BIOTECHNOLOGY
`
`Understanding and Modulating Opalescence and Viscosity
`in a Monoclonal Antibody Formulation
`
`BRANDEN A. SALINAS,1,2 HASIGE A. SATHISH,1,2,3 STEVEN M. BISHOP,3 NICK HARN,3 JOHN F. CARPENTER,2,4
`THEODORE W. RANDOLPH1,2
`
`1Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado
`
`2Center for Pharmaceutical Biotechnology, University of Colorado, Boulder, Colorado
`
`3MedImmune, LLC, Gaithersburg, Maryland 20878
`
`4University of Colorado Health Sciences Center, Denver, Colorado
`
`Received 8 February 2009; revised 27 March 2009; accepted 30 March 2009
`
`Published online 27 May 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21797
`
`ABSTRACT: Opalescence and high viscosities can pose challenges for high concentra-
`tion formulation of antibodies. Both phenomena result from protein–protein intermo-
`lecular interactions that can be modulated with solution ionic strength. We studied a
`therapeutic monoclonal antibody (mAb) that exhibits high viscosity in solutions at low
`ionic strength (20 cP at 90 mg/mL and 238C) and significant opalescence at isotonic
`ionic strength (approximately 100 nephelometric turbidity units at 90 mg/mL and 238C).
`The intermolecular interactions responsible for these effects were characterized using
`membrane osmometry, static light scattering, and zeta potential measurements. The net
`protein–protein interactions were repulsive at low ionic strength (4 mM) and attractive
`at isotonic ionic strengths. The high viscosities are attributed to electroviscous forces at
`low ionic strength and the significant opalescence at isotonic ionic strength is correlated
`with attractive antibody interactions. Furthermore, there appears to be a connection to
`critical phenomena and it is suggested that the extent of opalescence is dependent on the
`proximity to the critical point. We demonstrate that by balancing the repulsive and
`attractive forces via intermediate ionic strengths and by increasing the mAb concentra-
`tion above the apparent critical concentration both opalescence and viscosity can be
`simultaneously minimized. ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association
`J Pharm Sci 99:82–93, 2010
`Keywords:
`light scattering; protein delivery; protein formulation; viscosity; physical
`characterization
`
`INTRODUCTION
`
`For the treatment of chronic conditions with
`therapeutic proteins, patient-administered deliv-
`ery via subcutaneous injection is preferable.1
`
`Correspondence to: Branden A. Salinas (Telephone: 303-
`492-7471; Fax: 303-492-4341;
`E-mail: branden.salinas@gmail.com)
`
`Journal of Pharmaceutical Sciences, Vol. 99, 82–93 (2010)
`ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association
`
`Subcutaneous administration imposes a volume
`restriction of <1.5 mL, which in the case of some
`proteins, and particularly antibodies, requires
`protein concentrations that can surpass 100 mg/
`mL.2 In addition to accelerated aggregation rates
`at high protein concentrations,3 high-concentra-
`tion antibody formulations may exhibit undesir-
`able opalescence and high viscosity.4–6
`Opalescence introduces a potential safety issue
`because an opalescent solution is easily confused
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`MODULATING OPALESCENCE AND VISCOSITY IN A MAB FORMULATION
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`
`with turbid solutions, which can result from
`protein aggregation or other particulate forma-
`tion. Furthermore, it is challenging to develop
`placebo formulations for clinical studies that
`match the opalescence of the original protein
`formulation. Opalescence can arise in solutions
`that do not contain particulates; the cloudy
`appearance is simply a result of Rayleigh scatter-
`ing.4 Proteins are typically Rayleigh scatterers of
`visible light as they have diameters of <30 nm.
`Likewise, the high viscosities that can be
`exhibited by antibody solutions introduce several
`challenges. Manufacturing processes such as in-
`creasing protein concentration or buffer exchange
`with tangential flow filtration may become infea-
`sible. Also, the force and time required for
`subcutaneous injection of viscous formulations
`can result in increased pain on injection or even
`preclude this route of delivery altogether.5
`Protein–protein interactions play important
`roles in both viscosity and opalescence of protein
`solutions. Sukumar et al.4 describe how attractive
`monoclonal antibody (mAb) interactions can lead
`to opalescent solutions in the absence of any
`significant association between protein molecules.
