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`110.
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`111.
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`whee
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`113.
`
`114.
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`115.
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`116.
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`
`118.
`119.
`
`120.
`
`121.
`‘122.
`123.
`124.
`
`125.
`
`Yang YY, Wan JP, Chung TS, et al. POE PEG POEtriblock copolymeric microspheres containing
`protein. I. Preparation and characterization. J Control Release 2001; 75:115 128.
`Jansen JA, de Ruijter JE, Janssen PT, et al. Histological evaluation of a biodegradable Polyactive/
`hydroxyapatite membrane. Biomaterials 1995; 16:819 827.
`Deschamps A, Grijpma, DW, Geijen,
`J. Poly(ethylene oxide)/poly(butylenes terephthalate)
`segmented block copolymers:
`the effect of copolymer composition on physical properties and
`degradation behavior. Polymer 2001; 42:9335 9345.
`De Groot CJ, Van Luyn MJ, Van Dijk Wolthuis WN, et al. In vitro biocompatibility of biodegradable
`dextran based hydrogels tested with human fibroblasts. Biomaterials 2001; 22:1197 1203.
`Cadee JA, van Luyn MJ, Brouwer LA,et al. In vivo biocompatibility of dextran based hydrogels.
`J Biomed Mater Res 2000; 50:397 404.
`Stenekes RJ, Franssen O, van Bommel EM,et al. The use of aqueous PEG/ dextran phase separation
`for the preparation of dextran microspheres. Int J Pharm 1999; 183:29 32.
`Franssen O, Vandervennet L, Roders P, et al. Degradable dextran hydrogels: controlled release of a
`model protein from cylinders and microspheres. J Control Release 1999; 60:211 221.
`Cadee JA, de Groot CJ, Jiskoot W, et al. Release of recombinant humaninterleukin 2 from dextran
`based hydrogels. J Control Release 2002; 78:1 13.
`Constancis A, Meyrueix R, Bryson N, et al. Macromolecular colloids of diblock poly(amino acids)
`that bind insulin. J Colloid Interface Sci 1999; 217:357 368.
`Flamel Technologies webpage.
`Koch M, Steidle C, Brosman 5, et al. An openlabel study of abarelix in men with symptomatic
`prostate cancerat risk of treatment with LHRH agonists. Urology 2003; 62:877 882.
`Gefter M, Barker N, Musso G, et al. Pharmaceutical formulations for sustained drug delivery. U.S.
`patent and trademark office (US 6180608 B1). Praecis Pharmaceuticals, Inc., 2001.
`Praecis home page (http://www.praecis.com).
`Baxter BioPharma Solutions home page. Available at: http:/ /www-.baxterbiopharmasolutions.com.
`Durect home page. Available at: http://www-durect.com.
`Okumu FW, Dao le N, Fielder PJ, et al. Sustained delivery of human growth hormone froma novel
`gel system: SABER. Biomaterials 2002; 23:4353 4358.
`Qiu B, Stefanos 5, Ma J, et al. A hydrogel prepared byin situ crass linking of a thiol containing poly
`(ethylene glycol) based copolymer: a new biomaterial for protein drug delivery. Biomaterials 2003;
`24:11 18.
`injectable neutral solutions of chitosan form
`Chenite A, Chaput C, Wang D, et al. Novel
`biodegradable gels in situ. Biomaterials 2000; 21:2155 2161.
`Ruel Gariepy E, Chenite A, Chaput C, et al. Characterization of thermosensitive chitosan gels for the
`sustained delivery of drugs. Int J Pharm 2000; 203:89 98.
`Berrada M, Serreqi A, Dabbarh F, et al. A novel non toxic camptothecin formulation for cancer
`chemotherapy. Biomaterials 2005; 26:2115 2120.
`Yong CS, Choi J5, Quan QZ,et al. Effect of sodium chloride on the gelation temperature, gel strength
`and bioadhesive force of poloxamer gels containing diclofenac sodium. Int J Pharm 2001; 226:195 205.
`Wasan KM,Subramanian R, KwongM,et al. Poloxamer 407 mediated alterations in the activities of
`enzymes regulating lipid metabolismin rats. J Pharm PharmSci 2003; 6:189 197.
`Knight CG. Hydrophobic prodrugs in liposomes.In: Knight C, ed. Liposomes: from Physical Structure
`to Therapeutic Applicaiton. New York: Elsevier/North Holland Biomedical Press, 1981:381 390.
