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`Cal|#: Swag; “aeaax M3 gem » eaaaweam
`
`Journal Title: Journal of parenteral science and
`technology
`
`UN'VERS'TY 0F
`
`Nebraska
`LlnCOIIl
`Interiibrary Loan
`
`Volume: 45 Issue: 3
`Month/Year:
`1991
`
`Pages: 160-165
`
`Article Author: LEVINE, HL
`
`Article Title: THE USE OF SURFACE—
`TENSION MEASUREMENTS N THE DESIGN
`OF ANTIBODY—BASED PRODUCT
`FORMULATIONS
`
`ILL Number: -9224433
`
`4/21/2015 7:43 AM
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`AMGEN INC.
`
`Exhibit 1026
`
`Ex. 1026 - Page 1 of7
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`Ex. 1026 - Page 1 of 7
`
`AMGEN INC.
`Exhibit 1026
`
`

`

`RESEARCH AR TICLE
`
`The Use of Surface Tension Measurements in the Design of AntibodynBased
`Product Formulations
`
`HOWARD L. LEVINEA, TOM C. RANSOHOFF*, RUSSELL T. KAWAHATA, and W. C. MCGREGOR
`XOMA Corporation, Berkeley, California
`
`
`
`
`
`
`
`ABSTRACT: Many proteins in aqueous solution are sus
`ceptible to interfacial denaturation andprecipitation
`during mechanical agitation. With the large number ofprotein parenteral products currently in research or
`
`arefrequently used to stabilize parenteral products. While it is a commonly accepted technique, little has
`beenpublished about thepreczpitation andstabilizationprocesses in general. We describe the stabilization of
`antibody products in solution by preferential adsorption ofa water-soluble, non-ionic surfactant at the air-
`
`
`
`
`
` —-n-—-—----—.
`
`
`
`the protein may be further denatured from its surface -
`state and forced into entanglement and aggregation with
`other adsorbed proteins. Cumper and Alexander (5) sug-- -__-
`
`gested that primary aggregation occurs at the surface
`forming a non-ssurface active intermediate. This interme-
`diate is then subjected to further aggregation and precipi-
`tation in the liquid solution. Electron micrographs show-
`ing gamma-globulin aggregates and fibers induced by
`shaking (6) support this view of interfacial protein dena-
`turation.
`
`lntroduction and Background
`
`The development of a therapeutic protein product in—
`cludes the design of a formulation that provides for prod—
`uct stability under a variety of conditions (i.e., filling,
`shipment, storage, and administration). One important
`aspect of successful formulation development is the stabi-
`lization of proteins at air—liquid interfaces. We present a
`simple thermodynamic model which can be used to pre-
`dict the susceptibility of antibody-based products to sur-
`face denaturation based on surface tension data. The
`model can also be used to design formulations that prevent
`surface denaturation of the proteins.
`It is well known that proteins can adsorb to an air~liquid
`interface, denature, and then form aggregates or precipi-
`tates (l »—3). The driving force for adsorption of proteins or
`any surface-active component at the interface is a de—
`crease in the total free energy of the system. At the inter-
`face, a surface-active protein will unfold to some degree
`from its native solution conformation so that the more
`hydrophobic portions of the protein are exposed to the air
`phase. This arrangement provides a transition region from
`the non-polar air phase to the polar aqueous phase and
`shields the hydrophobic portions 'of the protein from the
`aqueous phase, causing a reduction in the surface tension
`and the total free energy of the system.
`MacRitchie (3) and Kaplan and Fraser (4) have shown
`that further conformational changes occur in proteins
`adsorbed at an air-liquid interface during compression
`and expansion of the interface. Therefore, when the inter-
`face to which the protein is adsorbed expands or contracts,
`-__,._-W__.._....__.________..._,
`
`Received November 28, 1989. Accepted for publication January 8,
`1991.
`
`* Current address: Dorr-Oliver, Inc, 612 Wheeler’s Farm Road, Mil-
`ford, CT 06460.
`A Author to whom correspondence should be addressed: 2910 Seventh
`Street, Berkeley, CA 94710.
`
`160
`
`The degree to which a protein adsorbs at an interface
`should provide an indicator of its surface stability in a
`liquid formulation. Since the surface tension of a protein ~~
`solution is related to the degree of adsorption of the prev -_
`tein at the interface, surface tension measurements may
`be useful in predicting the surface stability of protein
`products in liquid solutions and also in designing formula-
`tions that prevent surface~related denaturation.
`For practical purposes, predicting the surface instabil-
`ity of a protein product is far less useful than preventing it-
`Surface stabilization of protein products in liquid solu-
`tions is important in (i) shipment and storage of all protein
`products that are formulated and filled in liquid solutions,
`(it) reconstitution of lyophilized protein products, and
`(iii) dilution and administration of parenteral protein -
`products. All three of these cases involve protein solutions
`with liquid—air interfaces that may be exposed to signifi—
`cant surface stress due to shaking or vibration. Also, in all
`three cases even a small amount of aggregation or precipl“
`tation is either undesirable or completely unacceptable.
`One common method of protein solution stabilization IS
`the addition of a surface—active agent. Table I is a partial
`listing of therapeutic proteins containing surface-active
`stabilizers (7). One likely reason for the effectiveness Of
`these stabilizers is that they are thermodynamically fa-
`vored over the proteins for adsorption at the interface, and
`block the surface from the proteins in solution.
`
`‘
`
`
`
`
`
`
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`
`
`
`
`
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`
`
`Journal of Parenteral Science & Technology
`
`
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` Ex. 1026 - Page 2 of 7
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`Ex. 1026 - Page 2 of 7
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`

