MINIREVIEW
`
`Antibody Structure, Instability, and Formulation
`
`WEI WANG, SATISH SINGH, DAVID L. ZENG, KEVIN KING, SANDEEP NEMA
`
`Pfizer, Inc., Global Biologics, 700 Chesterfield Parkway West, Chesterfield, Missouri 63017
`
`Received 14 March 2006; revised 17 May 2006; accepted 4 June 2006
`
`Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20727
`
`ABSTRACT: The number of therapeutic monoclonal antibody in development has
`increased tremendously over the last several years and this trend continues. At present
`there are more than 23 approved antibodies on the US market and an estimated 200 or
`more are in development. Although antibodies share certain structural similarities,
`development of commercially viable antibody pharmaceuticals has not been straightfor-
`ward because of their unique and somewhat unpredictable solution behavior. This article
`reviews the structure and function of antibodies and the mechanisms of physical and
`chemical instabilities. Various aspects of formulation development have been examined
`to identify the critical attributes for the stabilization of antibodies. ß 2006 Wiley-Liss, Inc.
`and the American Pharmacists Association J Pharm Sci 96:1–26, 2007
`Keywords: biotechnology; stabilization; protein formulation; protein aggregation;
`freeze drying/lyophilization
`
`INTRODUCTION
`
`Protein therapies are entering a new era with
`the influx of a significant number of antibody
`pharmaceuticals. Generally, protein drugs are
`effective at low concentrations with less side
`effects relative to small molecule drugs, even
`though, in rare cases, protein-induced antibody
`formation could be serious.1 Therefore,
`this
`category of therapeutics is gaining tremendous
`momentum and widespread recognition both in
`small and large drug firms. Among protein drug
`therapies, antibodies play a major role in control-
`ling many types of diseases such as cancer,
`infectious diseases, allergy, autoimmune dis-
`eases, and inflammation. Since the approval
`of the first monoclonal antibody (MAb) product
`-OKT-3 in 1986, more than 23 MAb drug products
`have entered the market (Tab. 1). The estimated
`number of antibodies and antibody derivatives
`constitute 20% of biopharmaceutical products
`
`Correspondence to: Wei Wang (Telephone: (636)-247-2111;
`Fax: (636)-247-5030; E-mail: wei.2.wang@pfizer.com)
`
`Journal of Pharmaceutical Sciences, Vol. 96, 1–26 (2007)
`ß 2006 Wiley-Liss, Inc. and the American Pharmacists Association
`
`currently in development (about 200).2 The global
`therapeutic antibody market was predicted to
`reach $16.7 billion in 2008.3
`There are several reasons for the increasing
`popularity of antibodies for commercial develop-
`ment. First, their action is specific, generally
`leading to fewer side effects. Second, antibodies
`may be conjugated to another therapeutic entity
`for efficient delivery of this entity to a target site,
`thus reducing potential side effects. For instance,
`Mylotarg is an approved chemotherapy agent
`composed of calicheamicin conjugated to huma-
`nized IgG4, which binds specifically to CD33 for
`the treatment of CD33-positive acute myeloid
`leukemia. Another example is the conjugation of
`immunotoxic barnase with the light chain of the
`anti-human ferritin monoclonal antibody F11 as
`potential targeting agents for cancer immuno-
`therapy.4 Third, antibodies may be conjugated to
`radioisotopes for specific diagnostic purposes.
`Examples include CEA-Scan for detection of color-
`ectal cancer and ProstaScint for detection of
`prostate cancer. Lastly, technology advancement
`has made complete human MAb available, which
`are less immunogenic.
`
`@WILEY
`lnterScience®
`
`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 1, JANUARY 2007
`
`1
`
`Novartis Exhibit 2034.001
`Regeneron v. Novartis, IPR2021-00816
`
`

`

`5.5
`
`0.01%PS20
`
`10%a-Trehalose-
`
`10mg/mLsolution10mMHistidineHCl
`
`Dihydrate
`
`(wet)
`degeneration
`macular
`
`PS80
`
`Mannnitol
`9.6mg/0.8mL
`
`0.8mLCitricacid H2O
`NaCitrate,1.04mg/
`0.24mg/0.8mL
`DibasicNaPhos 2H2O;
`1.22mg/0.8mL
`NaPhos 2H2O;
`Monobasic
`
`(50mg/mL)
`solution
`
`TNF-alpha
`Blocks
`toDMARDs.
