Protein Denaturation by Combined Effectof Shear and Air-Liquid Interface

Yuh-Fun Maa and Chung C. Hsu

Department of Pharmaceutical Research and Development, Genentech, Inc.460 Point San Bruno Boulevard, South San Francisco, CA 94080;Telephone: 415-225-6349; fax: 415-225-3191; e-mail:maa.yuh-fun@gene.com

Received 27 March 1996; accepted 28 May 1996

Abstract: The effect of shear alone on the aggregation tem, it was concluded that neither high shear (.106)of recombinant human growth hormone (rhGH) and re- nor high shear rate (.105 sec21) alone had a significantcombinant human deoxyribonuclease (rhDNase) has effect on aggregation though it induced slight conforma-been found to be insignificant. This study focused

tional changes and clipping on rhGH (Maa and Hsu,on the synergetic effect of shear and gas-liquid inter-1996a). In this study, the combined effect of shear andface on these two model proteins. Two shearing systems,

the concentric-cylinder shear device (CCSD) and the air-liquid interface on proteins was investigated.rotor/stator homogenizer, were used to generate high Protein adsorption is a well-known phenomenon thatshear (.106) in aqueous solutions in the presence of affects a wide variety of processes involving two or moreair. High shear in the presence of an air-liquid inter- surfaces (Andrade, 1981, 1985; Horbett and Brash, 1986;face had no major effect on rhDNase but caused rhGH

Norde, 1986). The amphipathic nature of proteins, as ato form noncovalent aggregates. rhGH aggregation wasresult of a mixture of polar and nonpolar side chains,induced by the air-liquid interface and was found to in-

crease with increasing protein concentration and air-liq- causes protein molecules to be concentrated at inter-uid interfacial area. The aggregation was irreversible and faces (MacRichie, 1978). Protein denaturation at hy-exhibited a first-order kinetics with respect to the protein drophobic interfaces normally follows a mechanism byconcentration and the air-liquid interfacial area. Shear

which the protein molecule unfolds to expose its hy-and shear rate enhanced the interaction because of itsdrophobic regions to the interface, and then the un-continuous generation of new air-liquid interfaces. In the

presence of a surfactant, aggregation could be delayed folded molecule undergoes aggregation over its hy-or prevented depending upon the type and the concentra- drophobic regions. The aggregates eventually desorbtion of the surfactant. The effect of air-liquid interface on from the surface and, above a certain concentrationproteins at low shear was examined using a nitrogen

in the solution, precipitate (Cecil and Louis, 1970;bubbling method. We found that foaming is very detri-MacRichie, 1978; Thurow and Geisen, 1984). The inter-mental to rhGH even though the shear involved is low.

The use of anti-foaming materials could prevent rhGH action of proteins with solid surfaces has attracted moreaggregation during bubbling. The superior stability ex- attention than protein behavior at air-liquid interfaceshibited by rhDNase may be linked to the higher surface because of its importance in a number of fields, espe-tension and lower foaming tendency of its aqueous solu-

cially in blood-contacting medical devices (Andrade,tion. 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54:1981). Insulin denaturation at solid hydrophobic sur-503–512, 1997.

Keywords: aggregation by air-liquid Interface; foaming; faces is one of the most studied topics in this field (Bren-rhDNase; rhGH; shear; shear rate nan et al., 1985; Chawla et al., 1984; Thurow and

Geisen, 1984).As far as the air-liquid interface is concerned, it wasINTRODUCTION

reported that recombinant human growth hormoneProteins experience shear stress and are exposed to (rhGH) aggregates and precipitates upon shaking invarious interfaces during bioprocesses such as mixing, partially filled vials and that this phenomenon is inducedcentrifugation, filtration, and pumping. Since the air- by the air-liquid interface, not by the shear alone (Hagel-liquid interface is present in processes involving liquid ocher and Pearlman, 1989; Pikal et al., 1991). The obser-transfer and flow or in extreme cases such as spray vation that surface-induced rhGH aggregation could bedrying where large air-liquid interfacial areas are gener- depressed by adding a surfactant confirmed the ten-ated, it may present concerns to air-sensitive proteins. dency of rhGH molecules to be adsorbed to the air-In an earlier study where proteins were sheared in the liquid interface (Hagelocher and Pearlman, 1989). How-absence of an air-liquid interface using a concentric ever, these studies did not quantify either shear stresscylinder system and a rotor/stator homogenization sys- or air-liquid interfacial area.