`

`Genentech
`Ortho
`Cetus
`
`$0.08
`Polysorbate 80
`0.2
`Polysorbate 80
`
`SDS
`50.2
`
`ant. Finally, the surface tension behavior of protein-sur-
`actant systems is investigated and compared to the be-
`avior of standard binary surfactant systems (8).
`
`aterials and Methods
`
`MAb A, MAb B, MAb C, and MAb D are all highly
`urified murine monoclonal antibodies or antibody-ricin
`chain conjugates produced by XOMA Corporation
`Berkeley, CA). The approximate molecular weights of
`Ab A, MAb B, MAb C,rand MAb D are 200,000,
`00,000, 150,000, and 150,000 Daltons, respectively, Ri~
`in Toxin A chain was also produced by XOMA from
`astor beans, and the chicken egg albumin hydrolysate
`as obtained from Sigma (St. Louis, MO).
`
`
`ABLE 1. Protein Products Containing Surfactants
`
` Product Manufacturer
`Cs/Cp
`Surfactant
`
`/. W
`
`Alteplase (tPA)
`oKT—3
`
`{1,2
`
`
`In this paper, we demonstrate a correlation between the
`
`urface tension of purified proteins in aqueous formula-
`
`onS and the ease of denaturation and precipitation of
`
`hese proteins during shipping simulations. In addition,
`he effectiveness of surfactants at stabilizing proteins in
`
`olution during shipping simulationsis shown to depend
`
`
`
`roteins
`
`
`
`
`
`
`
`
`
`Surfactants
`
`Most of the polysorbate 80, or polyoxyethylene (20)
`
`orbitan monooleate used was Tween 80, N.F. Grade,
`rorn Spectrum Chemical (Gardena, CA). However, poly-
`
`orbate 80’s from other manufacturers—Emsorb 2722
`Emery Industries, Santa Fe Springs, CA), Lonzest
`
`MO-ZO (Lonza, Fair Lawn, NJ), and Tween 80, Protein
`
`rade (Calbiochem, La Jolla, CA)—were also tested.
`
`While differences in the UV absorbance spectra were
`bserved in the polysorbates from different sources, there
`
`ere no noticeable differences in their effectiveness at
`
`urface stabilization of proteins. Pluronic F-68, an oz-
`ydroxy-omega-hydroxy-poly(oxyethylene)poly(oxypro-
`
`ylene)poly(oxyethylene) block copolymer, was obtained
`
`rorn ICI Americas, Inc. (Wilmington, DE). BRIJ 700, a
`olyoxyethyleneglycol dodecyl ether, polyoxyethylene
`23) lauryl ether, was obtained from Calbiochem.
`
`
`
`urface Tension Measurements
`
`A Wilhelmy plate tensiometer was used to measure the
`urface tension of the solutions described above. This ten-
`iOmeter measures the difference between the force re-
`uired to suspend the plate in air, Fair, and the force
`Cquired to suspend the plate in a liquid solution, Fliq. The
`urface tension, 0, can be related to the difference in
`
`
`
`
`
`
`a = ~—~——-
`
`(Eq. 1)
`
`V01. 45, No.3 / May—June 1991
`
`
`
`
`
`
`
`
`where d and t are the length and thickness of the plate
`respectively.
`In our measurements, the balance was zeroed with the
`plate suspended in air, so that the measured force, F, is
`equal to (F1,q — Fair). The thickness of the plate is so small
`that it may be neglected in Eq. (1); plate lengths of either
`3/4 in. or 1 in. were used. The Wilhelmy plate apparatus
`was calibrated with deionized water to a surface tension of
`
`72 millinewtons/meter (mN/m). The accuracy of this
`method is approximately :I:0.5 mN/m.
`
`Shipping Simulations
`
`The shipping simulation was designed to mimic worst
`case shipping conditions for a liquid therapeutic protein
`product. Normally, a therapeutic protein formulated as
`an aqueous solution should be shipped in a mostly-filled
`vial that is oriented to minimize the area of the air-liquid
`interface and packaged to minimize vibration. For the
`shipping simulation, an aqueous solution of protein in a
`half-filled vial,.oriented horizontally to maximize the air-
`liquid interface, was shaken at 150 rpm on a Junior Orbi-
`tal shaker (Lab-Line Instruments, Inc., Melrose Park, IL)
`for 12—18 hours at room temperature (20—25°C). The
`cumulative surface stress imposed by this simulation was
`much greater than would be expected during shipment.
`Both before and after the shipping simulations, the
`protein solutions were analyzed for protein concentration,
`aggregation, and particulate level. Absorbance at 280 nm
`or HPLC assays were used to determine protein concen-
`tration. Absorbance readings were always taken on fil-
`tered solutions to prevent light-scattering interference
`from fine precipitate. The level of aggregation was deter-
`mined by size exclusion HPLC on a 7.5 X 300 mm TSK
`250 or TSK 400 column equilibrated in 20 mM sodium
`phosphate, 200 mM sodium sulfate, pH 6.8. Particulate
`levels were determined by visual observation and by parti—
`cle counting (HIAC~Royco).
`
`Results and Discussion
`
`Protein Surface Activity and Denaturation
`
`The shipping simulation is one method for testing the
`susceptibility of a protein in solution to surface denatur—
`ation and precipitation. Figure 1 shows a vial of an anti-
`body—based product, MAb A, after a shipping simulation.
`Clearly MAb A is very susceptible to surface denatur-
`ation.
`
`Interestingly, the rings of precipitate seen in Figure 1
`were formed only in slightly “overfilled” vials; 7.5 mL in a
`nominal 5 mL vial (Wheaton). An amorphous precipitate
`was seen after a shipping simulation with 5 mL in the 5
`mL vial. The fill volume, or more appropriately the sur«
`face-to-volume ratio, is obviously an important variable in
`surface denaturation of protein solutions. In fact, vials
`that were completely filled with the protein solution (i.e.,
`no air remaining) showed a dramatic reduction in surface
`denaturation and precipitation.
`The surface behavior of three antibodynbased products
`was investigated by measuring the surface tension of these
`products in solution as a function of protein concentration.
`
`161
`
`EX. 1026 - Page 3 of 7
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`Ex. 1026 - Page 3 of 7
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`

