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`Ye Q, Asherman ], Stevenson M, et al. DepoFoa m technology: a vehicle for controlled delivery of
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`supercritical fluid processing. Biomaterials 2000; 211158? 1593.
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`Regeneron Exhibit 1015.206
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`as a drug carrier for somatostatin. Int J [’lia rm 2001; 218:133 143.
`. Scholer N, Krause K. Kayser O, et al. Atovaquone nanosuspensions show excellent therapeutic effect in
`a new murine model of reactivated toxoplasmosis. Antimicrob Agents Chemother 2001; 45:1771 1779.
`Li LC, 7.1m 1., Song IF, Deng JS, et al. Effect of solid state transition on the physical stability of
`suspensions Containing bupivacaine lipid micmpa rticles. Pharm Dev Technol 2005; 101309 318.
`Papahadjopoulos D, Vail W], 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 cachleates. Methods Enzymol 2005; 391 :314 329.
`intravenous
`Sega rra l, Movshin DA, Zarif 1.. Pharmacokinetics and tissue distribution after
`administration of a single dose of amphotericin B cochleates, a new lipid based delivery system. ]
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`albicans infection in a mouse model. Antimicmb Agents Chemother 2000; 4411463 1469.
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`J Liposome Res 2001; 101339 356.
`Bracho G, [.astre M, del Campo J, et al. Proteolilmsome derived cochleate as novel adjuvant. Vaccine
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`an implantable osmotically driven system: the DUROS implant.
`In: Rathbone M, ed. Modified
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`Fowler ]E Jr., Gottesman JE, Reid CF, et al. Safety and efficacy of an implantable leuprolide delivery
`system in patients with advanced prostate cancer. J Urol 2000: ]64:'?30 734.
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`controlled release parenterals: AAPS workshop report, co sponsored by FDA and USP. Pharm Res
`2002; 19:1761 1768.
`Burgess DJ, Crommelin DJ, liussain AS, et al. Assuring quality and performance of sustained and
`controlled released parenterals. Eur J Pharm Sci 2004; 21:67.9 690.
`D’Souza 55, Del .uca PP. Methods to assess in vitro drug release from injectable polymeric
`particulate systems. Pharm Res 2006; 23:460 474.
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`time release from biodegradable microspheres. AAl’S [’harmSciTech 2005; 6:E553 E564.
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`protein release from PLCA based microparticles? [nt J Pharm 2008; 350:14 26.
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`Control Release 2001 ; 72:12? 132.
`Cheung RY, Kuba R, Rauth AM, et al. A new approach to the in vivo and in vitro investigation of
`drug release from locoregionally delivered microspheres. ] Control Release 2004; 100:121 133.
`Schliecker C, Schmidt C, Fuchs S, et al. in vitro and in vivo correlation of buserelin release from
`biodegradable implants using statistical moment analysis. ] Control Release 2004; 94:25 37.
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`kinetics: application to doxepin parenteral (i.m.) suspensions in lipophilic vehicles in dogs. Pharm
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`polymer systems for drug delivery and tissue engineering. Adv Drug Deliv Rev 2008; 60:373 387.
`Smit Tl- I, Thomas KA, lIoogeodoorn R], et a1. Sterilization and strength of 70/30 polylactide cages:
`e beam versus ethylene oxide. Spine 2007: 32:742 74?.
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`Peppas NA. Leobandung W. Stimuli sensitive hydrogels:
`chronolherapy. J Biomater Sci l’olym Ed 2004; 15:125 144.
`Smolensky MH, Peppas NA. Chmnobiology, drug delivery, and chrol‘iotherape'ul‘ics. Adv Drug
`Deliv Rev 2007; 59:828 851.
`Peppas NA, Huang Y, Torres Lugo M, et al. Physioochemical 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 fur drug delivery. Adv Drug Deliv Rev 2001;
`53:321 339.
`Kikuchi A, Okano T. Pulsatile drug release control using hyd rogels. Adv Drug Deliv Rev 2002; 54:
`53 77.
`Sershen 5, West I. Implantable. polymeric systems for modulated drug delivery. Adv Drug Deliv
`Rev 2002; 54:1225 1235.
`Murdan 5. Electro responsive drug delivery from hydrogels. ] Control Release 2003; 92:1 17.
`Maloney IM, Uhland 5A, Polito BF, et al. Electrothermally activated microchips for implantable drug
`delivery and biosensing. J Control Release 2005: 1091244 255.
`Avgoustakis K. Pegylated polyflactide) and polyUactide co glycolide) nanoparticles: preparation,
`properties and possible applications in drug delivery. Curr Drug Deliv 2004; 1:321 333.
`Abbas A0, Donovan MD. Salem AK. Formulating polyllactide co glycolidel particles for plasmid
`DNA delivery. ] Pharm Sci 2008; 97:2448 2461.
`De Laporte L, Shea LD. Matrices and scaffolds for DNA delivery in tissue engineering. Adv Drug
`Deliv Rev 2007; 591292 30?.