The objective of this study was to investigate thecombined effect of shear and air-liquid interface basedCorrespondence to: Y.-F. Maa

1997 John Wiley & Sons, Inc. CCC 0006-3592/97/060503-10

on quantitative information. To generate high shear, to avoid any air bubbles being trapped between thecylinders. All experiments were conducted at ambienttwo concentric cylinder-based shear devices were used

(Maa and Hsu, 1996a). To assess the effect of the air- temperature.liquid interface under low shear, protein solution wasbubbled with nitrogen to cause foaming. The other

Rotor/Stator Homogenizermodel protein used in this study, rhDNase, was rela-tively stable at the air-liquid interface. The surface prop- A Virtishear homogenizer (Model Templest IQ, Virtis)erties of these two proteins were determined to find a consisting of a digital display microprocessor control,correlation with protein denaturation at the air-liquid an overhead drive, and a homogenizing shaft (1 cm)interface. was used. The shaft tip is a rotor/stator assembly capable

of generating fine dispersions. This assembly resemblesa concentric-cylinder shear device but is an open system.MATERIALS AND METHODSThe dimensions of the rotor and the stator are: the outercylinder R0 5 0.4 cm; for the inner cylinder Ri 5 0.35

Proteins cm; the height of the cylinders H 5 1.8 cm; the gapvolume 0.212 cm. When the rotor rotates, the assemblyRecombinant human growth hormone (rhGH) with adraws the liquid in from the bottom and sends the liquidmolecular weight of 22.13 kDa was produced at Genen-out through the four 0.4-cm circular holes equally spacedtech, Inc. from bacterial fermentation of a strain of Esch-above the stator for circulation. The rotational speederrichia. coli. The protein contained the same 191 aminocan be steadily controlled in the range of 5,000 andacid residues as the natural, pituitary-derived rhGH.25,000 rpm. The emulsion vessel is a jacketed glass con-For shearing experiments, rhGH solution was preparedtainer with an inner cylinder of 6 cm in height 3 2.5by dissolving previously lyophilized, excipient-free pro-cm in inner diameter and a septum-capped side-port.tein into 5 mM sodium phosphate buffer (pH 7.4). Re-The hold-up volume of the inner tube was approxi-combinant human deoxyribonuclease (rhDNase), amately 55 mL. All homogenization was conducted atsugar-containing protein with a molecular weight of20 8C. Temperature control was achieved by circulating32.74 kDa, was also produced at Genentech, Inc. usingthe jacket of the vessel with 50/50 water/ethylene glycolthe Chinese hamster ovary cell line. rhDNase solutionmixture using a Lauda circulator (Model RMS).was prepared by dissolving previously lyophilized, pure

protein into a solution containing 150 mM NaCl and 1mM CaCl2 (pH of 6.3). All the solutions were prefiltered Foaming Experimentwith a 0.22 mm filter.

Two types of foam generation apparatuses were usedand are shown in Figures 1a and 1b. In Figure 1a, ap-

Reagents proximately 20 mL of a protein solution was chargedto a jacketed emulsion vessel with a septum-cappedTwo surfactants were used in this study, Polysorbate 20side-port. Through a 20 gauge needle (Precision Glide,(or Tween 20) (MW 5 1,227 Da, Karlshamns) and aBecton Dickinson), nitrogen was sent into the vessel atcopolymer of ethylene glycol and propyl glycol (Pluro-a flow rate of 30–50 mL/min through a pressure gaugenic F88, BASF). Tween 20 was prepared into a 20%(Fuji Silysia). As nitrogen bubbles passed through theaqueous solution with purified water, and Pluronic F88liquid, the solution started foaming. When the foamwas dissolved in the protein solution by mild agitation.grew to fill the vessel, it migrated to a glass receivingPluronic L-61 (BASF) was used as an antifoaming agentvessel via a piece of silicone tubing (0.250 ID 3 29 Long)and was prepared into a 25% aqueous solution.and collapsed in a foam collector.