`

`
`
`
`AttarShIpping‘
`
`Simulation (9%) 1
`
`
`
`Remaining
`
`Gamimm“)
`
`Figure 3—Product loss after a shipping simulation as a function of -
`surface activity. The percent protein remaining in solutiOn
`is plotted against the surface tension of a 0.1% (My)
`solution of the protein.
`
`'
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`75
`
`
`
`70
`
`7*
`0.01
`
`j.
`
`1
`
`55 T
`0-001
`
`Figure 2—Surface tension curves for three antibody products: (El) MAb A, (+) MAb B, and (0) MAb C
`
`Concentration (96)
`
`162
`
`Journal of Parenteral Science & Technology
`
`Ex. 1026 - Page 4 of 7
`
`Figure 1—Antibody precipitate formed by surface stress imposed
`during a shipping simulation.
`
`The surface tension curves, shown in Figure 2, demon-
`strate that MAb A is more surface active than MAb B,
`which is more surface active than MAb C.
`
`Based on our understanding of the surface-related de-
`naturation and precipitation of protein products, we
`would expect proteins that are more surface active to be
`more susceptible to surface denaturation. The susceptibil-
`ity to surface denaturation of six proteins (MAb A—D,
`Ricin Toxin A chain, and ovalbumin) was determined
`
`using the percent protein remaining in solution after a
`shipping simulation as an index of susceptibility. The
`surface tensions of the solutions before the shipping simu-
`lation (all solutions were initially 1 g/L) were also mea-_ __
`sured. Figure 3 shows the percent protein remaining after -
`shipping plotted as a function of solution surface tension.
`_
`The results indicate a correlation between the surface
`tension of a protein solution and its susceptibility to sur- “
`face denaturation and precipitation; specifically, protein
`solutions exhibiting lower surface tension appear to be‘
`more susceptible to protein denaturation and precipita-
`tion.
`There are a number of other important factors to con— -
`sider in this study. As shown by Hamaguchi (11), the
`force required for denaturation will vary substantially
`from protein to protein, depending on the ratio of cyste-
`ines to total amino acids, and this would be expected to
`
`-
`!
`
`-:
`
`
`
`
`Ex. 1026 - Page 4 of 7
`
`

`

`ffcct a protein’s susceptibility to surface denaturation.
`150, the shipping simulation used in this study was only a
`apshot in time; in reality, denaturation and precipita-
`on are time-dependent phenomena. Donaldson et a1. (2)
`ave shown that for long surface contact times, the kinet-
`105 cf interfacial protein denaturation depend upon the
`urface generation rate. More of these kinetic studies are
`* ceded. For instance, the formation of precipitate from
`denatured proteins is not yet well described in terms of
`ither mechanisms or kinetics.
`Despite the need for future work, the largely qualitative
`{correlation developed in this study works well with the
`roteins tested. This type of correlation not only allows for
`the prediction of susceptibility to surface denaturation, it
`also allows one to separate surface denaturation and pre-
`1cipitation of proteins from other types of precipitation.
`For instance, if precipitation is seen during the processing
`' of a protein product, it may be useful to know whether or
`T not the precipitation is surface-related. If the protein is
`i surface active, surface-related denaturation is a strong
`possibility, and careful attention should be given to avoid
`_ air~liquid interfaces. However, if the protein is not surface
`active, other precipitation mechanisms may be involved.
`
`; Surface Stabilization and Antibody-Based Products