`The opalescence is attributed to a simple inter-
`molecular attraction though this may be an
`oversimplification as it appears that the proximity
`to the liquid–liquid phase boundary and/or the
`critical point is important.7 Liu et al.6,8 detail an
`example of a mAb that reversibly self-associates
`and thus generates higher viscosity solutions
`relative to two nonassociating mAbs.
`Moon et al.,9 Yousef et al.,10 and Minton11 have
`all used membrane osmometry to characterize the
`physical behavior of model proteins such as bovine
`serum albumin and lysozyme at concentrations
`above 400 mg/mL. Osmometry is particularly
`amenable to high concentration studies as it is
`not subject to the optical limitations of other
`techniques such as analytical ultracentrifugation
`or light scattering. Various methods of interpret-
`ing osmotic pressure data have been developed.
`In the case of Moon et al.9 the protein–protein
`interactions are characterized via second virial
`coefficients for a binary protein mixture at con-
`centrations up to 100 mg/mL. Minton11–13 has
`presented a hard particle model for characterizing
`the osmotic pressures of proteins such as BSA and
`ovalbumin to concentrations above 400 mg/mL.
`Ross and Minton14 have also developed a model for
`the viscosity of protein solutions at high concen-
`trations by applying the Mooney equation for hard
`spheres to proteins. Yousef et al.10,15 character-
`
`ized the osmotic pressure of BSA and an IgG
`up to concentrations above 400 mg/mL with a
`free-solvent model that reveals information about
`protein hydration and protein–ion interactions.
`Light scattering is a complimentary method to
`membrane osmometry for determination of second
`virial coefficients as it reveals information about
`protein molecular weight and net protein–protein
`interactions in the given solvent. The second
`virial coefficient can be divided into a number of
`contributing components but is primarily influ-
`enced by hard-sphere repulsion, electrostatic
`repulsion or attraction, and van der Waals
`attractions.16 When measured with osmometry
`or light scattering, the second virial coefficient
`appears to include influences of cosolutes on
`protein nonideality.17 Thus,
`it should not be
`considered a measure of only protein–protein
`interactions as suggested by the standard statis-
`tical–mechanical definition of second virial coeffi-
`cients, but rather an overall indication of the
`protein’s nonideality in solution.17 Even so,
`second virial coefficients from osmometry or light
`scattering measurements provide a useful para-
`meter that has a statistical–mechanical basis and
`that is predictive of phase behavior and other
`events that derive from protein nonideality in
`solution.17
`Because of their long-range nature, electrostatic
`forces are expected to be a dominant influence
`on the intermolecular interactions responsible
`for opalescence and viscosity of protein solutions
`at pH values where the protein retains a signi-
`ficant effective charge. Ionic strength influences
`protein solution viscosities as well as the positions
`of solution phase boundaries and cloud points by
`modulating the electrostatic contribution to the
`intermolecular potential.5,6,18 In order to assess
`the extent of electrostatic interactions between
`protein molecules, it is essential to determine the
`effective charge of the protein. Recent evidence
`suggests that the apparent charge on a protein
`may not reflect the theoretical charge that is
`expected from the amino acid sequence, but rather
`can depend on the types and concentrations of
`ions in solution.19,20 Several methods have been
`employed to measure the electrophoretic mobility
`of proteins in the solutions of interest, from which
`the zeta potential, which is the potential at the
`slipping plane or diffuse layer boundary, can be
`estimated. These methods include capillary zone
`electrophoresis (CZE), membrane confined elec-
`trophoresis, and laser Doppler velocimetry.19,21,22
`The effective charge of a protein, as reflected in
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`SALINAS ET AL.
