(12)
`
`United States Patent
`DeFelippis et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 7,022,674 B2
`Apr. 4, 2006
`
`US007022674B2
`
`(54) POLYPEPTIDE COMPOSITIONS WITH
`IMPROVED STABILITY
`(75) Inventors: Michael Rosario DeFelippis, Carmel,
`IN (US); Michael Allen Dobbins,
`Lebanon, IN (US); Alby David
`Sharkinas, Indianapolis, IN (US); Alex
`Mark Prokai, Carmel, IN (US); Joseph
`Vincent Rinella, Ypsilanti, MI (US)
`
`(73) Assignee: Eli Lilly and Company, Indianapolis,
`IN (US)
`
`(*) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 509 days.
`(21) Appl. No.:
`10/130,836
`
`CA
`P
`EP
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`
`(22) PCT Filed:
`
`Dec. 5, 2000
`
`(86). PCT No.:
`
`PCT/USOO/32421
`
`S 371 (c)(1),
`(2), (4) Date: May 21, 2002
`
`(87) PCT Pub. No.: WO01/43762
`
`PCT Pub. Date: Jun. 21, 2001
`O
`O
`Prior Publication Data
`US 2003/02O78O2 A1
`Nov. 6, 2003
`
`(65)
`
`FOREIGN PATENT DOCUMENTS
`2242591
`7, 1998
`O s: R SEC
`O 885961 A1
`6, 1998
`WO 97/48414
`12/1997
`WO 98.21340
`5, 1998
`WO 99,2848O
`6, 1999
`WO 99,29336
`6, 1999
`WO 99,29337
`6, 1999
`WO 99.43708
`9, 1999
`WOOOO7617
`2, 2000
`WOOO.38652
`T 2000
`WOOOf 41546
`T 2000
`OTHER PUBLICATIONS
`Peter Klusmann, Novo Nordisk, Grounds for Appeal filed
`with the German Federal Patent Court Against the German
`Patent Office in Proceedings relating to the German Utility
`Model that Corresponds to this U.S. Appl. No. 10/130,836,
`filed Dec. 17, 2004.*
`Rohde, T.D., et al., “An Improved Glycerol/Insulin
`Formulation for Use in Implant not Pumps. Trans Ams. Soc
`Artif Intern Organs, vol. 33, 1987, pp. 316-318.
`Washabaugh M.W. et al., “Purification of Aqueous Ethylene
`Glycol, Analytical Biochemistry, vol. 134, 1983, pp. 144
`152.
`Bello J. et al., “Chemical Modification and Cross-Linking of
`Proteins by Impurities in Glycerol', Archives of
`Biochemistry and Biophysics, vol. 172, 1976, pp. 608-610.
`Robbins D.C. et al., “Antibodies to Covalent Aggregates of
`Insulin in Blood of Insulin-Using Diabetic Patients',
`Diabetes, vol. 36, 1987, pp. 838-841.
`Robbins D.C. et al., “Free Covalent Aggregates of
`Related U.S. Application Data
`(60) Provisional application No. 60 181,030, filed on Feb. thrustus .
`try Ependent Diabet
`8, 2000, provisional application No. 60/171,135, filed
`Ratner et al., “Persistent Cutaneous Insulin Allergy Result
`on Dec. 16, 1999.
`ing From High-Molecular-Weight Insulin Aggregates'.
`(51) Int. Cl.
`Diabetes, vol. 39, 1990, pp. 728-733.
`A6 IK 38/00
`
`(2006.01)
`
`(52) U.S. Cl. ............................................. 514/12: 514/3
`
`(58) Field of Classification Search .................. 514/12,
`514/21, 1; 424/455
`See application file for complete search history.
`
`(56)
`
`References Cited
`
`(Continued)
`Primary Examiner Christopher R. Tate
`Assistant Examiner Marcela M Cordero Garcia
`(74) Attorney, Agent, or Firm Thomas E. LaGrandeur;
`James A. Hoffmann; James J. Kelley
`
`(57)
`
`ABSTRACT
`
`U.S. PATENT DOCUMENTS
`4,076,758 A
`2/1978 Owsley et al.
`4,683,347 A
`7, 1987 Diaz et al.
`5,124,314 A
`6/1992 Cooper
`5,164.366 A 11/1992 Balschmidt et al.
`5,514,646 A
`5, 1996 Chance et al.
`5,618,913 A
`4/1997 Brange et al.