`notresponding
`
`5.2
`
`0.8mg/0.8mL
`
`4.93mg/0.8mLNaCl;
`
`0.69mg/0.8mL
`
`40mg/0.8mL
`
`RApatients
`
`PS20
`
`6
`
`1.8mg/20mL
`
`Dihydrate
`a-Trehalose
`400mg/20mL
`
`7.0–7.4
`
`8.48mg/mLNaCl
`
`L-Histidine
`6.4mg/20mL
`L-HistidineHCl,
`
`9.9mg/20mL
`
`reconstitution
`after
`21mg/mL
`440mg/vial,
`
`MonobasicNaPhos H2O
`7H2O;0.42mg/mL
`DibasicNaPhos
`
`1.88mg/mL
`
`mLsolution
`50mL;2mg/
`100mgMAbin
`
`5.7
`
`PS80
`
`Sucrose
`
`Na2EDTA
`0.056mg/3mL
`0.6mg/3mLKCl,
`
`6.8–7.4
`
`0.3mg/3mL
`
`24mg/3mLNaCl,
`
`Ascorbicacid
`NaCl,0.9–1.3mg/mL
`Maltose,0.9mg/mL
`1–2,9–15mg/mL
`5–6%Povidone,
`I131-MAb:
`w/vMaltose;
`
`HCl
`aceticacid,
`NaCl,glacial
`NaAcetate.3H2O,
`tetrahydrate,
`tartrate
`potassiumsodium
`chloride,
`Stannous
`0.29mg/vial
`
`monobasicKPhos
`0.6mg/3mL
`dibasicNaPhos,
`
`3.5mg/3mL
`
`(MAbvial)
`
`7.2
`
`145mMNaCl,10%
`
`10mMphosphate
`
`Tc99m
`1mLSalinew
`Reconstitutew
`MAb.
`Lyophilized
`
`1.25mg/vial
`
`solution
`
`30mg/3mL
`
`solution
`I131-MAb
`1.1mg/mL
`225mgvials;
`in35mgand
`MAbsolution
`Kit:14mg/mL
`
`HER2protein
`overexpress
`tumor
`cancerwhose
`breast
`
`Metastatic
`
`carcinoma
`colorectal
`expressing
`EGFR-
`
`cancer
`forcolorectal
`Imagingagent
`
`CD52-antigen
`leukemia,
`lymphocytic
`B-cellchronic
`
`lymphoma
`nonHodgkins
`follicular
`
`2
`
`WANG ET AL.
`
`6.2
`
`pH
`
`fillinvial)
`16mL
`(4mL,
`PS20
`
`16mLfillinvial)
`dihydrate(4mL,
`a-Trehalose
`
`fillinvial)
`(4mL,16mL
`anhydrous
`dibasicNaPhos
`1.2mg/mL
`NaPhosH2O;
`monobasic
`
`solution
`(25mg/mL)
`400mg/vial
`
`bindsVEGF
`rectum,
`ofcolonor
`carcinoma
`
`0.4mg/mL
`
`60mg/mL
`
`5.8mg/mL
`
`100mgand
`
`Surfactant
`
`Excipients
`
`Buffer
`
`MAbConc
`
`Indication
`
`Age-related
`
`injection
`Intravitreal
`
`fragment
`IgG1k
`
`2006Genentech
`
`Humanized
`
`Ranibizumab
`
`Lucentis
`
`8
`
`SC
`
`2002CATandAbbott
`
`Human
`
`Adalimumab
`
`Humira
`
`7
`
`148kDa
`IgG1k,
`
`IVinfusion
`
`1998Genetech
`
`Treatmentof
`
`IVinfusion
`
`2004ImCloneandBMS
`
`infusion
`
`IVinjectionor
`
`1996Immunomedics
`
`andBerlex
`Millenium
`
`IgG1k
`
`Humanized
`152kDa
`IgG1k,
`mouse
`human/
`Chimeric
`
`50kDa
`Fab,
`Murine
`
`150kDa
`IgG1k,
`
`Trastuzumab
`
`Herceptin
`
`6
`
`(lyo)
`
`Cetuximab
`
`Erbitux
`
`5
`
`Tc-99
`
`(lyo)
`
`Acrituomab;
`
`CEA-Scan
`
`4
`
`IVinfusion
`
`2001IlexOncology;
`
`Humanized,
`
`Alemtuzumab
`
`Campath
`
`3
`
`CD20positive
`
`IVInfusion
`
`2003Corixaand
`
`GSK
`
`IgG2l
`Murine
`
`I-131Tositumab
`Tositumomaband
`
`Bexxar
`
`2
`
`Metastatic
`
`IVinfusion
`
`2004Genetech
`
`Humanized
`
`Bevacizumab
`
`Avastin
`
`1
`
`BioOncology
`and
`
`149kDa
`IgG1,
`
`Route
`
`Company
`
`Year
`
`MAb
`
`Molecule
`
`Brandname
`
`#
`
`Table1.CommercialMonoclonalAntibodyProducts
`
`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 1, JANUARY 2007
`
`DOI 10.1002/jps
`
`Novartis Exhibit 2034.002
`Regeneron v. Novartis, IPR2021-00816
`
`

`

`ANTIBODY FORMULATION
`
`3
`
`(Continued)
`
`6.5
`
`0.7mg/mLPS80
`
`9mg/mLNaCl
`
`NaCitrate 2H2O
`
`10mg/mLsolution7.35mg/mL
`
`mL)PS80
`
`7.2
`
`0.001%(0.01mg/
`
`0.15MNaCl
`
`0.01MNaPhosphate