`Chen RR, Mooney D]. Polymeric growth factor delivery strategies for tissue engineering. I’harm Res
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`Kobsa S, 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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`8 Biophysical and biochemical characterization
`of peptide and protein drug product
`Tapan It. Das and James it. Carroll
`
`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 types are 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 antibody itself. 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 package includes a description of the characteri7ation 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 biotherapeu tic entity using a variety of analytical
`methods. Regulatory guidance on the characterization of biotherapeutic molecules can be
`found in several sources. The US. Food and Drug Administration (FDA),
`the European
`Medicines Agency [EMEAL 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.fdagov, www.emea.europa.eu).
`The International Committee on Harmonization (ICH) {www.iclmorg) 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 QSE 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 biotherapeu tic 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 impact on the safety and efficacy of the product needs to 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 ANIJ BIOCHEMICAL CtMI-IACIEHIZAHOII OI PH’HUE AND P80 IHN UHUG PHOUUCI
`
`195
`
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`1
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`Figure 1
`Portfolio of selected biothera
`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 con firmation 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 ensure that 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 may include 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, Q63, 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 characteri7ation, 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
`therapeutic biologics currently are administered via parenteral
`Most
`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. However it 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 which is
`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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`196
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`198
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`VOLUME 1' fUHMUMHOfl AMI) PACKAGING
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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 c]inical 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 other parts (for contents other than amino acid) of the
`biotherapeutic candidate. These liabilities are often referred to as "hot spots." There are some
`amino acids 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 when it is
`on the protein surface exposed to bulk solvent. Similarly, a surface—exposed pair of asparagine—
`glycine (Ash-(31y) when present in a loosely formed structural domain in the protein may be
`prone to deamidation under certain 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 [i.e., 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 [i 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 (Tyrl/ tryptophan (T113) 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 0n 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 Clreriiical chredctions
`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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`BIOPHYSICAL ANIJ BIOCHEMICAL CHM-MC IEHIZH'I 1'01“).I OI— PtPIItJE AM) PRO-“km UHUG PHOUUCI
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`199
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`
`
`Table 2 Protein and Peptide Degradation Hot Spots
`Type of degradation Occurrence in IgG and other proteins
`Labile groups
`Deamidation, Isomerization
`
`Asn Gly
`
`Asn Ser, Asn Asn,
`Asn Thr. Ash Lys.
`Asn His, Ash Asp
`
`Asp Pro
`Asp Gln
`
`Deamidalion, Isomerizalion
`
`Clipping (peptide bond)
`
`NNwE‘G in CH3 (IgG2a) (2)
`QN‘5“G in CL (Igeza) (2)
`LN3‘56 in CH2 (I961) (3)
`SN3’356 in CH3 (I961) (3)
`alums in CH3 (Igeza) (2)
`PENMNY in CH3 (3)
`vnwr in com of LC (4)
`SN329K in CH2 (5)
`D2" P275 (I961) (5)
`D K in hinge (I961) (s)
`H T in hinge (lgG1) (5)
`
`Asp Lys
`His Thr
`Ase
`Met
`
`Cys
`Trp
`
`Tyr
`
`Isomerization
`Oxidation
`
`Oxidation (to form disulfide)
`Oxidation
`
`Oxidation
`
`Pro
`Lys
`Fe HisIAspITyr
`His Fe (heme)
`Met Fe theme)
`
`Proline isomerization
`Glycation
`Metal bond breakage
`Metal bond breakage
`Metal bond breakage
`
`ems. in cons of HC (lgG1) (4)
`M34 in CDR1 of HC (I961) (6)
`I111‘[)1 in cons of HC (I961) (a)
`0‘05 in ones of HC (I962a) (2)
`w“, w“ in cone of HC (lgG1) (5)
`W105 in ones of He (lgG1) (6)
`Oxidation of lens protein forms
`dihydroxyphenylalanine, o and m Tyr, and
`di Tyr (7)
`Trans F"d isomer formation in B2 microglobulin (8)
`K“9 in LC (I961) (9)
`Iron loss by acidic pH, chelator in transferrin (10)
`Low pH Fe His breakage in hemoglobin (11)
`Labile Fe S (Met) bond in cytochrome c breaks
`under various conditions (12)
`Reaction with
`Amine and other reactive
`May form adducts such as carboxylate adduct with
`amino acids
`citrateIsuccinate (13)
`hutlerl'excipients
`Aggregation
`Various hydrophobic
`Potential hot spots for aggregation in IgG predicted
`using bioinformatics tools (14.15]
`segments
`
`Abbreviations: lgG, immunoglohulin: LC, light chain of lgG; 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, etc), 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
`
`the primary structure of a protein consists of its amino acid sequence. For
`Simply put,
`recombinant proteins, the amino acid sequence can be predicted from the cDNA used in 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 determines its 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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`VOLUME 1' HJHMULAHOFI' AMI) PACKAGWG
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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—terrninal extensions, splice. variants, and
`internal sequence extensions. Other changes to the primary structure may occur as a
`consequence of biochemical instability, 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 expected
`amino acid composition of the polypeptide. Recombinantly produced proteins have amino
`acid sequences predicted from the DNA sequence used in their production. The amino acid
`composition, therefore, is a predictable attribute, and can be confirmed using amino acid
`composition analysis. The technique can be broken down into three steps: complete hydrolysis
`of the polypeptide in to its constituent amino