`5,750,166 A
`5, 1998 Schellhaass
`5,951,993 A
`9, 1999 Scholz et al.
`5,969,175 A 10, 1999 Murao et al.
`6,034,054 A * 3/2000 DeFelippis et al. ............ 514.f4
`6,136,784. A 10, 2000 Litalien et al.
`6,268.343 B1
`7/2001 Knudsen et al. .............. 514/12
`6,358,924 B1* 3/2002 Hoffmann .......
`... 514/12
`6,551,992 B1 * 4/2003 DeFelippis et al. ............ 514/3
`
`
`
`The present invention provides means to improve the chemi
`cal stability of aqueous, parenteral pharmaceutical compo
`sitions comprising a polypeptide and glycerin. Reactive
`aldehydes are identified in commercial glycerins, and means
`for reducing Such are provided. Convenient means are
`provided to assay for reactive aldehydes in glycerin, and a
`strong linear correlation between the level of reactive alde
`hydes in glycerin and chemical stability of compositions
`comprising a polypeptide and glycerin is demonstrated. The
`invention includes aqueous compositions comprising a
`polypeptide and glycerin having improved chemical stabil
`ity compared to compositions previously known.
`
`23 Claims, 1 Drawing Sheet
`
`MPI EXHIBIT 1075 PAGE 1
`
`MPI EXHIBIT 1075 PAGE 1
`
`

`

`US 7,022,674 B2
`Page 2
`
`OTHER PUBLICATIONS
`Brange J, et al., “Chemical Stability of Insulin. 2 Formation
`of Higher Molecular Weight Transformation Products Dur
`ing Storage of Pharmaceutical Preparations’. Pharmaceuti
`cal Research, vol. 9, 1992, pp. 727-734.
`Schwendeman S.P. et al., “Stabilization of Tetanus and
`Diphtheria Toxoids Against Moisture-Induced Aggrega
`tion', Proc. Natl. Acad Sci. USA, vol. 92, 1995, pp. 11234
`11238.
`Fraenkel-Conrat H. et al., “The Reaction of Formaldehyde
`With Proteins. V. Cross-Linking Between Amino and
`Guanidyl Groups', JACS, vol. 70, 1948, pp. 2673-2684.
`Seetharama Acharya A. et al., “Reaction Of Glycolaldehyde
`With Proteins: Latent Crosslinking Potential of Oc—Hy
`droxyaldehydes, Proc. Natl. Acad. Sci. USA, vol. 80, 1983,
`pp. 3590-3594.
`Seetharama Acharya A. et al., “Cross-Linking of Proteins by
`Aldotriose: Reaction of the Carbonyl Function of the Keto
`Amines Generated in Situ With Amino Groups',
`Biochemistry, vol. 27, 1988, pp. 4522-4529.
`Brange J. Stability of Insulin, Kluwer Academic Publishers,
`Boston, 1994, pp. 23-36.
`Brange J., et al., “Formulation of Physically Stable Neutral
`Insulin Solutions for Continuous Infusion by Delivery
`Systems, Hormone Drugs, Published by the US
`Pharmacopoeial Convention, Rockville, Maryland, 1982,
`pp. 96-105.
`The European Pharmacopoeia Supplement 2000, Council of
`Europe, Strasbourg, France, 1999, pp. 747-750.
`The British Pharmacopoeia, British Pharmacopoeia Com
`mission, London, 1999, vol. 1, pp. 710-711.
`Dickinson R.G. et al., “A New Sensitive and Specific Test
`for the Detection of Aldehydes: Formation of 6-Mercapto
`3Substituted-s-Triazolo (4,3-b)-s
`Tetrazines”. Chemical
`Communications, 1970, pp. 1719-1720.
`Aldrich Technical Information Bulletin No. AL-145, Aldrich
`Chemical Co.; Hopps, H.B., Aldrichimica Acta 33:28-29,
`2OOO.
`Nash, T., “The Colorimetric Estimation of Formaldehyde by
`Means of the Hantzsch Reaction', Biochem. J., Vol. 55,
`1953, pp. 416-421.
`The International Pharmacipoeia. Third Edition, vol. 4,
`1994, pp. 176-181.
`Promotional Bulletin Entitled “Discover the Origins of
`Some of the World's Most Consistently Pure Products:
`Synthetic Glycerine Products', by Dow Chemical
`Company—Freeport, TX, USA, pp. 1-32.
`Sawicki E., et al., “The 3-Methyl-2-Benzothiazolone
`Hydrazone Test, Analytical Chemistry, Vol. 33, 1961, pp.