The apparatus in Figure 1b consisted of a foam pipetteShear Experiment and a foam receiver which are standard devices forConcentric Cylinder Shear Device (CCSD) determining foaming properties of surface-active agents

(Rose and Miles, 1953). The receiver was filled to theThis device was modified from a vortex flow filtration50-mL mark with the surfactant-containing solution.(VFF) system (Membrex) by removing the filter car-The pipette was filled to the 200 mL mark with solution.tridge to leave the inner cylinder as a solid rod. TheThe entire solution was then drained into the receiver atsystem consists of an electronic control unit and a rotarythe top and the foam height was read at 0 and 5 minutes.separation unit. The rotary unit is a closed system con-

taining two concentric cylinders with the gap volume ofapproximately 120 mL. The outer cylinder (L 3 ID 5 Surface Tension Measurement18 3 5 cm) remains stationary but the inner cylinder(L 3 ID 5 16 3 4 cm) can be rotated up to approxi- The surface tension of the protein solutions was deter-

mined using a tensiometer (Model 21, Fisher) equippedmately 2,000 rpm. Protein solution was loaded carefully

504 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 54, NO. 6, JUNE 20, 1997

Hepes, 150 mM NaCl and 1 mM CaCl2 at pH 7.0, wasalso pumped at a flow rate of 1 mL/min. The run timefor both systems was 15 minutes. Protein concentrationwas measured by optical absorption at 214 nm for rhGHand at 280 nm for rhDNase.

Scanning Microcalorimetry

Calorimetric measurements were carried out on a differ-ential scanning calorimeter (MC-2, MicroCal) equippedwith the standard DA-2 software package for data ac-quisition, analysis and deconvolution. The heart of thesystem is two side-by-side cells, sample and reference,adiabatically shielded in a reservoir. The temperatureof the reservoir was controlled by the recirculation ofa 50/50 water/ethylene glycol fluid from an externalbath. Using power feedback compensation, the enthalpychange involved in the sample due to the temperaturechange was measured in the sample cell, with the refer-ence cell providing a differential thermal standard. Allmeasurements in this study were performed using ascanning rate of 608C/hour. The working volume ofthe cells in this instrument was 1.22 mL. Sample andreference solutions were loaded into the cells by meansof a long needle.

Sodium Dodecyl Sulfate-Polyacrylamide GelElectrophoresis (SDS-PAGE)

To determine protein fragmentation and whether theprotein aggregates were covalently bound or non-cova-lently bound, silver-stained SDS-polyacrylamide gelelectrophoresis (SDS-PAGE, Hoefer Scientific Instru-ment) were performed on a Bio-Rad gel (4–15% gradi-ent, 0.375 M Tris-HCl, pH 8.8, 10 well comb) at 200 mVfor 40 min at the ambient temperature under both DL-

Figure 1. The schematic representation of a foam generation appara- dithiothreitol (DTT)-reduced and non-reduced condi-tus (a) and a foaming tendency measurement apparatus (b). Systemtions. Each sample was loaded at 1 mg per spot.(a) consists of a glass jacketed vessel with a septum-capped side port

for needle insertion, a nitrogen source, and a foam collector. System(b) consists of a foam receiver and a foam pipet.

RESULTS AND DISCUSSIONwith a 6 cm circumference platinum-iridium ring. Thereported value is an average of three measurements.

