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`Regeneron Exhibit 1015.206
`
`
`
`192
`
`VOLUME 1: FORMULA(ION AND PACKAGING
`
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`of current status and potential implications on drug product development. Biopharm Drug Dispos
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`drug release from locoregionally delivered microspheres. J Control Release 2004; 100:121 133.
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`
`Regeneron Exhibit 1015.207
`
`
`
`FORMULATION OF OEPOT DELIVERY SYSTEMS
`
`193
`
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`e beam versus ethylene oxide. Spine 2007; 32:742 747.
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`chtooothecapy. J 13iomater Sci Polyln Ed 2004; 15:125 144.
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`185. Sinha VR, Trehan A. Biodegradable n1icrospheres for parenteral delivery. Crit Rev Ther Drug
`Carrier Syst 2005; 22:535 602.
`
`Regeneron Exhibit 1015.208
`
`
`
`Biophysical and biochemical characterization
`of peptide and protein drug product
`Tapan K. Das and James A. 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 teclmologies for
`extending half-life such as conjtlgating 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 characterization of the biotherapeutic entity, wh icl1
`includes the Elucidation of Structure and Jmpurities sections, which, for biological entities can
`be quite complex. lt is expected that the applicant have a detailed understanding of the
`structure, heterogeneity, and stability of the biotherapeutic entity using a variety of ana lytical
`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 (!CH) (www.ich.org) provides guidance
`documentation agreed on by the regulatory agencies of the United States, Elurope, and
`Japan. The ICH guideline Q5 deals specificaJJy 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 Elqcidation 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 th.is section are used for confirmation of the predicted
`primary structure, higher order structtues, 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
`
`
`
`BIOPHYSICAL AND BIOCHEMICAL CHARACTERIZATION OF PEPTIDE AND PROTEIN DRUG PRODUCT
`
`195
`
`1600
`
`1200
`
`800
`
`400
`
`0
`
`,8
`
`I i '°
`I
`: - .. r IC
`:
`a
`... f
`;
`
`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.
`
`t:
`0
`
`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 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.
`
`PofenetJ Determination
`For biologics, in most cases, a relevant potency assay fur 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 hatch 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
`Most therapeutic biologics currently are administered via parenteral (intravenous or
`subcutaneous) route. The goal of biologics drug prodt1ct 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 }abilities 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 drng product formulation. "Formulation characterization" refers to
`
`Regeneron Exhibit 1015.210
`
`
`
`Downloaded from infonnaheallhcare com by McGill University on 01/15/13
`For personal use only
`
`Table 1 Examples of Biotherapeutics Class of Molecules Types Sources Technologies and Molecules
`
`Name of
`drug
`
`Name of active
`substance
`
`Class of molecule
`
`Technology
`
`Source
`
`ndication
`
`Genotropin
`
`Somatropin
`
`Protein
`
`Single polypeptide
`
`Somavert
`
`Pegvisomant
`
`Conjugated protein
`
`PEG- ntron
`Redipen
`Nplate
`
`Peginterferon 0'.·2b
`
`Conjugated protein
`
`Romiplostim
`
`Fusion protein
`
`Survanta
`
`Beractant
`
`Lipid-protein
`mixture
`
`DigiFab
`
`Lucentis
`
`Cimzia
`
`Enbrel
`
`Digoxin immune
`Fab
`Ranibizumab
`
`Certolizumab pegol
`(CDP-870)
`Etanercept
`
`Antibody fragment
`(Fab)
`Antibody fragment
`(Fab)
`Antibody fragment
`(Fab) conjugate
`Fusion protein
`
`Herceptin
`
`Trastuzumab
`
`Full length antibody
`
`Single polypeptide PEGylated
`at multiple sites
`Covalent conjugate of PEG to
`protein
`Fe-peptide fusion protein
`(peptibody)
`Natural bovine lung extract
`containing lipids and
`surfactant-associated
`proteins and added lipids
`digoxin-specific Fab
`
`Humanized gGt K
`
`Humanized antibody fragment
`Pegylated
`Dimeric fusion protein
`(extracellular portion of
`human tumor necrosis factor
`receptor linked to gG1 Fe
`Humanized gG1 K
`
`::0
`CD
`(Q
`CD
`:l
`CD ..,
`0
`:l
`m
`X
`:T
`C"
`;::;:
`
`~
`0
`~
`u,
`I\)
`~
`~
`
`Escherichia coli
`(rDNA)
`
`E. coli (rDNA)
`
`Growth hormone
`deficiency T urner
`syndrome and others
`Acromegaly
`
`E. coli (rDNA)
`
`Bovine lung
`extract
`
`Thrombocytopenic
`purpura
`Respiratory distress
`syndrome
`
`Ovine serum
`
`E. coli (rDNA)
`
`E. coli (rDNA)
`
`Digoxin toxicity or
`overdose
`Age-related macular
`degeneration
`Crohn's disease
`
`Mammalian cell
`(CHO) (rDNA)
`
`Rheumatoid arthritis
`plaque psoriasis and
`others
`
`Mammalian cell
`(CHO) (rDNA)
`
`Cancer (breast stomach
`pancreatic)
`
`E. coli (rDNA)
`
`nfections hepatitis C
`
`Schering-Plough
`
`..