`
`2mg/mLsolution
`
`CD20-antigen)
`lymphoma.(anti
`
`NonHodgkin’s
`
`complications
`clotrelated
`acuteblood
`Reductionof
`
`infusion
`
`5–7
`
`70.5
`
`6.0
`
`Phosphatebuffersaline
`
`0.5mg
`
`Imagingagentfor
`
`NaCl
`
`NaPhosphate
`
`vial
`powder/20-mL
`lyophilized
`equivalent
`protein-
`
`leukemia
`myeloid
`positiveacute
`ofCD33
`treatment
`calicheamicinfor
`Ablinkedto
`
`Dextran40,Sucrose,
`
`Monobasicanddibasic
`
`5mg
`
`Humanized
`
`PS80
`
`Sucrose
`
`DibasicNaPhos 2H2O
`MonobasicNaPhosH2O,
`
`6.1mg/10mL
`
`7.2
`
`0.5mg/10mL
`
`500mg/10mL
`
`2.2mg/10mL
`
`6.2
`
`123.2mg/vialSucrose3mg/vialPS20
`
`L-Histidine
`H2O;4.3mg/vial
`L-HistidineHCl
`
`6.8mg/vial
`
`reconstitution
`10mg/mLon
`20-mLVial,
`
`100mg/
`SWFI
`with1.3mL
`reconstitution
`after
`(100mg/mL)
`125mg/1.25mL
`
`150mgMAb/vial;
`(1mLpervial)
`solution
`conjugate/mL
`
`Phosphatebuffersaline
`
`0.5mg
`
`1mg/mLPS80
`
`43mg/5mLNaCl
`
`dibasicNaPhos
`NaPhos,9.0mg/5mL
`2.25mg/5mLmonobasic
`
`1mg/mLsolution
`(2mLpervial)
`solution
`conjugate/mL
`
`TNFalpha)
`(anti
`disease
`Crohn’s
`
`RAand
`
`ofLFA-1
`toCD11asubunit
`psoriasis,binds
`plaque
`tosevere
`
`Chronicmoderate
`
`cancer
`forprostate
`Imagingagent
`
`antigen)
`(antiCD3-
`rejection
`transplant
`acutekidney
`
`Reversalof
`
`ovariancancer
`colorectaland
`
`IVinfusion
`
`1997IDECand
`
`Genentech
`
`145kDa
`domain),
`region(Fab
`variable
`chain
`andheavy
`murinelight
`IgG1kwith
`human
`mouse/
`Chimeric
`48kDa
`murine,
`human-
`
`Rituximab
`
`Rituxan
`
`16
`
`IVinjectionand
`
`1994Centocor/Lilly
`
`Fab.Chimeric
`
`Abciximab
`
`ReoPro
`
`15
`
`IVinfusion
`
`1998Centocor
`
`regions)
`constant
`chain
`kappalight
`human
`chainand
`IgG1heavy
`tohuman
`corresponds
`murine,70%
`(app.30%
`TNFalpha
`against
`murineMAb
`human/
`Chimeric
`
`Infliximab
`
`Remicade
`
`14
`
`(lyo)
`
`Genentech
`
`IgG1k
`
`(lyo)
`
`SC
`
`2003Xomaand
`
`Humanized
`
`Efalizumab
`
`Raptiva
`
`13
`
`IVinjection
`
`1996Cytogen
`
`GYK-DTPA
`conjugatedto
`MurineIgG1k-
`
`pendetide
`capromab
`Indium-111
`
`ProstaScint
`
`12
`
`IVinjection
`
`1986OrthoBiotech
`
`Murine,IgG2a,
`
`Muromomab-CD3
`
`Orthoclone
`
`11
`
`170kDa
`
`OKT
`
`IVinjection
`
`1992Cytogen
`
`MurineIgG1k
`
`toGYK-DTPA
`conjugated
`
`pendetide
`Satumomab
`
`OncoScint
`
`10
`
`Wyeth
`
`calicheamicin
`with
`conjugated
`IgG4k
`
`IVinfusion
`
`2000Celltechand
`
`Humanized
`
`ozogamicin
`Gemtuzumab
`
`(lyo)
`
`Mylotarg
`
`9
`
`DOI 10.1002/jps
`
`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 1, JANUARY 2007
`
`Novartis Exhibit 2034.003
`Regeneron v. Novartis, IPR2021-00816
`
`

`

`7.1
`
`09%NaCl
`
`3.2mg/2mL
`
`CD20antigen.
`
`solution
`
`betaemission)
`cellulardamageby
`
`induces
`
`Ytterium-90
`(Kitwith
`
`6.9
`
`0.2mg/mLPS80
`
`4.6mg/mLNaCl
`
`DibasicNaPhos 7H2O
`Phos H2O;11mg/mL
`
`3.6mg/mLMonobasicNa
`
`0.5mgPS20
`
`145.5mgSucrose
`
`mgLHistidine
`
`LHistidineHCl H2O;1.8
`
`2.8mg
`
`Solution
`
`25mg/5mLMAb
`
`SWFI
`with1.4mL
`reconstitution
`1.2mLon
`Deliver150mg/
`
`complex
`IL-2receptor
`Tacsubunitof
`bindingtothe
`InhibitsIL-2
`transplants.