`In:
`Oussoren C, Storm G, Crommelin DJ, el al. Liposomes for local sustained drug release.
`Oussoren C, Storm G, Crommelin DJ, et al., eds. Sustained Release Injectable Products. Boca Raton:
`Interpharm/ CRC Press, 2000:137 180.
`Pacira Pharmaceuticals home page. Available at: http: / /www.pacira.com.
`. Mantripragada 5S. A lipid based depot (DepoFoamtechnology) for sustained release drug delivery.
`Prog Lipid Res 2002; 41:392 406.
`Ye Q, Asherman J, Stevenson M, et al. DepoFoamtechnology: a vehicle for controlled delivery of
`protein and peptide drugs. J Control Release 2000; 64:155 166.
`Westesen K, Bunjes, H, Koch, MHJ. Physicochemical characterization of lipid nanoparticles and
`evaluation of their drug loading capacity and sustained release potential. J] Control Release 1997;
`48:223 236.
`
`126.
`
`127.
`
`128.
`
`129.
`
`130.
`
`131.
`
`132.
`
`133.
`
`135.
`
`136.
`
`137.
`
`138.
`
`139.
`
`Del Curto MD, Chicco D, D’Antonio M,etal. Lipid microparticles as sustained release systemfor a
`GnRH antagonist (Antide}. J Control Release 2003; 89:297 310.
`Engwicht A, Girreser U, Muller BW. Characterization of co polymers oflactic and glycolic acid for
`supercritical fluid processing. Biomaterials 2000; 21:1587 1593.
`Reithmeier H, Herrmann J, Gopferich A. Lipid microparticles as a parenteral controlled release
`device for peptides. J Control Release 2001; 73:339 350.
`
`Regeneron Exhibit 1015.206
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`140.
`
`141.
`
`142.
`
`143.
`
`144.
`145.
`
`146.
`
`147.
`
`148.
`
`149.
`
`150).
`151.
`
`152.
`
`153.
`
`154.
`
`155.
`
`Reithmeier H, Herrmann J, Gopferich A. Development and characterization of lipid microparticles
`as a drug carrier for somatostatin. Int J Pharm 2001; 218:133 143,
`Scholer N, Krause K, Kayser O, et al. Atovaquone nanosuspensions show excellent therapeutic effect in
`a new murine modelof reactivated toxoplasmosis. Antimicrob Agents Chemother 2001; 45:1771 1779.
`Li LC, Zhu L, Song JF, Deng JS, et al. Effect of solid state transition on the physical stability of
`suspensions containing bupivacaine lipid microparticles. Pharm Dev Technol 2005; 10:309 318.
`Papahadjopoulos D, Vail WJ, Jacobson K, et al. Cochleate lipid cylinders: formation by fusion of
`unilamellar lipid vesicles. Biochim Biophys Acta 1975; 394:483 491.
`Zarif L. Drug delivery by lipid cochleates. Methods Enzymol 2005; 391:314 329.
`intravenous
`Segarra I, Movshin DA, Zarif L. Pharmacokinetics and tissue distribution after
`administration of a single dose of amphotericin B cochleates, a new lipid based delivery system. J
`Pharm Sei 2002; 91:1827 1837.
`Zarif L, Graybill JR, Perlin D, et al. Antifungal activity of amphotericin B cochleates against Candida
`albicans infection in a mouse model. Antimicrob Agents Chemother 2000; 44:1463 1469.
`Gould Fogerite 5, Kheiri, MT, et al. Cochleate delivery vehicles: applications in vaccine delivery.
`J Liposome Res 2001; 10:339 356.
`Bracho G, Lastre M, del CampoJ, et al. Proteoliposome derived cochleate as novel adjuvant. Vaccine
`2006; 24(suppl 2):52 530 31.
`Gould Fogerite 5, Mannino, RJ. Cochleate delivery vehicles: applications to gene therapy. Drug
`Deliv Technol 2003; 3.
`Croxatto HB. Progestin implants. Steroids 2000; 65:681 685.
`Chertin B, Spitz IM, Lindenberg T, et al. An implant releasing the gonadotropin hormonereleasing
`hormone agonist histrelin maintains medical castration for up to 30 months in metastatic prostate
`cancer. J Urol 2000; 163:838 844.