`93-96.
`Paz, M.A., et al., “Determination of Carbonyl Compounds
`with N-Methyl Benzothiazolone Hydrazone'. Archives of
`Biochemistry and Biophysics, vol. 109, 1965, pp. 548-559.
`Eberhardt M.A. et al., “A Colorimetric Procedure for the
`Determination of Aldehydes in Seawater and in Cultures of
`Methylotrophic Bacteria’, Marine Chemistry, vol. 17, 1985,
`pp. 199-212.
`Glutaraldehyde Test Kit, Model GT-1, Cat. No. 25872-00,
`by Hach, Loveland, CO, USA.
`Bailey B.W. et al., “New Spectrophotometric Method for
`Determination of Formaldehyde'. Analytical Chemistry,
`vol.43, 1971, pp. 782-784.
`Ziels N.W. et al., “Recovery and Purification of Glycerol,
`The Journal of the American Oil Chemists Society, Vol. 33.
`1956, pp. 556-565.
`
`Bello J., “The State of the Tyrosines of Bovine Pancreatic
`Ribonuclease in
`Ethylene Glycol and Glycerol'.
`Biochemistry, vol. 8, 1969, pp. 4535-4541.
`Riddick J.A. et al., “Organic Solvents”, Techniques of
`Chemistry, vol. 2. Third Edition, pp. 689-691.
`Stromquist D.M. et al., “C.P. Glycerol by Ion Exchange',
`Industrial and Engineering Chemistry, vol. 43, 1951, pp.
`1065-1070.
`Encyclopedia of Chemical Technology, Fourth Edition, Kirk
`Othmer, vol. 12, Glycerol, 1994, pp. 681-694.
`Remington’s Pharmaceutical Sciences, Mack Publishing
`Company, 18" Edition, 1990, Chapter 66, pp. 1316.
`Food Engineering, International Edition, Chilton Company,
`1997, p. 14.
`Knudsen L.B. et al., “Potent Derivatives of Glucagon-Like
`Peptide-1 with Pharmacokinetic Properties Suitable for
`Once Daily Administration”. Expedited Articles, Journal of
`Medical Chemistry, vol. 43, 2000, pp. 1664-1669.
`Shome B. et al., “A Reevaluation of the Amino Acid
`Sewuence of Human Follitropin B-Subunit, Journal of
`Protein Chemistry, vol. 7, 1988, pp. 325-339.
`The United States Pharmacopeia, The National Formulary,
`United States Pharmacopeial Convention, Inc. 2000, USP
`24, NF 19.
`Underberg W.J.M. et al., “Separation and Detection
`Tecniques for Peptides and Proteins in Stability Research
`and Bioanalysis”, Journal of Chromatography B. vol. 742,
`2000, pp. 401–409.
`Brange J. Galenics of Insulin, Springer-Verlag 1987.
`Du Chatinier W.M. et al., “Rapid Stability Indicating UV
`Assay of Methenamine Madelate in Tablets. Using Solid
`Phase Extraction'. Analytical Letters, vol. 22, 1989, pp.
`875-883.
`European Pharmacopoeia, Council of Europe, Strasbourg,
`1997, pp. 906-907.
`Roach P. et al., “Improved Postprandial Glycemic Control
`During Treatment with Humalog Mix25. A Novel
`Protamine-Based Insulin Lispro Formulation', Diabetes
`Care, vol. 22, 1999, pp. 1258-1261.
`Peter Schindler, Certificate fom Merck KGaA, Darmstadt,
`Germany (2001).
`Havelund S and Brange J. Abstract, Second Assisi
`International Symposium, Chemical Stabilization of Insulin
`Glycerol Mixtures, Novo Research Institute, Bagsvaerd
`Denmark (1986).
`Ja Rozandeal, Vector Control Methods for Use by
`Individuals and Communities, pps 505-513, (1997).
`J. Brange,et al., Chemical Stability of Insulin, Acta Pharm.
`4(3) 149-155 (1992).
`Ullmann's Encyclopeida of Industrial Chemistry, Fifth,
`Completely Revised Edition, vol. A12.
`S. Haveland and J. Brange, Poster Second Assisi
`International Symposium, Chemical Stabilization of Insulin
`Glycerol Mixtures, (1986).
`Peter Klusmann, Novo Nordisk, Grounds for Appeal filed
`with the German Federal Patent Court Against the German
`Patent Office in Proceedings relating to the German Utility
`Model that Corresponds to this U.S. Appl. No. 10/130,836.