`receivingrenal
`patients
`rejectionin
`acuteorgan
`Prophylaxisof
`
`FCeRI
`IgEreceptor
`bindingofIgEto
`
`202.5mg/vial,
`
`Asthma,inhibits
`
`forlungcancer
`
`4
`
`WANG ET AL.
`
`?
`
`10mg/mLsolutionPhosphatebuffersaline
`
`Imagingagent
`
`6.1
`
`3.0mg/15mL
`
`123mg/15mLNaCl
`
`PS80
`
`for15mL
`
`diBasicNaPhos 7H2O
`Phos H2O,7.24mg
`17.0mgMonobasicNa
`
`solution
`
`300mg/15mL
`
`MSrelapse
`
`5.6%Mannitol
`
`Glycine
`20mg40mg
`80mgMannitol;
`Sucrose;40mg,
`10mg,20mg
`
`0.8mg,1.61mgNaCl;
`
`3.0mMGlycine
`47mMHistidine,
`
`onreconstitution
`vial,100mg/mL
`
`50mgand100mg/
`
`(RSV)
`syncytialvirus
`oftheRespiratory
`Preventreplication
`
`antagonist
`receptor
`
`Na2HPO4
`
`KPhos;0.50mg,0.99mg
`3.61mg,7.21mgMonobasic
`
`reconstitution
`vial,4mg/mLon
`
`10mgand20mg/
`
`rejection,IL-2
`kidneytransplant
`Preventionofacute
`
`infusion
`
`pH
`
`Surfactant
`
`Excipients
`
`Buffer
`
`MAbConc
`
`Indication
`
`Route
`
`IVinfusion
`
`IDEC
`
`MurineIgG1k-
`
`Ibritumomab-
`
`Zevalin
`
`23
`
`Tiuxetan
`linkageto
`covalent
`thiourea
`
`Tiuxetan
`
`IVinfusion
`
`1997Roche
`
`Humanized
`
`Daclizumab
`
`Zenapax
`
`22
`
`144kDa
`IgG1,
`
`andTanox
`Novartis
`
`149kDa
`IgG1k,
`
`SC
`
`Genentechw
`
`Humanized
`
`Omalizumab
`
`(lyo)
`Xolair
`
`21
`
`DuPontMerck
`Ingelheimand
`
`IVinjection
`
`1996Boehringer
`
`MurineFab
`
`Nofetumomab
`
`Verluma
`
`20
`
`IVInfusion
`
`2004BiogenIDEC
`
`IgG4k
`
`Humainzed
`148kDa
`MAb1129,
`ofmurine
`IgG1k,CDR
`
`Natalizumab
`
`Tysabri
`
`19
`
`(lyo)
`
`IMinjection
`
`1998MedImmune
`
`Humanized
`
`Palivizumab
`
`Synagis
`
`18
`
`IVinjectionand
`
`1998Novartis
`
`144kDa
`IgG1k,
`Chimaric
`
`Basiliximab
`
`Simulect
`
`17
`
`(lyo)
`
`Company
`
`Year
`
`MAb
`
`Molecule
`
`Brandname
`
`#
`
`(Continued)
`
`Table1.
`
`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 1, JANUARY 2007
`
`DOI 10.1002/jps
`
`Novartis Exhibit 2034.004
`Regeneron v. Novartis, IPR2021-00816
`
`

`

`Development of commercially viable antibody
`pharmaceuticals has, however, not been straight-
`forward. This is because the behavior of antibodies
`seems to vary, even though they have similar
`structures. In attempting to address some of the
`challenges in developing antibody therapeutics,
`Harris et al.5 reviewed the commercial-scale
`formulation and characterization of therapeutic
`recombinant antibodies. In a different review,
`antibody production and purification have been
`discussed.2 Nevertheless, the overall instability
`and stabilization of antibody drug candidates
`have not been carefully examined in the litera-
`ture. This article, not meant to be exhaustive,
`intends to review the structure and functions
`of antibodies, discuss their instabilities, and sum-
`marize the methods for stabilizing/formulating
`antibodies.
`
`ANTIBODY STRUCTURE
`
`Antibodies (immunoglobulins) are roughly Y-shaped
`molecules or combination of such molecules (Fig. 1).
`Their structures are divided into two regions—the
`variable (V) region (top of the Y) defining antigen-
`binding properties and the constant (C) region
`(stem of the Y), interacting with effector cells and
`molecules. Immunoglobulins can be divided into
`five different classes IgA, IgD, IgE, IgM, and
`IgG based on their C regions, respectively desig-
`nated as a, d, e, m, and g (five main heavy-chain
`classes).6 Most IgGs are monomers, but IgA and
`IgM are respectively, dimmers and pentamers
`linked by J chains. IgGs are the most abundant,
`widely used for therapeutic purposes, and their
`structures will be discussed as antibody examples
`in detail.
`
`Primary Structure
`
`The structure of IgGs have been thoroughly
`reviewed.6 The features of the primary structure
`of antibodies include heavy and light chains,
`glycosylation, disulfide bond, and heterogeneity.
