMWPpeaks), relative to the total area of HMWP
`and semaglutide monomer peaks.
`Assay(II): Physical Stability of Semaglutide Compositions
`Assessed Via ThT
`
`The purposeofthis assay is to assess the physical stability
`of a GLP-1 peptide in aqueous solution.
`Low physical stability of a peptide or protein may lead to
`amyloid fibril formation. Fibrils are structurally well-or-
`dered, filamentous macromolecular structures formed by
`aggregation of soluble proteins and dominated by beta-sheet
`structure. Mature fibrils are insoluble and are resistant to
`
`degradation. For the sake of drug product quality and patient
`safety,
`it is desirable to minimize and control fibrillation
`events in pharmaceutical compositions of therapeutic pep-
`tides and proteins. Protein aggregation, including fibrilla-
`tion, can be assessed by visual
`inspection of a sample.
`Fibrillation can be assessed by the use of Thioflavine T
`(ThT), a small molecule indicator probe with a high speci-
`ficity for fibrils. ThT has a distinct fluorescence signature
`when bindingto fibrils compared to ThT in solution [Naiki
`et al. (1989) Anal. Biochem. 177, 244-249; LeVine (1999)
`Methods. Enzymol. 309, 274-284].
`Formation of a partially folded intermediate of the peptide
`is suggested as a general initiating mechanism for fibrilla-
`tion. A small amount of these intermediates nucleates to
`
`form a template onto which further intermediates may
`assembly and the fibrillation proceeds. The lag-time corre-
`spondsto the interval in which a critical amountof nuclei is
`generated and the apparent rate constant is the rate with
`whichthefibril itself is formed. The lag-time described in a
`ThT assay performed on a plate reader is therefore consid-
`ered indicative of the fibrillation tendency of a peptide
`composition in solution.
`Before performing the assay, ThT was added to the
`samples from a stock solution in H,O to a final concentration
`of 20 uM in samples. Sample aliquots of 200 ul of the
`composition comprising the GLP-1 peptide were placed in a
`96 well microtiter plate (optical 0.4 mL black Thermo
`Scientific Nunc) with a glass bead (2.8-3.2 mm, Whitehouse
`Scientific) placed in each well. Usually, eight replica of each
`sample were placed on the plate. The plate was sealed with
`sealing tape (Thermo Scientific Nunc).
`Incubation at given temperature, shaking and measure-
`ment of the ThT fluorescence emission were performed in a
`BMG FLUOStar Omega or a BMG FLUOStar Optima. The
`plate was incubated at 40° C. with double orbital shaking at
`300 rpm with an amplitude of 2 mm. Fluorescence mea-
`surement was performed using excitation through a 450 nm
`filter and measurement of emission through a 480 nm filter.
`The plate was measured every 20 minutes for a desired
`period of time. Between each measurement, the plate was
`shaken and heated as described.
`The threshold value was determined as the highest ThT
`fluorescence (in relative fluorescence units (RFU)) mea-
`sured on the plate at time 1 h 13 min, plus 100 RFU. The
`threshold value wasthen usedto calculate the lag time using
`the “time to threshold” method in the BMG FLUOstar
`software.
`
`Assay (III): Determination of Purity of Liraglutide
`Determination of purity was performed using high per-
`formance liquid chromatography (HPLC) using a Waters
`XTerra™ MS C18 column with a gradient elution of two
`mobile phases, where one mobile phase was an aqueous
`ammonium phosphate buffer (pH 8)/acetonitrile mixture and
`the other mobile phase wasacetonitrile in water. Detection
`was performed at 215 nm.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`8
`Assay (IV): Determination of Sum of Impurities of Sema-
`glutide
`
`Determination of purity of semaglutide is performed
`using reversed phase high performance liquid chromatogra-
`phy (RP-HPLC) using a Kinetex C18 column with an
`isocratic elution followed by a gradient elution of two
`mobile phases, where one mobile phase was an aqueous
`phosphate buffer/acetonitrile mixture and the other mobile
`phase was an aqueous acetonitrile/isopropanol mixture.
`Detection was performed at 210 nm. Purity of semaglutide
`is given as sum of impurities in % as the combined area of
`all chromatographic peaksrelative to semaglutide monomer
`peaks.
`
`Example 1: Semaglutide
`
`Compositions comprising semaglutide were tested in this
`experiment. The tested compositions contained semaglutide
`(as specified in Table 1), propylene glycol
`(14 mg/ml),
`disodiumhydrogenphosphate dihydrate (1.42 mg/ml), and
`optionally phenol (5.5 mg/ml) (as specified in Table 1), at
`pH 7.4 in an aqueous solution. These compositions were
`prepared as described herein in the section General Methods
`of Preparation. Chemical stability as expressed by HMWP
`was determined by Assay (1) described herein at start of the
`experimentand after storage at 25° C., 30° C. or at 37° C.