`WrightJ, Chester, AE, Skowronski, RJ, et al. Long term controlled delivery of therapeutic agents via
`an implantable osmotically driven system: the DUROS implant.
`In: Rathbone M, ed. Modified
`Release Drug Delivery Technology. New York: Marcel Dekker, 2003:657 669.
`Fowler JE Jr., GottesmanJE, Reid CF, et al. Safety and efficacy of an implantable leuprolide delivery
`system in patients with advanced prostate cancer. J Urol 2000; 164:730 734.
`Burgess DJ, Hussain AS, Ingallinera TS, et al. Assuring quality and performance of sustained and
`controlled release parenterals: AAPS workshop report, co sponsored by FDA and USP. Pharm Res
`2002; 19:1761 1768.
`Burgess DJ, Crommelin DJ, Hussain AS, et al. Assuring quality and performance of sustained and
`controlled released parenterals. Eur J Pharm Sci 2004; 21:679 690.
`. D'Souza SS, DeLuca PP. Methods to assess in vitro drug release from injectable polymeric
`particulate systems. Pharm Res 2006; 23:460 474.
`D'Souza 5S, Faraj JA, DeLuca PP. A model dependent approach to correlate accelerated with real
`time release from biodegradable microspheres. AAPS PharmSciTech 2005; 6:E553 E564.
`Gido C, Langguth P, Kreuter J, et al. Conventional versus novel conditions for
`the in vitro
`dissolution testing of parenteral slow release formulations: application to doxepin parenteral dosage
`forms. Pharmazie 1993; 48:764 769.
`Iyer SS, Barr WH, Karnes HT.Profiling in vitro drug release from subcutaneous implants: a review
`of current status and potential implications on drug product development. Biopharm Drug Dispos
`2006; 27:157 170.
`Giteau A, Venier Julienne MC, Aubert Pouessel A, et al. How to achieve sustained and complete
`protein release from PLGA based microparticles? Int J Pharm 2008; 350:14 26.
`UppoorVR. Regulatory perspectives on in vitro (dissolution)/in vivo (bioavailability) correlations. J
`Control Release 2001; 72:127 132.
`Cheung RY, Kuba R, Rauth AM,et al. A new approachto the in vivo and in vitro investigation of
`drug release from locoregionally delivered microspheres. ] Control Release 2004; 100:121 133.
`Schliecker G, Schmidt C, Fuchs 5, et al. In vitro and in vivo correlation of buserelin release from
`biodegradable implants using statistical moment analysis. ] Control Release 2004; 94:25 37.
`Gido C, Langguth P, Mutschler E. Predictions of in vivo plasma concentrations fromin vitro release
`kinetics: application to doxepin parenteral (i.m.) suspensions in lipophilic vehicles in dogs. Pharm
`Res 1994; 11:800 808.
`Chu DF, Fu XQ, Liu WH, et al. Pharmacokinetics and in vitro and in vivocorrelation of huperzine A
`loaded poly(lactic co glycolic acid) microspheres in dogs. Int J] Pharm 2006; 325:116 123.
`Dash AK, Haney PW, Garavalia MJ. Development of anin vitro dissolution method using microdialysis
`sampling technique for implantable drug delivery systems. J Pharm Sci 1999; 88:1036 1040.
`
`Lee:
`
`158.
`
`159.
`
`160.
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`161.
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`162.
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`163.
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`164.
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`165.
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`166.
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`Regeneron Exhibit 1015.207
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`193
`
`167.
`
`168.
`
`169.
`
`170.
`
`171.
`
`Akers MJ, Larrimore DS, Guazzo DM. Parenteral quality control: sterility, pyrogen, particulate, and
`package integrity testing. In: Swarbrick J, ed. Drugs and the Pharmaceutical Sciences. New York:
`Marcel Dekker, 2003:40 50.
`Staples M, Daniel K, Cima MJ, Langer R. Application of micro and nanoelectromechanical devices
`to drug delivery. Pharm Res 2006; 23:847 863.
`Davies OR, Lewis AL, Whitaker MJ, et al. Applications of supercritical CO2 in the fabrication of
`polymer systems for drug delivery and tissue engineering. Adv Drug Deliv Rev 2008; 60:373 387.