`
`Heavy and Light Chains
`
`IgGs contain two identical heavy (H, 50 kDa) and
`two identical light (L, 25 kDa) chains (Fig. 1).
`Therefore, the total molecular weight is approxi-
`mately 150 kDa. There are several disulfide bonds
`linking the two heavy chains, linking the heavy
`and light chains, and residing inside the chains
`(also see next section). IgGs are further divided
`
`ANTIBODY FORMULATION
`
`5
`
`Figure 1. Linear (upper panel) and steric (lower
`panel) structures of immunoglobulins (IgG).
`
`into several subclasses—IgG1, IgG2, IgG3, and
`IgG4 (in order of relative abundance in human
`plasma), with different heavy chains, named g1,
`g2, g3, and g4, respectively. The structural
`differences among these subtypes are the number
`and location of interchain disulfide bonds and the
`length of the hinge region. The light chains
`consist of two types—lambda (l) and kappa (k).
`In mice, the average of k to l ratio is 20:1, whereas
`it is 2:1 in humans.6 The variable (V) regions
`of both chains cover approximately the first
`110 amino acids, forming the antigen-binding
`(Fab) regions, whereas the remaining sequences
`are constant (C) regions, forming Fc (fragment
`crystallizable) regions for effector recognition and
`binding.6 The N-terminal sequences of both the
`heavy and light chains vary greatly between
`different antibodies. It was suggested that the
`conserved sequences in human IgG1 antibodies
`
`DOI 10.1002/jps
`
`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 1, JANUARY 2007
`
`Novartis Exhibit 2034.005
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`

`6
`
`WANG ET AL.
`
`are approximately 95% and the remaining 5%
`is variable and creates their antigen-binding
`specificity.5
`The V regions are further divided into three
`hypervariable sequences (HV1, HV2, and HV3) on
`both H and L chains. In the light chains, these are
`roughly from residues 28 to 35, from 49 to 59, and
`from 92 to 103, respectively.6 Other regions are the
`framework regions (FR1, FR2, FR3, and FR4). The
`HV regions are also called the complementarity
`determining regions (CDR1, CDR2, and CDR3).
`While the framework regions form the b-sheets,
`the HV sequences form three loops at the outer
`edge of the b barrel (also see Section 2.2).
`
`Disulfide Bonds
`
`Most IgGs have four interchain disulfide bonds—
`two connecting the two H chains at the hinge
`region and the other two connecting the two L
`chains to the H chains.6 Exceptions do exist. Two
`disulfide bonds were found in IgG1 and IgG4
`linking the two heavy chain in the hinge region
`but four in IgG2.7 In IgG1 MAb, HC is linked to
`the LC between the fifth Cys (C217) of HC and
`C213 on the LC. In IgG2 and IgG4 MAbs, it is the
`third Cys of HC (C123) linking to the LC.7 A
`disulfide bond between HC C128 and LC C214
`was found for mouse catalytic monoclonal anti-
`bodies (IgG2a).8
`IgGs have four intrachain disulfide bonds,
`residing in each domain of the H and L chains,
`stabilizing these domains. The intrachain disul-
`fide bonds in VH and VL are required in functional
`antigen binding.9 Native IgG MAbs should not
`have any free sulfhydryl groups.7 However,
`detailed examination of the free sulfhydryl groups
`in recombinant MAbs (one IgG1, two IgG2, and one
`IgG4) suggests presence of a small portion of free
`sulfhydryl group (approximately 0.02 mol per mole
`of IgG2 or IgG4 MAb and 0.03 for IgG1.7 In rare
`cases, a free cysteine is found. A nondisulfide-
`bonded Cys at residue 105 was found on the heavy
`chain of a mouse monoclonal antibody, OKT3
`(IgG2a).10
`
`Oligosaccharides
`There is one oligosaccharide chain in IgGs.6 This
`N-linked biantennary sugar chain resides mostly
`on the conserved Asn 297, which is buried
`between the CH2 domains.5,11 For example, the
`oligosaccharide resides on Asn-297 of the CH2
`domain of chimeric IgG1 and IgG3 molecules12
`
`but on Asn 299 in a monoclonal antibody, OKT3
`(IgG2a).10 The oligosaccharide, often microheter-
`ogeneous, is typically fucosylated in antibodies
`produced in CHO or myeloma cell lines5 and may
`lines.2,11 There are many
`differ in other cell
`factors that dictate the nature of the glycan
`microheterogenity on IgGs. These include cell
`line, the bioreactor conditions and the nature of
`the downstream processing. An additional oligo-
`saccharide can be found in rare cases. A human
`IgG produced by a human-human-mouse hetero-
`hybridoma contains an additional oligosaccharide
`on Asn 75 in the variable region of its heavy
`chain.13
`In addition, O-linked carbohydrates
`could also exist in this antibody.