Effect of Air-Liquid Interface under High Shear

rhGH and rhDNase solutions were sheared in a CCSDSize Exclusion Chromatography–High with a fill volume of 80 mL (²⁄₃ of full volume) and inPerformance Chromatography (SEC-HPLC)

a rotor/stator homogenizer with a fill volume of 20 mL(approximately ¹⁄₃ of full volume). The shear generatedrhGH samples were diluted to 1 mg/mL using a placebo

buffer solution and 50 mL was injected into a silica- in these two systems was calculated based on the equa-tions previously established (Maa and Hsu, 1996a). Fig-based Tosoh TSK 2000SW XL column (7.8-mm I.D. 3

30-cm L; particle size, 5 mm). The mobile phase con- ure 2 shows the monomer content of these two proteinsas a function of shear with the inner cylinder rotatingsisting of 50 mM sodium phosphate and 0.15N NaCl at

pH 7.2 was pumped at a flow rate of 1 mL/min. For at 1,500 rpm for the CCSD (shear rate 5 691 sec21) andthe rotor spinning at 15,000 rpm for the homogenizerrhDNase, each sample was also diluted to 1 mg/mL with

a placebo solution and 100 mL was injected into the (shear rate 5 1.09 3 105 sec21). Protein solutions practi-cally turned into an air-in-water emulsion during ho-same column. The mobile phase, a mixture of 5 mM

MAA AND HSU: AIR-LIQUID INTERFACE EFFECT ON PROTEINS 505

Figure 2. The shear effect on rhGH solution (2 mg/mL) sheared in a CCSD (s) and a rotor/stator homogenizer (h) and on rhDNase solution(5 mg/mL) sheared in a CCSD (d) and a rotor/stator homogenizer (j) in the presence of air-liquid interface.

mogenization. During shearing in the CCSD, the solu- was required by the CCSD than by the homogenizer.Although the total air-liquid interfacial area generatedtion turned foamy and the foam filled up the headspace.

In both shearing systems, the sheared rhGH solutions in the two systems was difficult to estimate, the air-liquid interfacial area of the homogenized emulsion wasbecame cloudy and the degree of turbidity increased

with shearing time due to formation of protein aggre- larger than that of the foamy bubbles in the CCSDbecause the homogenizer generated much finer air bub-gates. In contrast, rhDNase solution remained clear, and

the protein did not aggregate even at shear as high as bles. Due to the random nature of foaming, the size andthe number of foamy bubbles generated in the CCSD2 3 107. To cause the same extent of rhGH aggregation,

much higher shear (at least one order of magnitude) could not be reproducibly controlled, which presented

Figure 3. The monomer content for rhGH (2 mg/mL) sheared in a rotor/stator homogenizer at as a function of shear at the shear rate of6.89 3 104 (h) 1.09 3 105 (e) and 1.55 3 105 sec21 (n) with air-liquid interface, and at 1.55 3 105 sec21 without air-liquid interface (m). Thelinear regressed equation for (h) is y 5 99.1 2 0.61x, R 5 0.99; for (e) is y 5 99.0 2 1.90x, R 5 1.0; for (n) is y 5 96.6 2 5.30x, R 5 0.95.

506 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 54, NO. 6, JUNE 20, 1997

a major disadvantage of the CCSD system. The foaming d[aggregate]/dteffect on proteins will be discussed further below.

5 k[protein monomer]a[air–liquid interface]b (1)Figure 3 shows the effect of both shear and shear