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`the zeta potential, plays a key role in intermole-
`cular interactions.23
`This work concerns a therapeutic mAb that
`exhibits high viscosity and significant opalescence
`at concentrations above 30 mg/mL, both depen-
`dent on the solution conditions. At a typical liquid
`mAb formulation pH of 6, the ionic strength is the
`key solution parameter that affects both solution
`behaviors. Protein–protein intermolecular inter-
`actions are evaluated using membrane osmome-
`try and static light scattering in the concentration
`ranges where opalescence and high viscosity
`are exhibited. Zeta potentials are measured
`in order to assess the role of charge repulsion
`in the intermolecular interactions. A connection
`between the electrostatic nature of the mAb and
`viscosity is elucidated. Additionally, the connec-
`tion between opalescence and critical solution
`behavior, previously noted by Cromwell et al.,7
`is characterized. The dependence of viscosity and
`opalescence on ionic strength is opposite for this
`mAb. Furthermore, opalescence reaches a max-
`imum at some mAb concentration and then
`decreases at higher concentrations. This allows
`for the simultaneous minimization/optimization
`of the viscosity and opalescence of the final
`formulation via intermediate ionic strengths from
`which a high concentration, self-administered
`dosage form could be developed.
`
`MATERIALS AND METHODS
`
`The fully humanized mAb of the IgG1 subclass
`manufactured by MedImmune, Inc, Gaithers-
`burg, MD, will be referred to as the mAb and
`has a molecular weight of 148 kDa. The sample
`purity (>99%) was analyzed and confirmed by size
`exclusion chromatography and gel electrophor-
`esis. Protein concentrations were determined by
`UV absorption using an extinction coefficient of
`1.61 cm2/mg at 280 nm. All buffer conditions were
`achieved via exhaustive dialysis which consisted
`of a minimum of four buffer exchanges over a
`minimum of 24 h at volume ratio of buffer to
`sample of greater than 200:1. The final dialysate
`was reserved and used for all dilutions, blanks,
`and controls as needed.
`
`Viscosity
`
`Solution viscosities were measured with a Brook-
`field (Middleboro, MA) model DV-IIþ Pro cone/
`plate viscometer with spindle model CPE-40. The
`
`shear rate for all samples with a viscosity below
`20 cP was set to 600 s1 (80 rpm); for samples with
`viscosities above 20 cP a shear rate of 113 s1
`(15 rpm) was used. This is due to the force
`limitations on the spindle configuration used to
`keep the force on the spindle in the range
`suggested by the manufacturer. For solutions
`where the viscosity could be measured across a
`large range of shear rates, the mAb solution
`viscosity exhibited little to no dependence over the
`range 10–1000 s1 (data not shown). A solution
`volume of 0.5 mL was used for all samples and the
`temperature was controlled at 238C via a circulat-
`ing water bath.
`In order to interpret the results for the mAb
`solution viscosity, the Mooney hard-sphere visc-
`osity was calculated for an equivalently sized hard
`sphere using Eq. (1) 24
`
`
`
`
`
`hhs ¼ hs exp
`
`SF
`1 kF
`
`(1)
`
`where hhs is the viscosity of the equivalent hard-
`sphere solution, hs is the viscosity of the solvent, S
`is a shape parameter, k is a self-crowding factor,
`and F represents the volume fraction of the hard
`spheres. For these calculations, an S value of 3.6,
`as determined by Monkos25 for ovalbumin at room
`temperature, is used as well as a k value of 1.8,
`which is independent of temperature.
`The experimental viscosity data are fit using the
`Huggins equation given in Eq. (2) 26
`hsp ¼ h
` 1 ¼ hintc þ KHh2
`intc2
`hs
`
`(2)
`
`where hsp is the specific viscosity, h is the viscosity
`of the antibody solution, c is the antibody con-
`centration, hint is the intrinsic viscosity, and KH is
`the Huggins constant.
`
`Opalescence
`
`Opalescence was assessed by nephelometric
`turbidity (NTU) which was measured via light
`scattering at 908 on a spectrofluorometer (SLM
`AMINCO, Urbana, IL) equipped with a tempera-
`ture-controlled cell holder and 0.2 cm path length
`quartz cuvette. The excitation and emission were
`both set to 510 nm with a 4 nm bandwidth. All
`samples were measured at 238C and filtered with
`a Whatman Anotop 0.1 mm filter immediately prior
`to the measurement. The light scattering inten-
`sity was converted to NTU using a calibration
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`curve generated from AMCO Clear turbidity
`standards (GFS Chemicals, Columbus, OH).