`Smit TH, Thomas KA, HoogendoornRJ, et al. Sterilization and strength of 70/30 polylactide cages:
`e beam versus ethylene oxide. Spine 2007; 32:742 747.
`Bayer CL, Peppas NA. Advances in recognitive, conductive and responsive delivery systems. J
`Control Release 2008.
`Peppas NA, Leabandung W. Stimuli sensitive hydrogels:
`chronotherapy- J Biomater Sci Polym Ed 2004; 15:125 144.
`Smolensky MH, Peppas NA. Chronobiology, drug delivery, and chronotherapeutics. Adv Drug
`Deliv Rev 2007; 59:828 851.
`Peppas NA, Huang Y, Torres Lugo M,et al. Physicochemical foundations and structural design of
`hydrogels in medicine and biology. Annu Rev Biomed Eng 2000; 2:9 29.
`Qiu Y, Park K. Environment sensitive hydrogels for drug delivery. Adv Drug Deliv Rev 2001;
`53:321 339.
`Kikuchi A, OkanoT. Pulsatile drug release control using hydrogels. Adv Drug Deliv Rev 2002; 54:
`53 77.
`Sershen S, West J. Implantable, polymeric systems for modulated drug delivery. Adv Drug Deliv
`Rev 2002; 54:1225 1235.
`178.
`Murdan S. Electro responsive drug delivery from hydrogels. J Control Release 2003; 92:1 17.
`. Maloney JM, Uhland SA, Polito BF, et al. Electrothermally activated microchips for implantable drug
`delivery and biosensing. J Control Release 2005; 109:244 255.
`Avgoustakis K. Pegylated poly(lactide) and poly(lactide co glycolide) nanoparticles: preparation,
`properties and possible applications in drug delivery. Curr Drug Deliv 2004; 1:321 333.
`Abbas AO, Donovan MD,Salem AK. Formulating poly(lactide co glycolide) particles for plasmid
`DNA delivery. J PharmSci 2008; 97:2448 2461.
`De Laporte L, Shea LD. Matrices and scaffolds for DNA delivery in tissue engineering. Adv Drug
`Deliv Rev 2007; 59:292 307.
`Chen RR, Mooney DJ. Polymeric growthfactor delivery strategies for tissue engineering. Pharm Res
`2003; 20:1103 1112.
`Kobsa 5, Saltzman WM. Bioengineering approaches to controlled protein delivery. Pediatr Res 2008.
`Sinha VR, Trehan A. Biodegradable microspheres for parenteral delivery. Crit Rev Ther Drug
`Carrier Syst 2005; 22:535 602.
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`ideal carriers for chronobiclogy and
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`Regeneron Exhibit 1015.208
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`

`8|Biophysical and biochemical characterization
`of peptide and protein drug product
`Tapan K. Das and JamesA. Carroll
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`INTRODUCTION
`Classes of Biotherapeutics
`The biotherapeutics class of drugs that are commercially available encompass a range of
`compounds including recombinant or purified proteins, monoclonal antibodies (also
`proteins), peptides, conjugated or fused peptides, antibody conjugates, protein vaccines,
`oligonucleotides, protein-lipid complexes, enzymes, antibody fragments (Fabs), glycosylated
`proteins, and carbohydrates (Fig. 1). Additional molecule typesare in preclinical and clinical
`development.
`The biotherapeutics class contains a wide variety of recombinant proteins derived from
`microbial, mammalian, and yeast sources (Table 1). There are few products that are extracted
`from natural sources. The biotherapeutics class of drugs uses a variety of technologies for
`extending half-life such as conjugating to polyethylene glycol (PEG), fusion with antibody or
`Fab, and employing the antibodyitself. This is especially true for peptides and other small
`entities that would be cleared via the kidneys without a half-life enhancing strategy such as
`conjugation or fusion. Table 1 illustrates the wide variety of biotherapeutics entities on the
`market.
`
`Regulatory Guidance on Structural Characterization
`Regulatory approval of a biotherapeutic entity requires meeting the guidelines for chemistry,
`manufacturing, and controls (CMC) put forth by the relevant regulatory agency. A complete
`CMC packageincludes a description of the characterization of the biotherapeutic entity, which
`includes the Elucidation of Structure and Impurities sections, which, for biological entities can
`be quite complex.