rate on rhGH aggregation in the presence of air-liquid where [protein monomer] is the monomer concentra-interface during shearing by homogenization at three tion and [air-liquid interface] the total interfacial area.shear rates, 6.89 3 104, 1.09 3 105, and 1.55 3 105 sec21, The total interfacial area was determined by two factors,corresponding to the homogenization intensities of the air bubble size and the total air volume in the emul-7,000, 15,000, and 24,000 rpm, respectively. It was ob- sion. In this study we estimated the air bubble size usingserved that rhGH aggregation increased with increasing the equation previously established (Maa and Hsu,shear rate. However, in the absence of air-liquid inter- 1996b) for correlating the relationship between the sizeface, rhGH practically remained intact even at the high- of liquid droplets (dp) in a liquid-liquid emulsion andest shear rate, suggesting the critical role of the air- the homogenization intensity (v), dp Y v21.14. Using thisliquid interface. In principle, the air-liquid interfacial relationship, the ratio of dp at 24, 15, and 7 krpm wasarea increases with decreasing emulsion droplet size if calculated to be 1 : 2.38 : 4.07. The air volume in thethe air volume is constant. The increase in homogeniza- emulsion was determined using the following procedure.tion intensity results in smaller emulsion droplets and During homogenization at a specific rotational speed,in turn produces a greater air-liquid interfacial area. 1 mL of the emulsion was drawn from the side port ofThis again suggests that rhGH aggregation was induced the emulsification vessel using a 1-cc syringe. The air-by the air-liquid interface not by shear or shear rate in-liquid emulsion in the syringe was allowed for phasealone. This surface phenomenon can be envisioned as separation and the air volume (V) was obtained. Thean irreversible reaction: ratio of V at 24, 15, and 7 krpm was determined to be

1 : 1.5 : 2. Using this approach, in an emulsion containingSurface-sensitive protein 1 air–liquid interfaceair bubbles with a diameter of dp and a total air(reactant A) (reactant B)volume of V, the total air-water interfacial area can becalculated to be 6V/dp . Therefore, the ratio of the air-UUU➛

Shear

Shear rateAggregates

liquid interfacial area generated at 7, 15, and 24 krpm wasdetermined to be 1 : 3.6 : 8.1. The rate of rhGH mono-where the surface sensitive protein and the air-liquidmer disappearance was obtained from the slope ofinterface are the two reactants, and shear and shear ratelinear regressed lines in Figure 3 corresponding to theseare the promoting factors which enhance the air-liquidthree rotational speeds and their ratio was calculatedinterface renewal rate. The aggregation rate could be

expressed as: to be 1 : 3.1 : 8.7. The aggregation ratio matched well

Figure 4. The rhGH concentration effect on aggregation in the concentration of 0.5 (e), 2 (h) mg/mL sheared in a rotor/stator homogenizerat 24,000 rpm in the presence of air-liquid interface. The linear regressed equation for (e) is y 5 8.86 2 0.41x, R 5 0.96; for (h) is y 5

36.6 2 1.51x, R 5 0.94; for (s) is y 5 81.0 2 3.24x, R 5 0.99.

MAA AND HSU: AIR-LIQUID INTERFACE EFFECT ON PROTEINS 507

Table I. Foaming tendency and foam retention determined using awith the interfacial area ratio, suggesting possibly a firststandard ASTM method (D 1173-53).order reaction with respect to the interfacial area, i.e.

b 5 1 in Eq. (1). The effect of protein concentration Height at T 5 0 Height at T 5 5 minSample mM (cm) (cm)on the interfacial aggregation rate was investigated by

homogenizing rhGH solutions of three concentrations,rhGH 0.225 9.5 9.0

0.5, 2, and 5 mg/mL, at 24,000 rpm (shear rate 5 rhDNase 0.273 0.8 0.51.55 3 105 sec21). The data are shown in Figure 4. Log Tween 20 0.082 5.0 4.5

Tween 20 0.244 5.8 5.3[monomer] decreased linearly with time regardless ofTween 20 0.815 6.5 6.2the starting concentration, suggesting that the proteinTween 20 4.075 10.5 9.5concentration affects the aggregation rate also by theTween 20 8.150 12.5 12.0

first order, i.e. a 5 1 in Eq. (1).