`
`Membrane Osmometry
`
`The osmotic pressure of the mAb solutions was
`measured with a Wescor (Logan, UT) Colloid
`Osmometer Model 4420. A 10,000 MWCO mem-
`brane (product # SS-050) was used with the
`corresponding dialysate as the reference solution.
`All measurements were made at room tempera-
`ture (238C). The Osmocoll N standard (product
`# SS-025) was used for calibration. An initial
`sample injection of 300 mL followed by a minimum
`of two subsequent 50 mL injections were used until
`a stable pressure reading was obtained.
`To interpret the osmotic pressure data the
`osmotic virial expansion of the van’t Hoff equation
`is employed27
`
`Q
`
`P
`cRT
`
`¼ 1
`Mn
`
`þ ðSVCÞc
`
`(3)
`
`is the osmotic pressure, R is the uni-
`where
`versal gas constant, T is the absolute tempera-
`ture, Mn is a number-averaged molecular weight,
`and SVC is the osmotic second virial coefficient.
`For comparison of the osmotic pressure results to
`theoretical hard-sphere values, the Carnahan–
`Starling28 hard-sphere approximation is utilized
`
`P
`rhskBT
`
`¼ 1 þ j þ j2 j3
`ð1 jÞ3
`
`j ¼ p
`6
`
`rhss3
`
`(4)
`
`(5)
`
`where rhs is the number density of hard spheres,
`kB is the Boltzmann constant, j is the effective
`hard-sphere packing fraction, and s is the
`effective hard-sphere diameter.
`The hydrodynamic diameter of the mAb of
`10.2 nm, as measured by dynamic light scattering,
`was used for the effective hard-sphere diameter.
`
`Light Scattering
`
`Static light scattering is a complementary method
`to membrane osmometry for determination of
`the osmotic second virial coefficient. An Electro-
`Optics laser model 1145AP (Hsintien City, Tai-
`wan), a Brookhaven Instruments goniometer and
`cascade photodiode detector model BI-200SM
`and BI-APD (Holtsville, NY), respectively, were
`used to determine the excess Rayleigh ratios at a
`
`908 angle (scattering due to protein only) to the
`incident 633 nm light beam. The relationship used
`to determine the osmotic second virial coefficient
`is given here and is derived from the virial
`expansion of the ideal osmotic pressure equation29
`¼ 1
`þ 2ðSVCÞc
`Mw
`
`Kc
`R90
`
`(6)
`
`where Mw is the mass-averaged molecular weight,
`R90 is the excess Rayleigh ratio at 908, and the
`optical constant K is described by Eq. (7)
`0ðdn=dcÞ2
`K ¼ 4p2n2
`NAl4
`
`(7)
`
`where n0 is the refractive index of the solvent, dn/
`dc is the refractive index increment, and l is the
`wavelength of the incident beam. The Rayleigh
`ratio is given by Eq. (8)
`R ¼ Iur2
`¼ IuðconstantÞ
`IincVobs
`
`(8)
`
`here r is the distance from the observed volume to
`the detector, Iinc is the incident intensity of the
`laser beam, Iu is the measured intensity of the
`scattered light, and Vobs is the observed volume.
`A refractive index increment of 0.185 mL/g,
`estimated from a weight averaged contribution
`from the protein and carbohydrate portions of the
`mAb, was used for all light scattering analyses.30
`The constant can be determined from a system
`for which the Rayleigh ratio is known, in this
`case toluene at 633 nm (14 106 cm1). Once
`the constant is determined, then raw intensity
`measurements can be converted to Rayleigh ratios
`and the excess Rayleigh ratio is simply the
`Rayleigh ratio of the sample minus that of the
`solvent.