`
`~
`
`Company
`
`Pfizer
`
`Pfizer
`
`Amgen
`
`Mitsubishi Tanabe
`Ross (Abbott)
`
`<:'\ r-
`~
`BTG nternational Ltd -
`2l
`:J:,
`i\':
`~
`:i,.
`~
`0
`<:
`:i,.
`~
`~
`c:-,
`i:
`~
`~
`
`Nycomed
`Genentech and
`partners
`UCB and partners
`
`Amgen Wyeth
`Takeda
`
`Genentech and
`partners
`
`
`
`Downloaded from info.1maheallhcare com by McGill University on 01/15/13
`For personal use only
`
`Vectibix
`
`Panitumumab
`
`Full length antibody
`
`Humanized gG1 ic
`
`Gardasil
`
`Prevnar
`
`Human
`papillomavirus
`quadrivalent
`vaccine
`
`Pneumococcal 7-
`valent conjugate
`vaccine
`
`Protein vaccine
`(VLP)
`
`Vaccine
`(glycoconjugate)
`
`Fragmin
`
`Dalteparin sodium
`injection
`
`Carbohydrate
`
`Self-assembled VLP of capsid
`protein of HPV types 6 11
`16 and 18-adsorbed into
`aluminium-containing
`adjuvant
`Saccharides of capsular
`antigens of Streptococcus
`pneumoniae serotypes 4
`68 9V 14 18C 19F and
`23F each conjugated to
`diphtheria CRM197 protein
`Controlled depolymerization of
`sodium heparin
`
`Pulmozyme Dornase ex
`
`Fabrazyme
`
`Agalsidase p
`
`Enzyme
`(glycoprotein)
`Enzyme
`(glycoprotein)
`
`Recombinant human
`deoxyribonuclease
`Recombinant human
`cx-galactosidase A
`
`Mammalian cell
`(CHO) (rDNA)
`Saccharomyces
`cerevisiae
`(yeast) (rDNA)
`
`Cancer (colorectal)
`
`Prevention of several
`diseases caused by
`HPV
`
`Amgen
`
`Merck
`
`Serotype from soy mmunisation against
`peptone broth
`several diseases
`CRM197 from
`caused by S.
`pneumoniae
`Corynebacterium
`diphtheriae
`
`Wyeth (Pfizer)
`
`Porcine
`intestinal
`mucosa
`Mammalian cell
`(CHO) (rDNA)
`Mammalian cell
`(CHO) (rDNA)
`
`Deep vein thrombosis
`and others
`
`Pfizer
`
`Cystic fibrosis
`
`Fabry disease
`
`Genentech
`
`Genzyme
`
`Abbreviations gG immunoglobulin Fab antibody fragments PEG polyethylene glycol VLP virus-like particles mAb monoclonal antibody
`
`::0
`CD
`(Q
`CD
`:l
`CD ..,
`0
`:l
`m
`X
`:T
`C"
`;::;:
`
`~
`0
`~
`u,
`I\)
`~
`I\)
`
`)s
`
`~
`i
`~
`~ r--
`~
`~
`0
`~
`~
`§
`r--
`~
`~
`~
`~
`~
`~
`~ ij
`
`C")
`
`ls
`~
`~
`0
`61
`
`<: I
`~
`
`~
`
`..