`Physical stability as expressed by Thioflavin T (ThT) assay
`was determined by Assay (II) described herein.
`Theresults are given in Tables 2 and 3. Surprisingly, these
`results show that physical and chemical stability of sema-
`glutide were improved in compositions without phenol
`relative to those with phenol. Results shown in Table 3 are
`an average of 8 samples tested.
`
`TABLE1
`
`Compositions tested in Example 1
`
`Composition no.
`
`Description
`
`DoOoMwmnnawmBWN
`
`me
`
`Semaglutide 1 mg/ml, with phenol
`Semaglutide 1 mg/ml, without phenol
`Semaglutide 1.34 mg/ml, with phenol
`Semaglutide 1.34 mg/ml, without phenol
`Semaglutide 0.5 mg/ml, without phenol
`Semaglutide 0.5 mg/ml, with phenol
`Semaglutide 1.0 mg/ml, without phenol
`Semaglutide 1.0 mg/ml, with phenol
`Semaglutide 2.0 mg/ml, without phenol
`Semaglutide 2.0 mg/ml, with phenol
`
`TABLE 2
`
`Chemicalstability of semaglutide compositions, as expressed
`by content of high molecular weight proteins (HMWP), following
`storage at different temperatures. A lower HMWPconcentration
`corresponds to a better chemicalstability.
`HMWP(%
`
`Composition
`no.
`
`1
`2 (no phenol)
`3
`4 (no phenol)
`
`0 months
`
`25° C,
`6 months
`
`30° C.
`3 months
`
`37°C.
`3 months
`
`0.1
`0.1
`0.1
`0.1
`
`2.0
`0.3
`1.9
`0.3
`
`1.9
`0.3
`1.8
`0.4
`
`4.1
`0.5
`3.9
`0.6
`
`

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`10
`positions were prepared as described herein in the section
`General Methods of Preparation. Chemical stability as
`expressed by HMWPwas determined by Assay (I) and as
`expressed by sum of impurities was determined by Assay
`(IV) described herein at start of the experiment and after
`storage at 30° C. Physical stability as expressed by Thio-
`flavin T (ThT) assay was determined by Assay(II) described
`herein.
`
`The results are given in Table 7 and 8. In line with the
`results of Example 1,
`these results show that physical
`stability and chemical
`stability of
`semaglutide were
`improved in compositions without or with low phenol
`concentration relative to those with phenolat 5.5 mg/ml. The
`results show that physical stability and chemicalstability of
`semaglutide were also improved in compositions without
`phenol comprising either the buffer trisodiumcitrate dihy-
`drate or no buffer or isotonic agent sodium chloride, relative
`to those with phenol. Chemical and physical stability were
`improved for compositions with 0.1 mg/ml phenolrelative
`to compositions with 5.5 mg/ml phenol and similar to
`compositions with no phenol. This was demonstrated for
`compositions with pH 7.0-7.8 and semaglutide concentra-
`tion 0.1-10 mg/ml.
`
`TABLE 6
`
`Compositions tested in Example 3
`
`Content of composition
`
`
`
`sotonic
`agent
`
`PG**
`PG
`PG
`PG
`PG
`PG
`PG
`PG
`PG
`PG
`PG
`PG
`PG
`PG
`PG
`PG
`PG
`PG
`PG
`PG
`Citrate***
`Citrate
`None*
`None
`PG
`PG
`
`pH
`
`7.0
`7.0
`7.0
`TA
`TA
`TA
`78
`78
`78
`7.0
`7.0
`7.0
`TA
`TA
`TA
`78
`78
`78
`TA
`TA
`TA
`TA
`7A
`TA
`74
`TA
`
`9
`TABLE 3
`
`Physical stability of semaglutide compositions as
`expressed by Thioflavin T (ThT) assay. A longer lag
`time corresponds to a better physical stability.
`
`Composition no.
`
`Lag time (hours)
`
`5 (no phenol)
`6
`7 (no phenol)
`8
`9 (no phenol)
`10
`
`>117
`19
`>117
`35
`>117
`35
`
`Example 2 (Reference): Liraglutide
`
`The results of Example 1 are also surprising in view of the
`fact
`that
`the GLP-1 compound liraglutide—contrary to
`semaglutide—is less chemically stable in a composition
`without phenol. These results are shown in Table 5.
`The results in Table 5 were obtained as follows: Compo-
`sitions comprising liraglutide were tested. The tested com-
`positions contained liraglutide (as specified in Table 4),
`mannitol (36.9 mg/ml), disodium hydrogen phosphate (1.42
`mg/ml), and optionally phenol (as specified in Table 4), at
`pH 7.4 in an aqueous solution. These compositions were
`prepared as described herein in the section General Methods
`of Preparation. Chemic