`For the dynamic light scattering, the same
`equipment described above was employed as well
`as a BI-9000AT digital autocorrelator (Brookha-
`ven Instruments). A protein concentration of
`1 mg/mL was used for all samples. The resulting
`correlation functions were used to determine the
`diffusion coefficients from which the hydrody-
`namic diameters were calculated by the Stokes
`equation. The temperature for all light scattering
`measurements was controlled at 238C using a
`circulating bath temperature controller, and a
`solvent viscosity of 0.9 cP was used for all
`hydrodynamic diameter calculations. The mAb’s
`hydrodynamic diameter (10.2 0.8 nm) did not
`vary significantly with the solvent conditions used
`in this work.
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`Zeta Potential
`
`A Malvern Zetasizer Nano ZS (Malvern, UK)
`was used to measure the electrophoretic mobility
`of the antibody via laser Doppler velocimetry.
`Interactions with the electrodes of the instrument
`can result in an increased resistance and preclude
`an accurate measurement. As the Zetasizer
`applies a constant voltage, and the zeta potential
`is a constant current measurement, the current
`was monitored during measurement to ensure
`a constant current was achieved for the data
`reported. The zeta potential is calculated from
`Henry’s equation using the Smoluchoski approx-
`imation, which is valid for ionic strengths above
`1 mM22
`
`me ¼ 2"ksz
`3h
`
`(9)
`
`where me is the electrophoretic mobility, e is the
`dielectric constant or permittivity of the solution,
`ks is a model-based constant which from the
`Smoluchoski approximation is 1.5, and z is the
`zeta potential. An antibody concentration of
`2 mg/mL was used for all samples and the
`measurement was repeated on three samples at
`each condition and the errors are reported as the
`standard deviation. The temperature was con-
`trolled at 238C.
`The effective charge of an equivalent sphere
`can be estimated via the linearized Poisson–
`Boltzmann equation, also referred to as the
`Debye–Hu¨ ckel approximation, and is given by
`Eq. (10) 31
`
`Z ¼ 4p"rpð1 þ krpÞz
`
`e
`
`(10)
`
`where Z is the effective charge, rp is the effective
`sphere radius, k is the inverse electric double layer
`thickness or inverse Debye length, and e is the
`elementary charge.
`The electrostatic contribution to the osmotic
`second virial coefficient, also referred to as the
`Donnan term, can be calculated using Eq. (11) 16
`
`SVCelectrostatic ¼
`
`Z2
`4M2rsmions
`
`(11)
`
`where SVCelectrostatic is the electrostatic compo-
`nent of the second virial coefficient, M is the actual
`molecular weight of the protein, rs is the solvent
`density, and mions is the molal concentration of
`ions.
`
`Figure 1. The concentration dependence of the visc-
`osities of the mAb solutions at pH 6 and three ionic
`strengths. Solution conditions are: (*) 10 mM histidine;
`(&) 2.2 mM sodium phosphate; (*) 10 mM histidine,
`150 mM NaCl; (&) 2.2 mM sodium phosphate, 150 mM
`NaCl; (^) 10 mM histidine, 2 mM NaCl; (xxxx) viscosity
`of an equivalent hard sphere using the Mooney approx-
`imation (Eq. 1). Lines represent a fit to the polynomial
`in Eq. (2) where the solid lines correspond to the closed
`symbols, and the dashed lines correspond to the open
`symbols. Error bars represent standard deviation from
`three samples.
`
`RESULTS
`
`Viscosity
`
`ionic
`The mAb viscosity was measured at
`strengths of approximately 4 and 154 mM
`(neglecting the contribution of the polyelectrolytic
`protein); these solution conditions are referred to
`as low and high ionic strength. From Figure 1 it is
`clear that the viscosity is much higher in the low
`ionic strength buffer systems regardless of buffer
`type; however, the differences in viscosity were
`only measurable at mAb concentrations above
`50 mg/mL. The curves shown in Figure 1 are fits to
`the Huggins equation (Eq. 2). Addition of 150 mM
`NaCl to solutions containing the mAb at concen-
`trations of 80 mg/mL results in an approximately
`threefold decrease in the solution viscosity. In
`fact, in the presence of 150 mM NaCl, the solution
`viscosity is only slightly larger than that predicted
`for noninteracting hard spheres via the Mooney
`approximation (Eq. 1), and this excess viscosity is
`apparent only at the highest protein concentra-
`tions tested. Finally, the addition of 2 mM NaCl
`to the 10 mM histidine formulation results in a
`significant decrease in solution viscosity.