`It is expected that the applicant have a detailed understanding of the
`structure, heterogeneity, and stability of the biotherapeutic entity using a variety of analytical
`methods. Regulatory guidance on the characterization of biotherapeutic molecules can be
`found in several sources. The U.S. Food and Drug Administration (FDA),
`the European
`Medicines Agency (EMEA), and other regulatory agencies around the world often provide
`guidance documents on specific topics relating to the review and approval of drugs, and these
`can be excellent sources of information for applicants (www.fda.gov, www.emea.europa.eu).
`The International Committee on Harmonization (ICH) (www.ich.org) provides guidance
`documentation agreed on by the regulatory agencies of the United States, Europe, and
`Japan. The ICH guideline Q5 deals specifically with biotechnology products, and some
`information concerning characterization is available in this section, particularly Q5E on
`comparability. Q6B deals with specifications of biotechnology products, and provides further
`relevant information for biotherapeutic entities.
`
`Proof of Structure
`As part of the Elucidation of Structure section of a CMC package, a detailed analysis of the
`structure of the biotherapeutic is required. This evaluation is in addition to the normal batch
`release assays used for the product which ensure the safety and efficacy of each batch. The
`characterization assays included in this section are used for confirmation of the predicted
`primary structure, higher order structures, post-translational modifications, and degradation
`products that may form or increase on stability. The presence and levels of variant forms needs
`to be measured, and their impacton the safety and efficacy of the product needsto be assessed.
`The attributes investigated may be assessed using multiple analytical methods for each, as
`discussed in some detail below.
`
`Regeneron Exhibit 1015.209
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`BIOPHYSICAL AND BIOCHEMICAL CHARACTERIZATION OF PEPTIDE AND PROTEIN DRUG PRODUCT
`
`195
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`peutic class of drugs and drug candidates in
`various stages of development (data from
`PharmaCircle, March 2009). Numbers do not
`represent unique molecule types in any of the
`classes.
`
`The confirmation of primary structure may include assays that demonstrate the product
`has the expected amino acid sequence, such as amino acid sequencing, mass spectrometry
`(MS), and electrophoresis. These methods ensurethat there are no translation variants such as
`amino acid substitutions, terminal extensions, or unprocessed introns present in the product.
`Higher order structure may be assessed by biophysical and spectroscopic methods such as
`circular dichroism (CD) and fluorescence spectroscopy. This may include a determination of
`the disulfide bond connectivity, which can be critical for a protein to maintain its active
`conformation. Many post-translational modifications of proteins are possible, such as
`glycosylation. Other modifications mayinclude related species formed as a consequence of
`degradation, such as oxidation and deamidation. For conjugated products, variants due to the
`conjugation process and degradation products of these need to be assessed and understood. In
`total, biotherapeutics may include a heterogeneous mixture due to all of the variant forms
`possible, and the applicant needs to demonstrate an understanding of the species present.
`
`Potency Determination
`For biologics, in most cases, a relevant potency assay for the biological entity is required for its
`approval. The assay needs to demonstrate “the specific ability or capacity of a product to
`achieve a defined biological effect.” (ICH, Q6B, specifications: test procedures and acceptance
`criteria for biotechnological/biological products). One or more bioassays are typically included
`as part of batch release, and range from binding assays, cell-based assays, or in vivo animal
`assays. As part of characterization, it is expected that variant forms of the biological entity be
`assessed for potency. This involves isolation of the variant form and testing in the relevant
`bioassay(s)
`for
`the product. For species that
`form or increase on stability because of
`degradation, stress conditions can be used to generate sufficient material to perform potency
`assays.
`
`Formulation Characterization
`(intravenous or
`Most
`therapeutic biologics currently are administered via parenteral
`subcutaneous) route. The goal of biologics drug product formulation development is to
`minimize various degradation pathways to achieve a minimum shelf-life of 18 to 24 months at
`the intended storage condition. An emerging strategy in the biotherapeutics industry is to
`minimize investment in the early stages of preclinical and clinical development, and therefore,
`drug product formulation for early clinical
`trials may not be characterized in detail.