`German Original, English Translation, and Original Cited
`Supporting Documents. (Dec. 17, 2004).
`* cited by examiner
`
`MPI EXHIBIT 1075 PAGE 2
`
`MPI EXHIBIT 1075 PAGE 2
`
`

`

`U.S. Patent
`
`Apr. 4, 2006
`
`US 7,022,674 B2
`
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`MPI EXHIBIT 1075 PAGE 3
`
`MPI EXHIBIT 1075 PAGE 3
`
`

`

`US 7,022,674 B2
`
`1.
`POLYPEPTIDE COMPOSITIONS WITH
`IMPROVED STABILITY
`
`This application is a United States national stage appli
`cation filed under 35 U.S.C. S371 from International Appli
`cation No. PCT/US00/32421, filed Dec. 5, 2000, which
`claims benefit of U.S. Provisional Application 60/171,135,
`filed Dec. 16, 1999, Japanese Patent Application 377208/99,
`filed Dec. 28, 1999, and U.S. Provisional Application
`60/181,030, filed Feb. 8, 2000, each of which application is
`entirely incorporated herein by reference.
`
`10
`
`FIELD OF THE INVENTION
`
`This invention is in the field of human medicine. In
`particular, this invention is in the field of pharmaceutical
`compositions for treating various diseases, including diabe
`tes and hyperglycemia.
`
`15
`
`BACKGROUND OF THE INVENTION
`
`2
`9:727-734 (1992) concluded that covalent insulin dimers
`and polymers should be minimized to avoid these allergic
`reactions but no methods to achieve this goal were disclosed
`or Suggested.
`Three observations may be made about the problem of
`preparing reliably stable polypeptide compositions contain
`ing glycerin for parenteral administration. First, there has
`been a lack of a simple but accurate assay for determining
`the level of reactive aldehydes present in glycerin that lead
`to crosslinked polypeptide impurities. Second, there has
`been no teaching or Suggestion in the prior art that com
`mercial lots of glycerin manufactured from different sources
`should be evaluated to determine if certain sources are better
`than others in minimizing the polypeptide crosslinking reac
`tions. Third, there has been no convenient, efficient way of
`lowering the reactive aldehyde content of glycerin to elimi
`nate or minimize the aldehyde-induced crosslinking reac
`tions in aqueous, pharmaceutical polypeptide compositions.
`Each of these three observations will now be described in
`more detail.
`
`Measuring Reactive Aldehydes in Glycerin
`
`The lack of a simple, reliable method of measuring the
`reactive aldehyde impurities in glycerin that lead to forma
`tion of crosslinked polypeptide impurities has hindered
`Solution of the polypeptide crosslinking problem in formu
`lations containing glycerin.
`Formaldehyde can initiate crosslinking of polypeptides by
`a reactive imine link Schwendeman, S. P. et al., PNAS
`92:11234–11238 (1995) and Fraenkel-Conrat, H., et al.,
`JACS 70:2673–2684 (1948). Glyceraldehyde and glycola
`ldehyde react with amino groups in polypeptide Solutions,
`forming crosslinked polypeptides as described in Acharya,
`A. S., et al. PNAS 80:3590–3594 (1983) and Acharya, A.
`S., et al. Biochemistry 27:4522-4629 (1988).
`Aldehyde impurities in glycerol were speculated to be
`involved in formation of high molecular weight polymers in
`insulin formulations Brange J., et al., Pharm. Res.
`9:727-734 (1992); Brange, J., Stability of Insulin, Kluwer
`Academic Publishers, Boston, pp. 23–36 (1994); Brange, J.,
`et al., Hormone Drugs, Published by the US Pharmacopoeial
`Convention, Rockville, Md., pp. 95-105 (1982) but no
`methods to quantitate or remove the aldehyde impurities to
`improve chemical stability of the insulin formulations were
`disclosed.
`There are many assays for aldehydes in the literature, but
`their applicability to measuring the reactive aldehyde con
`tent of glycerin as a predictor of polypeptide crosslinking in
`pharmaceutical formulations is questionable.
`The European Pharmacopoeia Supplement 2000 Council
`of Europe, Strasbourg, France, pp. 747 751 (1999)
`describes an aldehyde test in its glycerol monograph. This
`test employs pararosaniline hydrochloride reagent and a 5
`ppm formaldehyde standard solution as the comparator.