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`Figure 2. Opalescence, measured by nephelometric
`turbidity, as a function of the mAb concentration at
`pH 6. Solution conditions are: (*) 10 mM histidine;
`(&) 2.2 mM sodium phosphate; (*) 10 mM histidine,
`150 mM NaCl; (&) 2.2 mM sodium phosphate, 150 mM
`NaCl. Lines are to guide the eye. Error bars from
`triplicate samples are smaller than the data points.
`
`Opalescence
`
`NTU, a measurement commonly used in determi-
`nation of water clarity that has been applied
`to protein opalescence, was utilized to quantify
`the degree of opalescence.4 Four of the buffer
`conditions used for the viscosity measurements
`were tested for opalescence (Fig. 2). To reduce the
`potential of scattering from insoluble particulates,
`the solutions were passed through a 0.1-mm
`syringe filter directly into the clean sampling
`cuvette and the scattering intensity was mea-
`sured immediately. The high ionic strength
`solutions are more opalescent than the low ionic
`strength solutions, although the differences are
`only apparent at the higher protein concentra-
`tions, in this case above 30 mg/mL. This ionic
`strength dependence is opposite to that observed
`for the viscosity. There is an approximate three-
`fold increase in opalescence with the addition of
`150 mM NaCl to solutions containing 80 mg/mL
`mAb. To further elucidate the effect of ionic
`strength, opalescence was measured as a function
`of mAb concentration in solutions containing
`10 mM histidine and either 0, 2, 5, 10, or
`150 mM NaCl at a pH of 6 (Fig. 5). The ionic
`strengths of the solutions are 4, 6, 9, 14, and
`154 mM NaCl, respectively. It is apparent that
`at the lower ionic strengths (<154 mM NaCl),
`opalescence reaches a maximum at mAb concen-
`trations in the vicinity 60 mg/mL.
`
`Figure 3. Osmotic pressure of the mAb at pH 6 mea-
`sured by membrane osmometry. (*) 10 mM histidine;
`(&) 2.2 mM sodium phosphate; (*) 10 mM histidine,
`150 mM NaCL; (&) 2.2 mM sodium phosphate, 150 mM
`NaCI; (^) predicted osmotic pressure of an equivalent
`hard sphere using the Carnahan–Starling approxima-
`tion with a hard-sphere radius equal to the hydrody-
`namic radius of the mAb of 5.1 nm (Eq. 4). Remaining
`lines are linear regressions of the corresponding data
`sets and the short dashed lines correspond to the open
`symbols. Error bars correspond to 0.2 mmHg which is
`the specified sensitivity limit of the osmometer and is
`typically larger than the sample to sample variation.
`
`Osmometry and Light Scattering
`
`In order to probe the protein–protein intermole-
`cular interactions in the four solution conditions
`of interest, osmotic pressure measurements were
`used to generate osmotic virial plots (Fig. 3).
`Similarly, light scattering intensities at 908 were
`used to produce the Debye plots (Fig. 4). The
`resulting molecular weights (number-averaged
`when determined from osmotic pressure measure-
`ments and weight-averaged when determined by
`light scattering) and the osmotic second virial
`coefficients from the linear regressions of the data
`are presented in Table 1. All of the measured
`molecular weights are near that expected for
`the monomer (148 kDa) and there is little cur-
`vature over the concentration range, measured
`suggesting that the antibody is monomeric and
`there is not a significant amount of antibody
`self-association. Additionally, the two low ionic
`strength buffer
`conditions generate positive
`slopes, indicating net pair-wise repulsion between
`antibody molecules in the corresponding buffer
`systems. The two high ionic strength buffer
`conditions result in negative slopes, indicative
`of net pair-wise attraction between antibody mole-
`cules in the given solution. For the net-repulsive
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`Figure 4. Debye plot generated from light scattering
`measurements of the mAb at pH 6 in various solutions:
`(*) 10 mM histidine; (&) 2.2 mM sodium phosphate;
`(*) 10 mM histidine, 150 mM NaCl; (&) 2.2 mM sodium
`phosphate, 150 mM NaCl. The long dash, short dash
`line represents the predicted light scattering from an
`equivalent hard sphere with a radius equivalent to the
`hydrodynamic radius of the mAb of 5.1 nm. Remaining
`lines are linear regressions of
`the corresponding
`data sets and the dotted lines correspond to the open
`symbols. Error bars from standard deviation of three
`measurements are smaller than the symbols.