`
`As described in the Introduction and Background Sec-
`tion, surfactants are frequently used to stabilize or solubi-
`lize proteins in aqueous solutions (7, 12). Table I lists
`:three therapeutic proteins with surfactants included in
`their formulations. While it is commonly accepted that
`L surfactants are effective stabilizers, little work has been
`_ devoted towards understanding the mechanism by which
`1 they stabilize. Figure 4 illustrates the effectiveness of
`} polysorbate 80, a non-ionic surfactant, at stabilizing MAb
`
`
`
`A. The figure shows two vials after a shipping simulation,
`both with the same initial concentration of MAb A; the
`vial on the right contains polysorbate 80 and is clearly free
`of precipitate, while the vial on the left has no surfactant
`and is similar to the vial in Figure 1, containing a signifi-
`cant amount of precipitated protein.
`Adsorption theory predicts that if a surfactant is more
`surface active than a protein product, the surfactant will
`adsorb more strongly at the interface. If the surfactant is
`significantly more surface active than the protein, the
`surfactant will almost completely block the interface from
`the protein as depicted in Figure 5. This action will pro-
`vide an essentially hydrophilic surface for the protein (i.e.,
`the hydrophilic heads of the surfactant molecules), which
`will prevent the protein from adsorbing at the interface
`and protect it from surface-related denaturation and pre-
`cipitation.
`The effectiveness of polysorbate 80 at stabilizing MAb
`A was determined as a function of surfactant concentra-
`tion using shipping simulations at a MAb A concentration
`of 1 g/ L. The results of these experiments, which are
`shown along with the surface tension curve for polysor-
`bate 80 in Figure 6, suggest that a polysorbate 80 concen-
`tration of approximately 0.1% is needed to completely
`stabilize MAb A during surface stress. Based on the sur-
`face tension curve in Figure 6 and the 55 mN/m surface
`tension of a l g/L solution of MAb A, we would predict
`effective stabilization at polysorbate 80 concentrations of
`less than 0.01%. The need for significantly more surfac-
`tant for effective stabilization of MAb A is not easily
`
`explained.
`Another potential use for surface tension measure-
`ments is to aid in the screening of surfactants for use as
`surface stabilizers for protein products in liquid solutions.
`The surface tension curves of three non-ionic surfactants
`with high HLB numbers are shown in Figure 7. We fo-
`cused on this group of surfactants because our experience
`indicated that ionic surfactants or surfactants with very
`low HLB numbers sometimes accelerated denaturation or
`aggregation of antibody-based products. The three sur—
`factants shown in Figure 7 exhibited different degrees of
`surface activity, from polysorbate 80, which displayed a
`
`Monolayer
`
`
`
`
`
`Figure 4—Surface stabilization of an antibody during a shipping simu-
`Iation. The vial on the left contains MAb A formulated
`without polysorbate 80. The vial on the right contains the
`same antibody formulated with polysorbate 80. Both vials
`were photographed after a standard shipping simulation.
`
`
`
`Proteins and Surfactants
`in Air-Liquid Systems
`
`Figure 5——A schematic representation of the ability of surfactants to
`protect proteins from surface denaturation.
`
`Vol. 45, No. 3 / May—June 1991
`
`163
`
`EX. 1026 - Page 5 0f 7
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`
`
`Ex. 1026 - Page 5 of 7
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`