`The British Pharmacopoeia 1999 British Pharmacopoeia
`Commission, London, pp. 710–711 (1999) discloses a test
`for aldehydes and reducing Substances in glycerin using
`pararosaniline hydrochloride and visual comparison with a
`standard solution containing 5 ppm of formaldehyde.
`The “Purpald' reagent, 4-amino-3-hydrazino-5-mer
`capto-1,2,4-triazole Dickinson, R. G., et al., Chem. Com
`mun. p. 1719 (1970) reacts with aldehydes and has been
`used for determination of formaldehyde in air, glycols,
`vaccines, resins and plastic products and detection of acetal
`dehyde in liver tissue sections and fruit Aldrich Technical
`
`Many polypeptide pharmaceutical compositions are uti
`lized for the treatment of diseases in humans and other
`mammals. Due to their high lability following oral delivery,
`polypeptide drugs must generally be delivered by parenteral
`routes. Chief among these routes are subcutaneous, intra
`muscular and intravenous.
`Polypeptide drug products are traditionally Supplied to
`pharmacies, hospitals and patients as solutions, Suspensions,
`or lyophilized products. In liquid form, each polypeptide
`drug formulation requires a certain minimum level of chemi
`cal and physical stability for a defined length of time
`governed by treatment regimen, patient convenience, patient
`safety and regulatory guidelines.
`To avoid pain or possible tissue damage, liquid polypep
`tide drug compositions are designed to provide tonicity or
`osmolarity close to that of the bodily fluids at or surrounding
`the site of administration. Excipients such as glycerin,
`dextrose, mannitol, lactose and salts such as Sodium chloride
`are often used for this purpose. Examples of polypeptide
`drug products employing glycerin as an isotonicity agent
`include those comprising as active agent human insulin,
`insulin lispro, insulin aspart and glucagon.
`Glycerin has also been used in pharmaceutical composi
`tions as a solubilizer, wetting agent, emulsifier, Solvent,
`bulking Substance, antioxidant, chelating agent and preser
`vative Spiegel, A. J., et al., J. Pharm. Sci. 52:917–927
`(1963); Wang, Y-C. J. et al., J. Parenteral Drug Assoc.
`34:452–462 (1980); Remington's Pharmaceutical Sciences,
`Mack Publishing Company 18" Edition, p. 1316 (1990); Li,
`S., et al., J. Pharm. Sci. 85:868–872 (1996); Sieger, G. M.,
`et al., U.S. Pat. No. 4,016,273, issued 5 Apr. 1977; Heinz, D.
`N., WIPO publication WO98/29131, 9 Jul. 1998).
`For some polypeptide formulations, physical instability
`precludes the use of salts for isotonicity, a problem often
`Solved by employing glycerin. Glycerin, however, is known
`to contribute to chemical instability in polypeptide products.
`In particular, impurities present in glycerin, Such as alde
`hydes, are believed to initiate covalent crosslinking reac
`tions leading to polypeptide dimers and polymers. See, for
`example, Bello, J., et al. Arch. Biochem. Biophys. 172:
`608–610 (1976). For insulin products, such dimers and
`polymers have been linked to antigenicity and cutaneous
`allergy as described in Robbins, D. C., et al. Diabetes
`36:838–841 (1987); Robbins, D. C., et al. Diabetes
`36:147–151 (1987); and Ratner, R. E., et al. Diabetes
`39:728-732 (1990). Brange, J., et al. Pharm. Res.
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`MPI EXHIBIT 1075 PAGE 4
`
`MPI EXHIBIT 1075 PAGE 4
`
`

`

`US 7,022,674 B2
`
`3
`Information Bulletin No. AL-145, Aldrich Chemical Co.;
`Hopps, H. B., Aldrichimica Acta 33:28–29 (2000).
`The reaction of formaldehyde with acetylacetone to form
`a colored product was described by Nash, T. Biochem. J.
`55:416–425 (1953). This reagent appeared to be fairly
`specific for formaldehyde, as interference from acetaldehyde
`was only 1% on a molar basis.
`The glycerol monograph of The International Pharmaco
`poeia Third Edition, WHO, 4:176–181 (1994), described a
`test for aldehydes and reducing Substances using fuchsin/
`Sulfurous acid solution. Color intensity was compared to a
`0.2 M solution of potassium permanganate.