`
`conditions the amount of light scattered is near
`or slightly less than that predicted for a hard-sphere
`solution of monomers, whereas for the net-attrac-
`tive conditions the intensity of scattered light is
`larger than that expected based on hard-sphere
`predictions. There is approximately a threefold
`difference in the Rayleigh ratios or intensity of
`scattered light at 40 mg/mL between the low and
`high ionic strength solutions for each buffer type.
`
`Zeta Potential and Charge Estimates
`
`To determine the role of electrostatics in the
`solution behavior of this mAb, zeta potentials
`
`were measured for the mAb in the low and high
`ionic strength buffer systems. The results are
`presented in Table 2. The antibody in the two low
`ionic strength solutions exhibits a zeta potential
`and order of magnitude higher than that observed
`in the high ionic strength solutions. In fact,
`the electrophoretic mobility of the mAb in both
`150 mM NaCl buffer systems was barely percep-
`tible by the laser Doppler velocimetry technique
`used, suggesting that the molecule possesses a
`very low effective charge under these conditions.
`The effective charge estimates, using the Debye–
`Hu¨ ckel approximation (Eq. 10), are approximately
`threefold lower in the high ionic strength solu-
`tions compared to effective charges in the low ionic
`strength solutions. Interestingly, the theoretical
`surface charge for the mAb at pH 6 is þ15.
`Although the zeta potential is still positive, even
`the 4 mM ionic strength solutions reduce the
`effective charge at the slipping plane by a factor of
`4. The effective charge at the slipping plane or
`diffuse layer boundary is what contributes to
`the intermolecular interactions and this can be
`reflected in the estimate of the electrostatic
`contribution to the second virial coefficient.16
`The electrostatic contribution to the second virial
`coefficient is significant for the low ionic strength
`buffer conditions. In fact, it is on the same order as
`the difference in the measured second virial
`coefficients for the mAb at the low and high ionic
`buffer systems. The electrostatic contribution to
`the second virial coefficient in the high ionic
`strength solutions is insignificant when compared
`to the total second virial coefficient. This that the
`switch from net-repulsive to net-attractive second
`virial coefficient values at higher ionic strengths
`is due to decreases in protein effective charge and
`increased electrostatic screening of the remaining
`charge at high ionic strength.
`
`Table 1. Second Virial Coefficients (SVC) and Molecular Weights Are Reported as Determined by Linear
`Regression of the Data in Figures 3 and 4
`
`Formulation
`
`10 mM histidine
`2.2 mM sodium phosphate
`10 mM histidine, 150 mM NaCl
`2.2 mM sodium phosphate, 150 mM NaCl
`
`Osmometry
`SVC 105
`(mL mol/g2)
`5.8 2.5
`13.0 1.9
`4.3 2.0
`2.4 1.1
`
`MW
`(kDa)
`124 17
`137 14
`163 21
`140 9
`
`Light Scattering
`SVC 105
`(mL mol/g2)
`4.93 0.65
`10.2 1.2
`4.71 0.89
`5.0 1.3
`
`MW
`(kDa)
`174 10
`141 13
`174 11
`125 9
`
`All measurements were conducted at a pH of 6. Units of the SVC are mL mol/g2 and the units of MW are kDa. Errors represent 95%
`confidence intervals from the regressions.