`

`
`
`
`
`
`
`0.1 at.
`
`3/0..
`
`/
`
`c,
`
`Polysorbate 80
`
`i
`
`
`
`(mN/m)
`
`—
`
`
`40 “r
`T
`r
`“
`0.001
`0.01
`0.1
`
`MAb A
`(%)
`
`
`
`1
`
`Polysorbate 80 Concentration (%)
`Figure 6—Stabilization of MAb A by polysorbate 80. The percent MAb A remaining in solution after a shipping simulation as a function at
`polysorbate 80 concentration is plotted (<> ) along with the surface-tension curve for polysorbate 80 (El). The solid line labelled a MAb A
`represents the surface tension of a 0.1% solution of MAb A without surfactant presen .
`'
`
`relatively high activity to BRIJ 700 which was nearly
`independent of concentration.
`A comparison of the effectiveness of these surfactants
`at stabilizing MAb A is shown in Table II. Clearly, the
`more surface active surfactants, Pluronic F68 and poly-
`
`sorbate 80, provided better surface stabilization than ‘
`BRIJ 700. Moreover, we have shown that common protein
`stabilizing agents such as dextrose, mannitol, and albu-
`min, which display little or no surface activity, provided
`no added surface stabilization for our protein products.
`
`60 .....
`
`
`
`- A
`
`
`
`A
`
`--
`
`3-
`
`
`Figure 7—Surface tension curve for three non-ionic surfactants: (El) Polysorbate 80, (+) Pluronic F-68, and (0) BRIJ 700.
`
`__________
`55-.
`v \
`-
`
`\
`
`- ~<>~~ ~ e
`
`,
`____ ~ '.-’ ‘BR_IJ 700 I;
`‘ ‘ ‘ ~
`
`.
`
`A
`_
`50-
`w
`-
`
`._
`-
`45 "
`
`O- (mN/m)
`
`\ +
`\\
`
`Pluronic F-68
`\
`
`\
`
`\
`
`+
`
`\
`
`\ +
`
`Polysorbate80
`
`D
`
`l
`I
`I
`i
`l
`l
`l
`l
`.
`I
`
`:
`i
`l
`
`
`
`
`
`
`
`i
`i
`
`r
`r
`~— 3~ J.
`0.01
`0.1
`1
`
`.4
`40+
`0.001
`
`Concentration (%)
`
`164
`
`Journal of Parenteral Science & Technology}
`
`Ex. 1026 - Page 6 of 7_,
`
`Ex. 1026 - Page 6 of 7
`
`