`In a promotional bulletin entitled “Discover the Origins of
`Some of the World's Most Consistently Pure Products:
`Synthetic Glycerine Products” by Dow Chemical Company
`(Freeport, Tex., USA), pp. 10–11, UV spectroscopy is used
`to compare OPTIMTM Glycerine 99.7% USP with less pure
`glycerin samples. No quantitative assessment of the level of
`aldehydes or other organic impurities is provided.
`Glyceraldehyde reacts with 3-methyl-2-benzothiazoli
`none hydrazone hydrochloride (MBTH). In Sawicki, E., et
`al. Anal. Chem. 33:93-96 (1961) this reagent was shown
`to react with DL-glyceraldehyde, but only measurement of
`formaldehyde in auto exhaust fumes and polluted air was
`disclosed. Paz, M. A., et al. Arch. Biochem. Biophys.
`109:548–559 (1965) showed that L-glyceraldehyde reacted
`with MBTH and disclosed an assay to detect trace quantities
`of aldehydes in the presence of ketones, keto acids and
`various types of pyranose carbohydrates during biochemical
`reactions. Eberhardt, M. A., et al. Marine Chemistry
`17:199–212 (1985) disclosed the use of MBTH to measure
`aldehydes, especially formaldehyde, in seawater and bacte
`rial cultures. MBTH is utilized in a commercial assay using
`glutaraldehyde, or 1,5-pentanedial Glutaraldehyde Test Kit
`Model GT-1, Hach (Loveland, Colo., USA) as a standard.
`This test uses a color wheel for measuring glutaraldehyde
`levels as low as 1 mg/L.
`Bailey, B. W., et al. Anal. Chem. 43:782–784 (1971)
`showed the reagent p-phenylenediamine reacted with form
`aldehyde, acetaldehyde and benzaldehyde but was highly
`selective for formaldehyde. It was used to measure low
`concentrations of formaldehyde in air.
`We have surprisingly discovered a novel MBTH Test
`using glyceraldehyde as a standard that can be effectively
`used to accurately determine the level of reactive aldehydes
`present in glycerin Samples. We have also discovered that
`the level of crosslinking in polypeptide formulations con
`taining glycerin is strongly correlated in a linear relationship
`with the level of reactive aldehyde in the glycerin used to
`prepare the formulations as measured by the aforementioned
`assay. Thus, our novel MBTH Test may be used to readily
`predict the relative chemical stability of aqueous, parenteral
`polypeptide compositions comprising glycerin and may also
`be employed to select suitable lots of glycerin for use in
`preparing Such compositions.
`
`4
`Aldehydes in glycerin form by autocatalytic or thermal
`oxidation, as noted in Mohr, J., et al. Canadian Patent
`Application 2.242,591, published 13 Jul. 1998. As reported
`by Ziels, N. W. J. Amer. Oil Chemists Soc. 33:556–565
`(1956), the processes used to commercially manufacture
`and purify glycerin have a great impact on the final purity of
`the glycerin, regardless of the starting material. Glycerin has
`been manufactured from many sources, including animal fat,
`plants, fermentation, chemical synthesis from Smaller
`organic molecules and from propylene. Methods of manu
`facturing glycerin from these and other sources are well
`known to those skilled in the art. However, what influence
`the source has on the level of reactive aldehydes found in
`lots of commercially manufactured glycerin and on the
`ultimate chemical stability of aqueous, parenteral polypep
`tide compositions comprising glycerin has not been explored
`or determined.
`Rohde, T. D., et al. Trans. Am. Soc. Artif. Intern. Organs,
`33:316–318 (1987) disclosed a new insulin formulation for
`use in implantable pumps containing about 80% glycerin in
`which the animal-rendered glycerin used in previous for
`mulations was replaced with glycerin from an unspecified
`synthetic source that was further purified by the authors
`using a mixed bed ion exchange column. The new and
`previous formulations also differed in pH, a key factor
`influencing extent of crosslinking reactions in insulin for
`mulations. In treating diabetic patients, a longer flow cycle
`and lower insulin usage with the new formulation Suggested
`improved stability, which was attributed to the difference in
`pH and the synthetic glycerin’s extra purification.
`Using the MBTH Test described herein, we have most
`Surprisingly discovered that commercial glycerin lots manu
`factured from non-animal sources contain lower levels of
`reactive aldehydes than animal-derived glycerin. This was
`demonstrated for glycerin derived from plants and propy
`lene. Glycerin derived from propylene has particularly low
`levels of reactive aldehydes. We also discovered that com
`mercially manufactured glycerin lots derived from plant and
`propylene sources have a much lower average reactive
`aldehyde content per month of age than glycerin lots derived
`from animal sources, which suggests the level of reactive
`aldehydes increases faster over time in animal derived
`glycerin than in plant and propylene derived glycerin.