Effect of the Surfactant and 1 mg/mL) sheared in a CCSD at 1,500 rpm. Thedata indicate that both surfactants significantly reducedProtein molecules are surface sensitive because of theirthe aggregation rate and that Pluronic F88 is more effec-amphipathic nature. The generally accepted mechanismtive than Tween 20 in protecting rhGH from aggregationfor rhGH aggregation at the air-liquid interface is pro-at the air-liquid interface.tein unfolding at the interface, which exposes the hy-

drophobic patches of the protein, facilitating protein- Surface Properties of rhGH and rhDNaseprotein interactions. If another surface-active materialsuch as a surfactant were present to compete with the Since rhGH is relatively unstable at the air-liquid inter-

face compared to rhDNase as shown in Figure 2, weprotein and occupy the interface, there would be lessinterface available for the protein, thereby reducing pro- decided to determine the air-liquid interfacial properties

of the two proteins. It is interesting to find that these twotein aggregation. Katakam et al. (1995) investigated theeffect of surfactants on the physical stability of rhGH protein solutions have very different foaming properties

based on the two methods illustrated by Figures 1a andusing three different non-ionic surfactants and foundthat surfactants can prevent interfacially-induced aggre- 1b. When the protein solution was slowly bubbled with

nitrogen at a flow rate of 30 mL/min, using the devicegation at or above their critical micelle concentration.Two surfactants, polysorbate 20 and Pluronic F88, shown in Figure 1a, rhGH solution foamed so exces-

sively that the foam grew and migrated via silicone tub-were tested in this study. Figure 5 shows the time courseof decrease in monomer content of rhGH solution con- ing into a foam collector where the foam bubbles col-

lapsed. This process resulted in liquid transportation.taining Tween 20 (0.1 mg/mL) and Pluronic F88 (0.1

Figure 5. The protective effect of the surfactant on rhGH aggregation. 2mg/mL of rhGH solution (s), the same solution containing 1 mg/mL of Pluronic F88 (h), 0.1 mg/mL of Pluronic F88 (e), and 0.1 mg/mL of Tween (n). All solutions were sheared in a CCSD at 1500 rpm.

508 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 54, NO. 6, JUNE 20, 1997

Figure 6. Surface tension of rhGH (s), rhDNase (h), and Tween 20 (n) solutions over a range of concentrations.

However, rhDNase solution foamed only slightly re- surface tension (Myers, 1988). The surface tension ofthe rhGH and rhDNase solutions at different proteingardless of the rate of bubbling, suggesting that rhGH

has a better foaming tendency than rhDNase. This was concentrations were determined using a tensiometer.The results are summarized in Figure 6. The surfaceconfirmed by an ASTM method (Figure 1b) (Rose and

Miles, 1953). After the solution in the pipette (located tension for rhGH decreased quickly from 74 dyne/cm(protein free) to approximately 50 dyne/cm for rhGHat the top of the foam receiver) was completely drained,

the height of the foam generated in the receiver was as low as 0.04 mM and remained unchanged over a widerange of protein concentration. For rhDNase, its surfacemeasured immediately and 5-minute after draining. Ta-

ble 1 summarizes the results for the solutions of rhGH tension decreased to around 68 dyne/cm over the con-centration ranging from 0.045 to 0.136 mM. Tween 20(0.225 mM), rhDNase (0.273 mM), and Tween 20 (0.2 to

8 mM). Our quantitation demonstrated rhGH’s foaming behaved like rhGH but the surface tension decreasedeven lower, 35 dyne/cm. The result further suggests thattendency is an order of magnitude greater than

rhDNase’s. In addition, the rhGH foam was stable after rhGH is more surface active than rhDNase.Table II summarizes the aggregation data for rhGH5 minutes whereas rhDNase’s collapsed. The foam

height for Tween 20 increased with concentration. and rhDNase after N2 bubbling with or without a surfac-tant or an antifoaming agent. Under N2 bubbling atTween 20 had the same foaming tendency as rhGH at

a concentration between 0.8 and 4 mM, suggesting that 30–50 mL/min for 45 minutes, rhGH solution migratedto the foam collector (Figure 1a) due to foaming. TherhGH had a higher foaming tendency than Tween 20

at the same molar concentration. solution in the foam collector was cloudy and containedmore soluble aggregates (68.5%) than the bubbled solu-One of the major characteristics of a surfactant to act

as a foaming agent is its effectiveness in reducing the tion (2.8%) which remained clear. The decrease in rhGH

Table II. Soluble aggregation of rhGH (0.09 mM) and rhDNase (0.273 mM) solutions after N2 bubbling at an air flow rate of 30–50 mL/min.

