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22 BioPharm MAY 2002
Process Development
Freezing Biopharmaceuticals Using Common Techniques — and theMagnitude of Bulk-Scale Freeze-Concentration
Large-scale freezing and thawingis commonly used inbiopharmaceutical manufacturingbut is not well understood.Freeze–thaw variations can existwithin or between batches, andnonuniform processes raiseserious validation concerns.
Freezing is a processing step used bymost biopharmaceutical companies.Yet little attention is generally given tothis important step, even thoughreports suggest that processes for
freezing protein solutions require detailedattention (1).
Freezing Processes ReviewedFrozen storage of drug products is generallypreferred over liquid storage for severalreasons including increased product stabilityand shelf life, decreased microbial growth,and elimination of foaming during transport.Chemical reaction rates are typically slowedat low temperatures, and immobilizing amacromolecule in the glassy state can slowits physical degradation from aggregation (2).In addition, freezing is often necessary andpractical during batch processing. Freezingcan be used to lyse cells, to store final bulkdrug substances before fill-and-finishoperations, and to introduce a hold step forpooling batches of intermediates. Improvingoperational logistics to maximize throughputis critical (3). Freezing an intermediateenables decoupling of processes such asfiltration and column purification, allowingfor more flexibility in production. Manybiopharmaceutical products are lyophilized,and freezing is the first step of that process.With freezing in widespread use,understanding the process is essential.
Historically, freezing was considered aconservative product storage method.Although freezing is often the safest andmost reliable method of product storage, ithas inherent risks. Freezing can inducestress in proteins through cold denaturation,by introducing ice–liquid interfaces (4–6)(7,8) and by the freeze-concentration(cryoconcentration) of solutes when thewater crystallizes (9). This stress can alsolead to pH shifts if the buffer salts crystallize(10,11). Freeze-concentration can cause
Serena Donnelly Webb,Jonathan N. Webb, Timothy G. Hughes, David F. Sesin, and Aimee C. Kincaid
Serena Donnelly Webb and Jonathan N. Webbwere senior research scientists at IntegratedBiosystems, Inc., when this article was written.David F. Sesin was director, and Aimee C. Kincaid was a research engineer in theapplications science department, and correspondingauthor Timothy G. Hughes is vice president ofbusiness development and technical and professionalservices (TAPS) at Integrated Biosystems, Inc., 445Devlin Road, Napa, CA 94558, 707.226.9300,fax 707.226.9303, [email protected],www.integratedbio.com.
phase separation of excipients and loss ofnative protein structure on subsequentdrying (12–14). Minimizing these stressesshould be the primary objective whenfreezing any biopharmaceuticalintermediate, bulk drug substance, or finalformulation. Two types of freeze-concentration can be defined: amorphousphase and bulk-scale or progressiveconcentration.
Amorphous phase freeze-concentration. Freezingcauses unavoidable dehydration of theamorphous phase when water moleculescrystallize as ice. Fewer liquid watermolecules present increase the concentrationof solutes locally, regardless of the methodof freezing. This phase separation of water,occurring at a microscopic scale, is calledamorphous phase freeze-concentration.Although concentrations reached during theamorphous phase can stress biologicalmacromolecules, the kinetics of the processare relatively rapid (fusion typically occursin minutes). If necessary, producttemperatures can be lowered below glasstransition temperatures or to temperatureswhere undesirable molecular reactions (suchas oxidation, deamidation, and aggregation)are minimized.
Bulk-scale or progressive freeze-concentrationoccurs on a macroscopic scale (15).Depending on conditions during freezing,pure ice crystals that form on cooled surfacescan grow slowly into the solution, increasingthe concentration of any solutes in theremaining liquid phase. Progressive freeze-concentration is exacerbated in containerswith poor heat transfer, and the resultingmacromolecular stress can persist for hoursor days.
Freezing conditions. The extent of freeze-concentration is affected by freezingconditions: The freeze-concentration ofsolutes (or conversely, solute moleculeentrapment in the growing ice front) is
24 BioPharm MAY 2002
Process Development
strongly influenced by mixing and the icefront’s rate of advance (16,17). Slow advanceof the ice front and rapid mixing near the icefront enhance progressive freeze-concentration (18). Therefore, to uniformlyentrap solute molecules and to preventprolonged freeze-concentration stress in abiopharmaceutical solution, it is critical tomaintain rapidly advancing ice fronts and toavoid mixing the solution during the fusionprocess.
Storage and thawing. After freezing, productsare stored in different types of freezer forvarying lengths of time (days, weeks, oryears). When material is needed for furtherprocessing, the containers are thawed attemperatures ranging from refrigeratedconditions (2–8 °C) to as high as 30 °C.Although thawing is commonly overlookedin process designs, at least one exampleshowed that a slow thaw in sodiumphosphate buffer can cause detrimentaleffects on a model protein, -galactosidase(19). During a thaw, conditions would besimilar to those present at the end of thefreeze because bulk-scale freeze-concentration is maintained until all watermolecules are released from the ice matrixinto the amorphous phase. Mixing duringthe thaw will minimize effects of any freezeconcentration that occurred during thefreeze.
Biopharmaceutical MethodsFreezing and thawing methods differ amongbiopharmaceuticals companies and products.Furthermore, the processes themselves are notwell characterized, and little relevant scale-down testing is practiced. Product containersrange from tiny plastic tubes to large bottles,bags, or carboys, and each may hold volumesfrom a few milliliters to a maximum of about20 liters. Freezing is commonly carried out inupright or chest freezers available inlaboratory and process development areaswith set-point temperatures ranging from 20 °C to 80 °C.