`Additionally, long-term stability data may be rarely available in early stage. Howeverit is
`necessary to make an assessment of potential chemical and physical labilities that may impact
`long-term stability. A part of this assessment can be achieved by Preformulation work whichis
`a combination of experimental and bioinformatics studies conducted in early stage prior
`to nominating a drug product
`formulation. “Formulation characterization” refers to
`
`Regeneron Exhibit 1015.210
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`

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`Regeneron Exhibit 1015.211
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`sginoajoyypuesaiGojouy9a,seqinossadA|,sajnoajoyyJoSs2/5sonnedeuauyloigjosajdwexy4aIqeL
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`Regeneron Exhibit 1015.212
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`characterization of drug product formulation using biochemical and biophysical methods for
`adequate understanding of structural and functional correlations to stability in a stage
`appropriate manner. It should be noted that depending on the type of biologics candidate and
`its stability profile, it may be necessary to conduct additional formulation characterization
`studies especially when stability is poor and/or stability-bioactivity correlation is complex.In
`later stages of clinical development as well as for biologics license applications (BLA) it is
`expected that extensive formulation characterization studies are conducted.
`
`Determination of Hot Spots
`An important and first step in formulation characterization is to determine the potential
`liabilities in the amino acid sequence and otherparts (for contents other than amino acid) of the
`biotherapeutic candidate. These liabilities are often referred to as “hot spots.” There are some
`aminoacids or groups of amino acids that exhibit common occurrences of chemical or physical
`degradation events such as oxidation and deamidation. For example,
`the amino acid
`methionine (Met) undergoes oxidation, especially in the presence of oxygen and whenitis
`on the protein surface exposed to bulk solvent. Similarly, a surface-exposed pair of asparagine-
`glycine (Asn-Gly) when present in a loosely formed structural domain in the protein may be
`prone to deamidation undercertain formulation conditions (1).
`
`Linear sequence vs. folded structure. Determination of hot spots may not be trivial for all
`protein types. Prediction of lability of an amino acid based on primary structure [ie., amino
`acid linkage (Table 2)] does not work well for folded proteins because surface exposure and
`flexibility in the three-dimensional structure are among the important criteria dictating
`propensity of degradation. For certain classes of biotherapeutics where adequate correlation
`between structural and chemical degradation is available,
`it might be possible to more
`accurately predict hot spots. For example, immunoglobulins (IgGs) of a given subtype may
`contain common hot spots in the conserved part of the sequence (Table 2). Similarly,
`degradation behavior of a nonconserved amino acid in a conserved structural motif in IgGs
`may be partially predicted on the basis of structural flexibility of the motif (unordered vs.
`helical or B sheet). While these approaches are quite useful in enlisting the common hot spots
`for chemical degradation, they may not predict physical degradation (aggregation) hot spots or
`unique chemical degradation events [e.g., tyrosine (Tyr)/tryptophan (Trp) oxidation].
`The determination of hot spots needs information on folded structure but many
`biotherapeutic candidates will not have its crystal structure or other solution-based
`(e.g., NMR) structure available. In the absence of structure, homology modeling may be
`beneficial to derive qualitative structure using bioinformatics tools. In a recent study, Wang
`et al. (14) employed a novel use of bioinformatics tools to delineate common sequence
`segments across several antibodies and hypothesized that such segments may contribute to
`aggregation propensity on the basis of certain physicochemical properties of the contributing
`amino acids in these segments (rich in aliphatic/aromatic residues). Using full antibody
`atomistic molecular dynamics simulations, Chennamsetty et al. (15) identified the antibody
`regions prone to aggregation by using a technology called spatial aggregation propensity.
`Development of such bioinformatics tools is a good first step in understanding aggregation
`propensity, however it remains to be experimentally tested how accurately and widely such
`tools can be used for reliable prediction appropriate for drug development.