`
`~
`
`
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`VOLUME 1: FORMULATION AND PACKAGING
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`characterization of drug prodttct 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. ln
`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 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 grottps 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 (Asn-Gly) 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 ]ability 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 structttral motif in IgGs
`may be partially predicted on the basis of structural flex ibility of the motif (unordered vs.
`helical or P sheet). While these approaches are quite useful in enlisting the common hot spots
`for chemical degradatiou, 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
`Following determination of hot spots as described above, the next step in formulation
`characterization is to experimentally determine the major degradation pathway(s) and to
`trnderstand 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 stmctures 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
`
`Regeneron Exhibit 1015.213
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`BIOPHYSICAL AND BIOCHEMICAL CHARACTERIZATION OF PEPTIDE ANO PROTEIN DRUG PRODUCT
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`199
`
`Table 2 Protein and Peptide Degradation Hot Spots
`
`Labile groups
`
`Type of degradation
`
`Occurrence in lgG and other proteins
`
`Asn Gly
`
`Deamidation, lsomerization
`
`Asn Ser, Asn Asn,
`Asn Thr. Asn Lys,
`Asn His, Asn Asp
`
`Deamidalion, lsomerization
`
`Asp Pro
`Asp Gin
`
`Asp Lys
`His Thr
`Asp
`Met
`
`Cys
`Trp
`
`Tyr
`
`Clipping (peptide bond)
`
`lsomerization
`Oxidation
`
`Oxidation (to form disulfide)
`Oxidation
`
`Oxidation
`
`Pro
`Lys
`Fe His/Asp/Tyr
`His Fe (heme)
`Met Fe (heme)
`
`Amine and other reactive
`amino acids
`Various hydrophobic
`segments
`
`Proline isomerization
`Glycation
`Metal bond breakage
`Metal bond breakage
`Metal bond breakage
`
`Reaction with
`buffer/excipients
`Aggregation
`
`NN386G in CH3 (lgG2a) (2)
`QN156G in CL (lgG2a) (2)
`LN316G in CH2 (lgG1) (3)
`SN385G in CH3 {lgG1) (3)
`RN423$ in CH3 (lgG2a) (2)
`PEN390NY in CH3 (3)
`VN3°T in CDR1 of LC (4)
`SN329K in CH2 (5)
`D274 p275 (lgG1) (5)
`D K in hinge (lgG1) (5)
`HT in hinge {lgG1) (5)
`
`D 102G in CDR3 of HC (lgG1) (4)
`M34 in CDR1 of HC (lgG1) (6)
`M101 in CDR3 of HC (lgG1) (6)
`c105 in CDR3 of HC (lgG2a) (2)
`W54, W55 in CDR2 of HC (lgG1) (6)
`W105 in CDR3 of HC (lgG1) (6)
`Oxidation of lens protein forms
`dihydroxyphenylalanine, o and m Tyr, and
`di Tyr (7)
`Trans p32 isomer formation in ~2 microglobulin (8)
`K49 in LC {lgG1) (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)
`May form adducts such as carboxytate adduct with
`citrate/succinate (13)
`Potential hot spots for aggregation in lgG predicted
`using bioinformatics tools (14, 15)
`
`Abbreviafions: lgG, immunoglobulin; LC, light chain of lgG; HC, heavy chain of lgG; 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 ,"lide 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 used in its
`productiou. 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 determi11es its ability to maintain its ftmction. 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
`tmderstanding its overall stmcture and properties.
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`Regeneron Exhibit 1015.214
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`200
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`VOLUME 1: FORMULATION AND PACKAGING
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`During production of recombinant proteins, several modifications to the primary
`structure are possible. These include errors in transcription or translation, generatil1g such
`variant forms as am.ino 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 biochem.ical instability, such as deamidation or oxidation. Al