`

`
`
`
`
`
`
`
`
`
`
`35 —r-—--—-————-——-—--—r——-
`0.001
`0.01
`
`Concentration (°/o)
`
`Figure 8—Surface tension curves in antibody-product systems. The surface tension of various concentrations of MAb A is plotted for C“ values of
`O (+), 0.1 (0). 1(A), and 00 (E1).
`
`
`
`TABLE II. Surface Stabilization of MAb A: Effectiveness of
`
`Three Nonionic Surfactants
`
`MAb A Remaining (%[
`
`
`
`
` Surfactant a (mN/M) Case 1* Case II**
`
`Polysorbate 80
`Pluronic F-68
`
`BRIJ 700
`
`43.5
`49.8
`54.4
`
`95
`98
`70
`
`97
`99
`83
`
`* Case I: 0.1% MAb A (a = 58.4 mN/m) + 0.1% surfactant.
`*-‘ Case II: 0.01% MAb A (a = 67.9 mN/m) + 0.1% surfactant.
`
`Finally, the concept of multicomponent adsorption at
`
`
`
`blocking the interface and preventing protein adsorption
`at the surface.
`
`References
`
`1. W. Ramsden, “Separation of solids in the surface layers of solutions
`and suspensions,” Proc. Roy Soc. (London), Ser. B72, 156 (1903).
`2. T. L. Donaldson, E. F. Boonstra, and J. M. Hammond, “Kinetics of
`protein denaturation at gas-liquid interfaces,” J. Coll. Int. Sci., 74,
`443 (1980).
`3. F. MacRitchie, “Proteins at interfaces,” Adv. Prot. Chem., 32, 283
`(1978).
`4. K. J. Kaplan and M. J. Fraser, “Formation of fibres from protein
`monolayers,” Nature, 171, 559 (1953).
`5. C. W. N. Cumper and A. E. Alexander, “The surface chemistry of
`proteins,” Trans. Faraday Soc., 46, 235 (1950).
`6. A. F. Henson, J. R. Mitchell, and P. R. Musselwhite, “The surface
`coagulation of proteins during shaking,” J. Coll. Int. Sci., 32, 162
`(1970).
`7. Y. J. Wang and M. A. Hanson, “Parenteral formulations of proteins
`and peptides: Stability and stabilizers,” J. Parenter. Sci. Technol.,
`42, Suppl. ZS (1988).
`8. C. M. Nguyen and J. F. Scamehorn, “Thermodynamics of mixed
`monolayer formation at the air-water interface,” J. Coll. Int. Sci.,
`123, 238 (1988).
`9. A. W. Adamson, Physical Chemistry of Surfaces, 4th ed., Wiley,
`New York, 1983.
`10. J. Berg, unpublished notes (1980).
`11. K. Hamaguchi, “Studies on protein denaturation by surface chemi-
`cal method. I. The relationships between monolayer properties and
`urea denaturation of lysozyme,” J. Biochem., 42, 449 (1955).
`12. S. Hershenson, T. Stewart, C. Carroll, and Z. Shaked, “Formula-
`tion of recombinant interferon-B (Betaseron TM) using Laureth 12,
`a novel nonionic surfactant,” presented at the 186th Annual ACS
`Meeting, Los Angeles (1988).
`
`
`C* m Cp/Cs. The results shown in Figure 8 indicate that
`
`polysorbate 80, which was much more surface active than
`MAb A, dominated the surface tension behavior of the
`
`multicomponent system. Only at very low total antibody
`
`Concentrations with C* = 1 did the MAb A / polysorbate
`80 surface tension curve deviate from the polysorbate 80
`
`Curve. This type of behavior has been observed in binary
`
`surfactant systems (8) and suggests a protein contribution
`
`to the surface behavior of the system at low surfactant
`
`Concentrations and higher protein-to-surfactant ratios.
`
`These data provide more support for our hypothesis that
`the surfactant inhibits surface denaturation of proteins by
`
`
`
`VOL 45, No. 3/ May—June 1991
`
`165
`
`7
`
`Ex. 1026 - Page 7 of 7
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`Ex. 1026 - Page 7 of 7
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