`Furthermore, we discovered that aqueous, parenteral
`pharmaceutical compositions of polypeptides comprising
`glycerin derived from propylene have improved chemical
`stability compared to similar compositions prepared with
`animal derived glycerin.
`
`Lowering Reactive Aldehyde Levels in Glycerin
`
`Finally, no simple, efficient method for lowering the level
`of reactive aldehydes in glycerin to improve the chemical
`stability of pharmaceutical polypeptide compositions com
`prising glycerin has been disclosed.
`Bello, J. Biochemistry 8:4535–4541 (1969) and Bello,
`J., et al. Arch. Biochem. Biophys. 172:608–610 (1976)
`sought to prevent crosslinking in a protein solution contain
`ing glycerin by purifying the glycerin. The glycerin was first
`treated with the reducing agent sodium borohydride. The
`reduction step was followed by treating the glycerin with
`MB-3 resin to remove inorganic salts, and finally by distil
`lation in vacuo. There was no indication of the level of
`reactive aldehydes before or after this treatment. The low
`ered crosslinking achieved by this glycerin purification was
`short-lived.
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`
`Glycerin Derived from Various Sources
`
`Another hindrance to Solving the polypeptide crosslinking
`problem in formulations containing glycerin has been the
`failure to recognize the importance of considering the Source
`from which commercial glycerin is manufactured and the
`process by which the glycerin is manufactured. In particular,
`there has been no teaching or Suggestion that commercial
`lots of glycerin manufactured from different sources should
`be evaluated to determine if certain sources are better than
`others in minimizing the polypeptide crosslinking reactions.
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`5
`Various glycerin purification techniques were also
`described in Riddick, J. A., et al. Techniques of Chemistry
`II: Organic Solvents, Physical Properties and Methods of
`Purification, Wiley-Interscience, New York, pp. 689-690
`(1970), Diaz, Z. et al. U.S. Pat. No. 4,683,347, issued 28
`Jul. 1987, Stromquist, D. M., et al. Ind. Eng. Chem.
`43:1065-1070 (1951) and Ziels, referenced earlier, but
`none involved lowering the level of reactive aldehydes by
`contacting the glycerin with a polymeric resin comprising
`free amino groups.
`Washabaugh, M. W., et al. Anal. Biochem. 134: 144-152
`(1983) described a cumbersome procedure for lowering
`aldehyde levels in ethylene glycol which involved reducing
`with sodium borohydride, diluting 4-fold with water and
`passing the aqueous solution though four chromatography
`columns containing different resins. Aldehydes in the Solu
`tion were reduced by 86% as quantified using MBTH and a
`glycolaldehyde standard.
`Mohr, J., et al., referenced earlier, described purification
`of ethylene glycol by contacting the solution with a reducing
`phosphorous compound. Aldehyde levels, as measured with
`MBTH, were lowered. Murao, et al. U.S. Pat. No. 5,969,
`175, issued 19 Oct. 1999 described a method for purifying
`a nitrile containing an aldehyde by contacting the nitrile with
`a cation exchange resin carrying a polyamine. There was no
`suggestion either of these methods would be useful for
`lowering the reactive aldehyde content of glycerin to
`improve the chemical stability of polypeptide compositions
`comprising glycerin.
`We have discovered a simple, efficient method of lower
`ing the level of reactive aldehydes in glycerin samples that
`provides improved chemical stability to polypeptide com
`positions comprising glycerin. This method avoids the use of
`35
`reducing agents, avoids necessarily diluting the glycerin and
`is compatible with direct use of the purified glycerin in
`formulations containing polypeptides.
`The discoveries described above have been combined to
`provide novel preparations of polypeptide compositions for
`parenteral administration comprising glycerin that have
`improved chemical stability compared to polypeptide com
`positions previously known. These stabilized polypeptide
`compositions provide increased safety to patients who use
`them to treat their disease or condition.
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`US 7,022,674 B2
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`6
`Another aspect of the invention is an aqueous, parenteral
`pharmaceutical composition comprising a polypeptide and
`glycerin wherein the glycerin is derived from a non-animal
`SOUC.
`Another aspect of the invention is an aqueous, parenteral
`pharmaceutical composition comprising a polypeptide and
`glycerin, wherein the glycerin has a reactive aldehyde con
`tent of less than 8 ppm.