`
`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 1, JANUARY 2010
`
`DOI 10.1002/jps
`
`Ex. 2006-0007
`
`

`
`MODULATING OPALESCENCE AND VISCOSITY IN A MAB FORMULATION
`
`89
`
`Table 2. The Measured Zeta Potentials for the mAb in the Four Buffer Conditions of Interest at pH 6, as well as the
`Corresponding Calculations of Effective Charge and the Electrostatic Component of the Second Virial Coefficient
`(SVC)
`
`Formulation
`
`10 mM histidine
`2.2 mM sodium phosphate
`10 mM histidine, 150 mM NaCl
`2.2 mM sodium phosphate, 150 mM NaCl
`
`Zeta Potential
`(mV)
`7.4 0.4
`6.9 0.3
`0.6 0.9
`0.6 0.8
`
`Effective
`Charge
`
`3.8
`3.6
`1.1
`1.1
`
`SVCelectrostatic  105
`(mL mol/g2)
`
`6.8
`6.8
`0.01
`0.01
`
`Errors represent standard deviation from three measurements.
`
`DISCUSSION
`
`Charges on the surface of antibodies result in
`long-range intermolecular forces that predomi-
`nate at low ionic strengths and are the primary
`source of the high solution viscosity. The Debye
`length at a 4 mM ionic strength is approximately
`4.8 nm, whereas the average hydrodynamic sur-
`face-to-surface antibody spacing is of the same
`order, approximately 9 nm at an antibody con-
`centration of 50 mg/mL and 5 nm at 100 mg/mL.
`This is the primary source of the large increases in
`viscosity near 100 mg/mL in the low ionic strength
`solutions. Charge repulsion and the related
`electric double layer have been noted as the
`source of high viscosity for proteins and other
`polyelectrolytes.32 As the intrinsic fluorescence
`and far ultra-violet circular dichroism (far UV-
`CD) signals are unaffected by NaCl for this mAb
`(data not shown) the mAb conformation appears
`to be independent of ionic strength. With the
`assumption that the antibody structure is not
`significantly affected by ionic strength, there are
`two main contributions to viscosity, which are
`prevalent at low ionic strengths and are referred
`to as electroviscous effects. The first is resistance
`to movement from the double layer surrounding
`each protein molecule. The concentration of
`ions in the electric double layer is significantly
`different than in the bulk solution, the electric
`double layer then acts as an effective increase
`in the hydrodynamic radius of the molecule. The
`second results from the charge repulsion from
`the double layers of other molecules, which is
`directly related to the zeta potential.
`Saluja et al.33 have shown these electroviscous
`effects to be important for antibody solutions
`at low ionic strengths (4 mM) utilizing high-
`frequency rheology measurements. At
`ionic
`strengths above 10 mM, the electroviscous effects
`become less and less significant as the electric
`
`double layer becomes more similar to the bulk
`solution, the effective charge on the protein is
`reduced, and Debye length is shortened due to
`charge screening. The high ionic strength solution
`conditions yield viscosities very near
`those
`expected for the equivalent hard spheres. This
`suggests that the short-range van der Waals
`forces responsible for intermolecular attraction
`contribute significantly less to the solution
`viscosity than the long-range, repulsive electro-
`static forces. It has been reported that reversible
`association increases antibody solution visco-
`sity6,8; however, electrostatic effects were not
`directly investigated in that work. The conclusion
`was drawn because the extent of reversible
`association and the viscosity decreased with
`increasing ionic strength. The electroviscous
`effects were not investigated or identified as
`possible contributors to the reduction in viscosity.
`The dominant source of the high viscosity for this
`mAb is repulsive and electrostatic in nature.
`Opalescence is a phenomenon of Rayleigh
`scatters and the increased light scattering of
`attractive antibody solutions has been attributed
`to an increased apparent or effective molecular
`weight.4 In a recent study of other opalescent
`mAb formulations, Cromwell et al.7 demonstrated
`through measurement of critical exponents that
`critical density fluctuations, related to proximity
`to mixture critical points, result in extensive
`Rayleigh scattering from monomeric antibody
`molecules under solution conditions that produce
`net protein–protein attraction. Opalescence has
`long been a phenomenon linked to phase behavior
`near critical points and is thus often referred to as
`critical opalescence. Some of the earliest work on
`the relationship