Surfactant Antifoaming Bubbling time Soluble aggregation Soluble aggregationProtein added added (min) in solution (%) in foam (%)

rhGH no no 45 2.8 68.5rhGH Tween 20 no 45 2.2 6.8rhGH no Pluronica (2) 45 0.2 no foamrhDNase no no 45 0 no foamrhDNase no no 4560 0 no foam

aPluronic L-61.

MAA AND HSU: AIR-LIQUID INTERFACE EFFECT ON PROTEINS 509

concentration due to insoluble aggregation was deter- wheremined to be approximately 15% for the foamed solution.

Fb 5 prwD3g/6 (2b)For the bubbled solution, rhGH concentration de-creased from 2 mg/mL to 1.5 mg/mL as determined Fg 5 praD3g/6 (2c)by UV at 280 nm. This decrease was due to rhGH

Ff 5 CDrwV2yA/2 (2d)concentrating in the film of the foam and subsequently

transferring to the collector. In the presence of 2 mg/ and rw and ra were the densities of water and air, gmL of Tween 20, the solution on the foam side was clear was the acceleration due to gravity, CD was the dragand the amount of soluble aggregates was significantly coefficient which could be expressed as 0.4 1 40Re21

reduced. In the presence of an anti-foaming agent, Plur- in the semi-turbulent flow region (Norde, 1986), andonic L-61, neither significant foaming nor aggregation A 5 the projected area of the bubble in a plane 908 towas observed in the bubbled solution. N2 bubbling of the bubble motion 5 pD2/4. Based on Eqs. 2a–d, therhDNase solution generated no foam and had no effect terminal velocity was expressed ason rhDNase aggregation even after 76 hours bubbling.

V2y 5 4gD(rw 2 ra)/[3rw(0.4 1 40/Re)] (3)This experiment further demonstrated that rhDNase

was more stable than rhGH at the air-liquid interface For rw 5 0.9982 g/mL and ra 5 0.001204 g/mL at 208Cand that foaming would be very detrimental to air-liquid and 760 mm Hg, g 5 980 cm/sec2, and D 5 0.5 cminterface-sensitive proteins. The effect of this shear asso- (estimated), Re 5 VyraD/ma , and ma 5 184 3 1026 g/ciated bubbling process was estimated using a simplified cm/sec, Vy was calculated to be 9.8 cm/sec. At thismodel where a rigid spherical particle of a diameter D velocity, the Reynolds number was calculated to be 32moved up against friction at a terminal velocity (Vy) in and was confirmed to be in the semi-turbulent zone.a semi-turbulent flow region, i.e. 0.2 , Reynolds number The shear rate, g, over the surface of the air bubble, 1000 (Bird et al., 1960; Masters, 1991; Stokes, 1850). was assumed to arise from the frictional force and isThe major assumption was to neglect the effect of defor- defined as Ff/A 5 makgl. Eq. (2d) was rearranged tomation which an air bubble of the same shape and size obtain kgl:might be subjected to during bubbling (Coulson and

kgl 5 CDrwV2y/(mw) (4)Richardson, 1968). In this model, the sum of all the

forces acting on the bubble was zero. The buoyancyTherefore, kgl was calculated to be 800 sec21 using mw 5

force (Fb) was offset by the force of gravity (Fg) and0.01 g/cm/sec. After the bubble travelled a distance

the frictional force by the liquid (Ff):of 5 cm (Q P 0.5 sec), the averaged shear, kgQl, wascalculated to be 400. The shear rate associated with theFb 5 Fg 1 Ff (2a)

Figure 7. SM thermograms for rhDNase (5 mg/mL) before ( ) and after (----) shearing in a CCSD for 16 hours in the presence of air-liquid interface.

510 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 54, NO. 6, JUNE 20, 1997

transportation of the bubble via the tubing into the foam ence on rhDNase conformation. For rhGH, it is difficultto evaluate the effect of air-liquid interface using the SMcollector was presumably much smaller than that during

bubbling, but the time the protein exposed to the inter- technique because the protein was highly aggregated. Inthe presence of Tween 20, the SM thermogram for rhGHface was much longer in the tubing than during bubbling.