`Another aspect of the invention is a process for preparing
`an aqueous, parenteral pharmaceutical composition com
`prising, combining water, a polypeptide and non-animal
`derived glycerin.
`Another aspect of the invention is a process for preparing
`an aqueous, parenteral pharmaceutical composition com
`prising, combining water, a polypeptide and glycerin having
`a reactive aldehyde content of less than 8 ppm.
`Another aspect of the invention is a process for lowering
`the level of reactive aldehydes in glycerin comprising,
`contacting glycerin with a solid drying agent and a poly
`meric resin comprising free amino groups.
`The compositions of the present invention may be in the
`form of a solution or in a suspension in which the polypep
`tide remains partially or completely insoluble in the com
`position. The compositions may also be formed from two
`pack type manufactured products in which a polypeptide in
`solid form is combined with a separate diluent solution
`comprising glycerin prior to parenteral administration.
`The polypeptide in the pharmaceutical compositions of
`this invention may be chemically synthesized or produced
`biosynthetically using recombinant DNA techniques.
`The invention includes the use of a composition of the
`present invention as a medicament or for use in preparing a
`medicament for the treatment of diseases in mammals.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 shows the linear relationship (R=0.90) between
`analyses of commercial lots of non-animal derived glycerin
`determined by the MBTH Test of the present invention and
`the Modified 10x-GST Test.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`The term “glycerin” refers to the chemical propane-1,2,
`3-triol, CAS Registry Number 56-81-5). The empirical
`formula for glycerin is C(3)H(8)O(3), and it has the structure
`OH CH-CH(OH) CH-OH. In some literature
`reports, the term “glycerol' is used to refer to the chemical
`compound, glycerin” refers to purified commercial prod
`ucts containing 95% or more of glycerol, and 'glycerine' is
`used as a commercial name for products whose principal
`component is glycerol. For the present specification, glyc
`erin, meaning the chemical propane-1,2,3-triol, may be
`incorporated into the aqueous, parenteral pharmaceutical
`compositions of the present invention by using any solution
`comprising the chemical propane-1,2,3-triol. The glycerin
`concentration in the pharmaceutical polypeptide composi
`tions of the present invention is defined in terms of milli
`grams of propane-1,2,3-triol per milliliter of the composition
`and is less than 500 mg/mL.
`Glycerin was first discovered in 1779 by Carl W. Scheele,
`who produced it by heating olive oil with litharge. Since that
`time, at least five distinct sources of materials have been
`used to produce glycerin.
`One source of glycerin is animals. Tallow or fats from
`animals such as cattle and sheep are esterified and then
`
`BRIEF SUMMARY OF THE INVENTION
`
`Accordingly, one aspect of the present invention is the use
`50
`of non-animal derived glycerin as the glycerin component in
`an aqueous, parenteral pharmaceutical composition com
`prising a polypeptide and glycerin, to improve the chemical
`stability of the composition. The improved chemical stabil
`ity is directed to a reduction in the level of covalently
`55
`crosslinked polypeptides formed because a lower level of
`reactive aldehydes is present in the glycerin used in prepa
`ration of the composition. More specifically, the present
`invention provides for the use of glycerin derived from
`plants or propylene to improve the chemical stability of
`aqueous, parenteral pharmaceutical compositions compris
`ing a polypeptide and glycerin.
`Another aspect of the invention is the use of glycerin that
`has a reactive aldehyde content of less than 8 ppm as the
`glycerin component in an aqueous, parenteral pharmaceuti
`cal composition comprising a polypeptide and glycerin, to
`improve the chemical stability of the composition.
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`US 7,022,674 B2
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`7
`saponified in a process that generates glycerin as a by
`product of soap manufacturing. Animal fats are also hydro
`lyzed or saponified directly to generate glycerin.
`A second commercial source of glycerin is plants. Gen
`erally, oils derived from coconut, palm, canola, Soy or other
`plants are used to generate glycerin by methods comparable
`to those used with animal fats.
`A third source of glycerin is fermentation. Glycerin is
`fermented from natural Sources such as beet molasses or
`using microorganisms modified with recombinant DNA
`10
`technology such as those described by Bulthuis, B. A., et al.
`WIPO publication WO98/21340, 22 May 1998 and by
`Nair, R. V., et al. WIPO publication WO98/28480, 10 Jun.
`1999).
`A fourth Source of glycerin is chemical sy