`
`Physical and Chemical Degradations
`the next step in formulation
`Following determination of hot spots as described above,
`characterization is to experimentally determine the major degradation pathway(s) and to
`understand the mechanism of degradation. Unlike small molecule drugs, protein-based
`biotherapeutics candidates have added complexity of several degrees of structure such as
`secondary, tertiary and quaternary structures that are critical to its stability and intended
`function. The degradations observed and/or predicted can be categorized into two types
`chemical and physical degradations. Majority of the degradations cited in Table 2 are of
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`Table 2. Protein and Peptide Degradation Hot Spots
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`
`
`Labile groups Occurrence in IgG and other proteins Type of degradation
`
`
`
`
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`Asn Ser, Asn Asn,
`Asn Thr, Asn Lys,
`Asn His, Asn Asp
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`Deamidation, Isomerization
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`Asn Gly Deamidation, Isomerization=NN*SG in CH3 (IgG2a) (2)
`QN™®®G in CL (IgG2a) (2)
`LN?"§G in CH2 (IgG1) (3)
`SN*"5G in CH3 (IgG1) (3)
`RN*2°S in CH3 (IgG2a) (2)
`PEN“°°NY in CH3 (3)
`VNO°T in CDR1 of LC (4)
`SN?"K in CH2 (5)
`D??4 p75 (igG1) (5)
`Clipping (peptide bond)
`Asp Pro
`Asp Gin DKin hinge (IgG1) (5)
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`H T in hinge (IgG1) (5)
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`Asp Lys
`His Thr
`Asp
`Met
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`Cys
`Trp
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`Tyr
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`Isomerization
`Oxidation
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`Oxidation
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`D'°G in CDR3 of HC (IgG1) (4)
`M* in CDR1 of HC (IgG1) (6)
`M"*" in CDR3 of HC (IgG1) (6)
`Oxidation (to form disulfide) C'°° in CDR3 of HC (IgG2a) (2)
`Oxidation
`w**| wW®* in CDR2 of HC (IgG) (6)
`w'® in CDR8 of HC (IgG1) (6)
`Oxidation of lens protein forms
`dihydroxyphenylalanine, o and m Tyr, and
`di Tyr (7)
`Trans P™ isomer formation in 2 microglobulin (8)
`K** in LC (IgG1) (9)
`Iron loss by acidic pH, chelatorin transferrin (10)
`Low pH Fe His breakage in hemoglobin (11)
`Labile Fe S (Met) bond in cytochrome c breaks
`under various conditions (12)
`May form adducts such as carboxylate adduct with
`Reaction with
`Amine and other reactive
`citrate/succinate (13)
`buffer/excipients
`amino acids
`Potential hot spots for aggregation in IgG predicted
`Aggregation
`Various hydrophobic
`segments using bioinformatics tools (14,15)
`
`
`Pro
`Lys
`Fe His/Asp/Tyr
`His Fe (heme)
`Met Fe (heme)
`
`Proline isomerization
`Glycation
`Metal bond breakage
`Metal bond breakage
`Metal bond breakage
`
`Abbreviations: |gG, immunoglobulin; LC, light chain of IgG; HC, heavy chain of IgG; Tyr, tyrosine; Met, methionine.
`
`chemical nature, whereas physical degradation includes aggregation, particulate formation,
`and related structural degradation events associated with adsorption, misfolding, denaturation
`(by heat, chemicals, chaotropes, ete.), partial misfolding, nucleating species, and sometimes
`chemical degradation. Physical degradation is complex and may involve a wide variety of
`causative factors that may involve protein-protein interaction, native state conformational
`distortion, air-water interfacial
`tension, and conformational changes induced by solvents,
`additives, and processing. Therefore, a multitude of biophysical
`tools (in addition to
`biochemical characterization) is often necessary to achieve a comprehensive formulation
`characterization.
`
`ASSESSMENT OF PRIMARY STRUCTURE
`Simply put,
`the primary structure of a protein consists of its amino acid sequence. For
`recombinant proteins, the amino acid sequence can be predicted from the cDNA usedin its
`production. This basic attribute of a protein determines the entirety of its biophysical and
`biochemical properties. The amino acid sequence of a protein determines its ability to fold
`properly, and thus determinesits ability to maintain its function. Therefore, a small change in
`the primary structure, depending on its location, may have a range of effects on a protein’s
`activity, from no effect to a very large impact. The amino acid sequence can also impact the
`chemical and physical stability of a protein, even when there is no measurable impact on
`activity. Thus, confirming the amino acid sequence of a protein is
`fundamental
`to
`understanding its overall structure and properties.
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`Regeneron Exhibit 1015.214
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`During production of recombinant proteins, several modifications to the primary
`structure are possible. These include errors in transcription or translation, generating such
`variant forms as amino acid substitutions, N- and C-terminal extensions, splice variants, and
`internal sequence extensions. Other changes to the primary structure may occur as a
`consequence of biochemicalinstability, such as deamidation or oxidation. All of these variant
`forms can have large impacts on the properties of the protein, and need to be detected and
`controlled during production and storage.
`
`Amino Acid Composition Analysis
`One of the most basic assessments of primary structure is the confirmation of the exp