`for correct
`Proper glycosylation is critical
`functioning of antibodies.11 It was demonstrated
`that removal of the oligosaccharide in IgGs (IgG1
`and IgG3) made them ineffective in binding to C1q,
`in binding to the human FcgRI and activating C;
`and generally more sensitive to most proteases
`than their corresponding wild-type IgGs (one
`exception).12 This is because the binding site on
`IgG for C1q, the first component of the complement
`is localized in the CH2 domains.11
`cascade,
`Furthermore, the glycosylation can affect the
`antibody conformation.12
`Oligosaccharides in other regions can also play
`a critical role. Removal of an oligosaccharide in a
`Fv region of the CBGA1 antibody resulted in a
`decreased antigen-binding activity in several
`ELISA systems.13 In addition, this oligosaccharide
`might play critical role in reducing the antigenicity
`of the protein.14
`The sugar composition of the oligosaccharide is
`also critical in antibody functions. It has been
`shown that a low fucose (Fuc) content in the
`complex-type oligosaccharide in a humanized
`chimeric IgG1 is responsible for a 50-fold higher
`antibody-dependent cellular cytotoxicity (ADCC)
`compared with a high Fuc counterpart.15
`
`Heterogeneity
`
`Purified antibodies are heterogeneous in struc-
`ture. This is true for all monoclonal antibodies
`(MAbs) due to differences in glycosylation pat-
`terns, instability during production, and terminal
`processing.5 For example, five charged isoforms
`were found in recombinant humanized monoclo-
`nal antibody HER2 as found by capillary iso-
`electric focusing (cIEF) and sodium dodecyl
`sulfate–capillary
`gel
`electrophoresis
`(SDS–
`CGE).16 Six separate bands were focused under
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`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 1, JANUARY 2007
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`DOI 10.1002/jps
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`

`IEF for two mouse monoclonal antibodies IgG2a
`(k) and IgG1 (k).17 A mature monoclonal antibody,
`OKT3 (IgG2a),
`contain cyclized N-terminus
`(pyroglutamic acid, 17 D) in both H and L chains,
`processed C-terminus (no Lys, 128 D) of the H
`chains, and a small amount of deamidated form.10
`Similar observation was also reported for a huma-
`nized IgG1 (k).18 In rare cases, gene cross-over may
`lead to formation of abnormal heavy chains. For
`example, a purified monoclonal anti-IgE antibody
`contains a small amount of a variant H chain,
`which had 16 fewer amino acid residues than the
`normal H chain (position is between Arg108 of the
`L chain and Ala124 of the H chain).19
`
`Secondary and Higher-Order Structure
`
`The basic secondary and higher-order structural
`features of IgGs have been reviewed.6 Only a
`small portion of the three-dimensional structures
`of IgGs has been solved.20 The antibody’s secon-
`day structure is formed as the polypeptide chains
`form anti-parallel b-sheets. The major type of
`secondary structure in IgGs is these b-sheets
`and its content is roughly 70% as measured by
`FTIR.21 The light chain consists of two and the
`heavy chain contains four domains, each about
`110 amino acid long.6,20 All these domains have
`similar folded structures—b barrel, also called
`immunoglobulin fold, which is stabilized by a
`disulfide bond and hydrophobic interaction (pri-
`mary). These individual domains (12 kDa in
`size) interact with one another (VH and VL; CH1
`and CL; and between two CH3 domains except the
`carbohydrate-containing CH2 domain) and fold
`into three equal-sized spherical shape linked by a
`flexible hinge region. These three spheres form a
`Y shape (mostly) and/or a T shape.22
`The less globular shape of IgGs is maintained
`both by disulfide bonds and by strong noncovalent
`interactions between the two heavy chains and
`between each of
`the heavy-chain/light-chain
`pairs.23 Through noncovalent interactions, a less
`stable domain becomes more stable, and thus, the
`whole molecule can be stabilized.24 A detailed
`study indicates that the interaction between two
`CH3 domains are dominated by six contact
`residues, five of these residues (T366, L368,
`F405, Y407, and K409)
`forming a patch at
`the center of the interface.25 These noncovalent
`interactions are spatially oriented such that
`variable domain exchange (switching VH and VL;
`inside-out
`IgG;
`ioIgG)
`induces noncovalent
`multimerization.26
`
`ANTIBODY FORMULATION
`
`7
`
`The six hypervariable regions in CDR (L1, L2,
`L3, H1, H2, and H3) form loops of a few predictable
`main-chain conformations (or canonical forms),
`except H3 loop, which has too many variations in
`conformation to be predicted accurately.27,28
`There is a slight difference in the loop composition
`and shape between the two types of light chains.20
`However, no functional difference was found in
`antibodies having l or k chain.6
`
`Basic Functions of Antibodies
`
`The basic functions of antibodies have been
`reviewed.6 There are two functional areas in
`IgGs—the V and C regions. The V regions of the
`two heavy and light chains offer two identical
`antigen-binding sites. The binding of the two sites
`(bivalent) can be independent of each other and
`does not seem to depend on the C region.29 The
`exact antigen-binding sites are the CDR regions
`with participation of the frame work regions.30
`Binding of antigens seems through the induced-
`fit mechanism.31,32 The induced-fit mechanism
`allows multispecificity and polyreactivity. It has
`been suggested that about 5–10 residues usually
`contribute significantly to the binding energy.32
`The C regions of antibodies have three main
`effector functions (1) being recognized by receptors
`on immune effector cells,
`initiating antibody-
`dependent cell cytotoxicities (ADCC), (2) binding
`to complement, helping to recruit activated pha-
`gocytes, and (3) being transported to a variety of
`places, such as tears and milk.6 In addition, C
`domains also modulate in vivo stability.23,29,33 The
`function of Fc is affected by the structure of Fab.