Therefore, shear plays a very minor role and rhGH sheared with the air-liquid interface was identical tothat for rhGH sheared without the air-liquid interfaceaggregation is mostly due to air-liquid interfacial inter-

actions. (data not shown), probably because rhGH had littlechance to be exposed to the interface.

Effect on Protein ConformationSDS-PAGE

We employed scanning microcalorimetry to examineFigure 8 shows the silver-stained SDS-PAGE for rhGHany conformational changes due to air-liquid interfacialbefore and after shearing in the homogenizer for 10interaction. In an earlier study (Maa and Hsu, 1996a),minutes at 24,000 rpm under both the reducing and non-scanning microcalorimetry was used to provide informa-reducing conditions. It is worth noting that Lanes 2 andtion on protein structural changes with respect to4 show a similar band pattern in the high molecularchanges in thermal properties. Since the protein mole-weight region, suggesting that the aggregates in Samplecule is composed of several folded domains, its scanning4 (shear in the presence of air-liquid interface) werecalorimetry data is influenced by domain-domain inter-mainly non-covalently bound in nature. Low molecular-actions. The understanding of these interacting domainsweight fragments (around 14,000 daltons and lower)relies on theoretical models, which makes data interpre-were observed in Lanes 3 and 5 for both non-reducingtation subjective. To avoid this complexity, here, weand reducing gels, particularly in the latter, suggestingfocused only on the protein’s denaturation temperaturebackbone clipping due to shear (Maa and Hsu, 1996a).and the shape of the transition peak. If protein mole-rhGH sheared in the presence of air-liquid interfacecules unfold or experience conformational changes from(Lanes 4 and 4R) was not clipped probably because thetheir native states in the presence of a destabilizer, weaggregated form was less susceptible to shear force thanmight observe that the denaturation temperature down-the monomeric rhGH in the solution.shifts or the shape of the transition peak changes. We

used these changes to determine the combined influenceof shear and air-liquid interface on protein conforma- CONCLUSIONStions. Figure 7 shows that shearing in the CCSD at 1,500

We have examined the effect of air-liquid interface onrpm in the presence of air-liquid interface did not shiftthe stability of two model proteins, rhGH and rhDNase.the SM thermogram peak temperature. A slight differ-rhGH denatured at the air-liquid interface, especiallyence in peak splitting was the same as what was observedunder high shear whereas rhDNase was relatively stable.earlier (Maa and Hsu, 1996a) when no air-liquid inter-This was due to the fact that rhGH was more prone toface was involved. This suggests that the combined effectbe adsorbed to the air-liquid interface than rhDNase asof shear and air-liquid interface has no significant influ-exhibited by lower surface tension and higher foamingtendency. Also, rhGH aggregated, particularly at highprotein concentrations and a large air-liquid interfacialarea. Shear and shear rate enhanced the interaction ratebecause they increased the air-liquid interface renewalrate. Foaming was very detrimental to surface activeproteins even though the shear involved was low. There-fore, precautions to prevent foaming from occurringshould be taken during protein processing. rhGH aggre-gation could be prevented by the presence of a surfac-tant or an anti-foaming agent.

The authors are grateful to Dr. Tue Nguyen for projectsupport and to Ms. Phuong-Anh Nguyen for SDS-PAGEanalysis.Figure 8. Silver-stained SDS-PAGE for rhGH before and after

shearing in the homogenizer at 24,000 rpm for 10 minutes. Lane 1 isthe molecular weight marker. Lane 2 is for rhGH before shearing.

ReferencesLances 3 and 4 are for rhGH sheared in the absence and in thepresence of air-liquid interface, respectively. Lane 5 is for 1% Tween20-containing rhGH sheared in the presence of air-liquid interface. Andrade, J. D. 1982. In Interaction of the Blood with Natural and

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