`Variable domain exchange (switching VH and VL;
`inside-out IgG; ioIgG) affected Fc-associated func-
`tions such as serum half-life and binding to protein
`G and FcgRI.26
`The hinge region provides flexibility in bivalent
`antigen binding and activation of Fc effector
`functions.26 Two chimeric IgG3 antibodies lacking
`a genetic hinge but with Cys residues in CH2
`regions was found to be deficient in their inter-
`molecular assembly, and both IgG3 DHþ Cys and
`IgG3 DHþ 2Cys lost greatly their ability to bind
`FcgRI and failed to bind C1q and activate the
`complement cascade.34
`
`Alternative Forms of Antibodies
`
`In addition to species-specific antibodies, other
`antibody forms are generated to meet various
`needs. In the early development of antibody
`therapies, antibodies were made from murine
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`DOI 10.1002/jps
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`8
`
`WANG ET AL.
`
`sources. However, these antibodies easily elicit
`formation
`of
`human
`anti-mouse
`antibody
`(HAMA). Therefore, humanized chimeric antibo-
`dies were generated. Chimeric monoclonal anti-
`bodies (60–70% human) are made of mouse
`variable regions and human constant regions.2
`Such antibodies can still
`induce formation of
`human anti-chimeric antibody (HACA). Highly
`humanized antibodies, CDR-grafted antibodies,
`are made by replacing only the human CDR with
`mouse CDR regions (90–95% human).2 These
`antibodies are almost the same in immunogeni-
`city potential as completely human antibodies,
`which may illicit formation of human anti-human
`antibody (HAHA).
`Other alternative forms of antibodies have also
`been generated and these different forms have
`been reviewed.35 Treatment with papain would
`cleave the N-terminal side of the disulfide bonds
`and generate two identical Fab fragments and
`one Fc fragment. Fab0s are 50 kDa (VHþ CH1)/
`(VLþ CL) heterodimers linked by a single disul-
`fide bond. Treatment with pepsin cleaves the
`C-terminal side of the disulfide bonds and pro-
`duces a F(ab)0
`2 fragment. The remaining H chains
`were cut into several small fragments.6 Cleavage
`by papain occurs at the C-terminal side of His-
`H22836 or His-H227.37 Reduction of F(ab0)2 will
`produce two Fab0.23
`Fv fragments are noncovalent heterodimers
`of VH and VL. Stabilization of the fragment by a
`hydrophilic flexible peptide linker generates single-
`chain Fv (scFvs).2 Fragments without constant
`domains can also be made into domain antibodies
`(dAbs). These scFvs are 25–30 kDa variable domain
`(VHþ VL) dimers joined by polypeptide linkers of at
`least 12 residues. Shorter linkers (5–10 residues) do
`not allow pairing of the variable domains but allow
`association with another scFv form a bivalent dimer
`(diabody) (about 60 kDa, or trimer: triabody about
`90 kDa).38 Two diabodies can be further linked
`together to generate bispecific tandem diabody
`(tandab).39 Disulfide-free scFv molecules are rela-
`tively stable and useful for intracellular applica-
`tions of antibodies—‘‘intrabodies.’’38 The smallest of
`the antibody fragments is the minimal recognition
`unit (MRU) that can be derived from the peptide
`sequences of a single CDR.2
`
`ANTIBODY INSTABILITY
`
`Antibodies, like other proteins, are prone to a
`variety of physical and chemical degradation path-
`
`ways, although antibodies, on the average, seem to
`be more stable than other proteins. Antibody
`instabilities can be observed in liquid, frozen, and
`lyophilized states. The glycosylation state of an
`antibody can significantly affect its degradation
`rate.40 In many cases, multiple degradation path-
`ways can occur at the same time and the degrada-
`tion mechanism may change depending on the
`stress conditions.41 These degradation pathways
`are divided into two major categories—physical
`and chemical instabilities. This section will explore
`the possible degradation pathways of antibodies
`and their influencing factors.
`
`Physical Instability
`
`Antibodies can show physical instability via two
`major pathways—denaturation and aggregation.
`
`Denaturation
`
`Antibodies can denature under a variety of
`conditions. These conditions include temperature
`change, shear, and various processing steps.
`Compared with other proteins, antibodies seem
`to be more resistant to thermal stress. They may
`not melt completely until temperature is raised
`above 708C,21,42,43 while most other mesophilic
`proteins seem to melt below 708C.44 Shear may
`cause antibody denaturation. For example, the
`antigen-binding activity of a recombinant scFv
`antibody fragment was reduced with a first-order
`rate constant of 0.83/h in a buffer solution at a
`shear of approximately 20,000/s.45
`Lyophilization can denature a protein to var-
`ious extents. An anti-idiotypic antibody (MMA
`383) in a formulation containing mannitol, sac-
`charose, NaCl, and phosphate was found to loose
`its in vivo immunogenic properties (only 10–20%
`of normal response rate) upon lyophilization.46
`Since the protein showed no evidence of degrada-
`tion after lyophilization, no change in secondary
`structure by CD (29% b-sheet, 14% a-helix, and
`57% ‘‘other’’), the loss of activity was attributed to
`the conformational change. Indeed, tryptophan
`fluorescence properties were different between the
`lyophilized and unlyophilized antibodies.46
`
`Aggregation
`
`Antibody aggregation is a more common manifes-
`tation of physical instability. The concentration-
`dependent antibody aggregation was considered
`the greatest challenge to developing protein
`formulations at higher concentrations.47 This is
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`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 1, JANUARY 2007
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`DOI 10.1002/jps
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`because protein aggregates generally have
`reduced activity and more importantly, greater
`immunogenicity potential because of the multi-
`plicity
`of
`epitopes
`and/or
`conformational
`changes.14,48 Immunoglobulin aggregates have
`been shown to cause serious renal failure49 and
`anaphylactoid reactions such as headache, fever,
`and chills.50 Therefore, the aggregate level in
`commercial
`intravenous immunoglobulin pro-
`ducts is limited to less than 5% based on the
`WHO standards.
`Aggregates can form easily both in liquid and
`solid states under a variety of conditions (Tab. 2).
`Since protein aggregation is often the consequence
`of protein–protein interactions, a process influ-
`enced by diffusion rate and geometric constraints
`of the interaction sites, such factors would influ-
`ence significantly the aggregation rate, including
`protein concentration change, viscosity,
`ionic
`strength, pH, and temperature.51 Other con-
`ditions may also affect protein aggregation,
`including shaking, long-term storage, freeze-thaw
`process, lyophilization process, etc.
`Increasing the concentration of antibodies often
`increases the aggregation tendency of the protein.
`It was demonstrated that increasing the IgG1
`concentration (in 10 mM citric acid, 100 mM NaCl
`pH 5.5) from 2.7 mg/mL to 50 mg/mL almost
`linearly increased the Nephelometric Unit (NU)
`from 2 to 40.52 Since EP defines a clear solution as
`having equal or less than three NU, most antibody
`solutions in the study are opalescent except those
`at 5 mg/mL or less. However, as the weight average
`molecular weight was found to be 0.9–1.3 times
`that expected of a monomer (about 149 kDa),
`minimal protein–protein association (and readily
`reversible) is suggested.52 Shaking can accelerate
`antibody precipitation and the shape of the
`precipitates may depend on the sample volume in
`a container.53 It appears that protein solutions
`exhibiting lower surface tension are more suscep-
`tible to protein denaturation and precipitation.53
`Low-temperature treatment may induce aggre-
`gation of antibodies. The reversible low-tempera-
`ture-induced aggregation (below 378C) of serum
`cryoglobulins is well known.54 Human IgM cryo-
`globulin preparations easily precipitate or gel at
`temperatures below 10–128C and the process is
`reversible at a higher temperature.20 In a recent
`study, aggregation of IgG1 at low temperature at
`above 18 mg/mL was reversible as measured by
`light scattering.52 The low-temperature-induced
`aggregation of cryoglobulins is poorly understood
`and was thought to involve sites within both Fab
`
`ANTIBODY FORMULATION
`
`9
`
`and Fc.20 The authors of this article believe that
`low temperature reduces the hydrophobic interac-
`tion, which is the major force in protein folding.
`Without enough hydrophobic interaction at low
`temperature, hydrophobic regions of antibodies
`become more exposed to solvent and lead to
`increased intermolecular hydrophobic interaction,
`leading to aggregation.
`In close relation to the low-temperature effect,
`freeze-thaw process often induces protein aggre-
`gation. However, freeze-thaw-induced antibody
`aggregation does not seem to be a major issue,
`partly due to the reversibility of antibody aggre-
`gates. Freeze-thawing of a chimeric (L6) antibody
`solution at pH 7.2 for as many as 35 times led to
`formation of dimers (not larger aggregates).41
`Maximum amount of dimers (about 20%) was
`observed at pH 6.5 and minimum was below 5.5 or
`above 8.5 (less than 2%). The freeze-thaw-induced
`aggregation is apparently reversible after treat-
`ment at 378C for a few hours.41 Freeze-thawing of
`rhuMAb anti-CD20 three times (freezing to either
`20 or 708C, thawing to 58C) did not lead to
`significant change in aggregation, no significant
`loss of monomer by RP-HPLC or SEC-HPLC.55 On
`the other hand, isolated Fabs seems to be more
`prone to freeze-thaw-induced aggregation relative
`to full-length antibodies. For example, Lee56 found
`that freeze-thawing of a scFv (MW 27,000 d) at
`1.45 mg/mL in sodium phosphate buffered saline
`(PBS) at pH