Other freezing methods, such as manualplacement of product containers into liquidnitrogen or dry ice/alcohol baths, can belabor-intensive and create logistical,handling, and safety issues during largebatch processing.
Liquid nitrogen systems and cold bath submersions.One system for freezing involved rotation ofstainless steel cans filled with product in
chambers cooled by liquid nitrogen (20).This process reportedly increasedaggregation for a recombinant 20-kD proteinwhen compared with IBI controlled freezing(20). Aggregation may be the result ofextensive exposure to air–liquid interfacesor to increased solute concentrations, both ofwhich would be intensified by motionduring fusion.
To prevent extreme variations in freezingtimes and the effects of load on freezers,manual placement of containers into liquidnitrogen or dry ice/alcohol baths beforeloading into a freezer might sound like agood idea, at least for small batches ofproduct. However, freezing by submersionin liquid nitrogen followed by placement ina 40 °C freezer resulted in about a30–70% decrease in activity for lactatedehydrogenase and about a 60% decrease inactivity of -galactosidase compared withless rapid freezing methods (1). Further,submersion methods have been associatedwith significant increases in soluble andinsoluble aggregates (21,22).
Several possible mechanisms may accountfor degradation when freezing bysubmersion. Placing containers in liquidnitrogen has been shown to increase icesurface area (23), and denaturing of nativeprotein structure can be ascribed to unfoldingon ice surfaces observed usingphosphorescence techniques (6). Fast coolingcan trap air that is then released duringthawing to denature proteins at the air–liquidinterface (24,25). Other drawbacks includeenvironmental, containment, and safety issuesthat result from working with large quantitiesof liquid nitrogen, dry ice, or solvents.
Container BSAa Sodium for Freezing (mg/mL) Citrateb(mM)
1-L bottles 11.8 1571-mL Eppendorf tube 1.52-mL sample vial 1.115-mL centrifuge tube 1.3
50-mL centrifuge tube 1.2Wedge (normal fast) 1.3 17
Wedge (slow) 4.4 59
aInitial concentration was 1.0 mg/mLbInitial concentration was 10 mM
Table 1. Maximum concentrations of BSA andsodium citrate measured in bottles, smallcontainers, and the wedge (at both normalfast freeze, 4.3 h, and slow freeze, 13 h)
Sampling BSAb Sodium Depth (cm)a (mg/mL) Citratec (mM)
5 1.2 174 1.3 163 1.2 142 1.1 131 1.0 11
aSampling depth refers to the distance of thesample from the surface of the ice.
bInitial concentration was 1.0 mg/mL cInitial concentration was 10 mM
Table 2. Concentrations of BSA and sodiumcitrate at various depths at the last point tofreeze in the CryoWedge 20
Figure 1. The CryoWedge models a slice ofthe CryoVessel, keeping all heat transfersurfaces the same as in the vessel. It canbe used in scale-down studies tosuccessfully model thermal behavior oflarge volumes.
Figure 2. CryoWedge 20 after sampling iscomplete; the area displaying the onlysignificant increase in concentration (thelast point to freeze) is highlighted in red.Samples were removed at multiple depthsperpendicular to the ice surface (1-cmincrements). Arrowed lines represent thecharacteristic length (from active heatsurfaces to center point, 5.75 cm) for icefront velocity calculations.
Process Development
Freezing in plastic bags. Plastic bags are usedoccasionally for product freezing, althoughproblems exist with the bags as currentlyconfigured. At low temperatures, someplastics become brittle, leading to bagbreakage and product loss. Thin plasticlayers provide no insulation from sub-zerotemperatures, creating handling difficulties.Volume expansion of the ice causessignificant mechanical stress. Bags do offersome advantages over other rigid containers,such as off-the-shelf sterility and asepticconnections. For a given volume, bags cansignificantly reduce resistance to heattransfer and freeze distances. However,uncontrolled freezing of multiple bags cancreate variations in freezing patterns andsubsequent concentration gradients similarto that observed in bottles.
Thawing. Containers are thawed incontrolled conditions (from 2–8 °C to ashigh as 30 °C) and in uncontrolledconditions, such as at room temperature.Mixing during the thaw is normallydiscouraged because if done improperly, itcan introduce excessive air–liquidinterfaces. When done properly, mixingduring thawing ensures producthomogeneity and minimizes bulk-scalefreeze-concentration effects. Thawcompletion is determined visually.
Test ParametersConsidering the variety of methods used forfreezing and thawing, an exhaustive study ofeach method’s effects on proteins would beimpractical. Therefore, we studied a fewcommon methods by varying container size,freezer temperature, and freezer load. Wecharacterize the freezing process by totalfreezing time, the extent of concentrationgradients in the frozen product, and totalthawing time. We compared those resultswith those achieved using a wedge(CryoWedge 20 from IntegratedBiosystems, Inc., www.integratedbio.com).
The wedge uses controlled, rapid rates offreezing and thawing and is a scale-downtool for the company’s CryoVessel productline (26–28). Figure 1 illustrates the vessel’sdesign, which increases the heat transfersurface area and reduces the characteristicfreeze distances, allowing rapid temperaturechanges in large volumes of solution. Activeheat transfer surfaces include the externaljacket and core heat exchanger. Stainlesssteel fins, that subdivide the maincompartment into equal sections or wedges,increase the surface area for heat transfer.All sections are geometrically identical andhave similar thermal environments. Thedesign effectively scales down large-volumethermal behavior by freezing a “slice” ofproduct in the wedge.
Materials and MethodsThe biomolecular solution. Bovine serum albumin(BSA) fraction V (Fisher Scientific,www.fishersci.com) was dissolved in 18 MΩ deionized water at a concentration of1 mg/mL in 10 mM sodium citrate buffer(sodium citrate and citric acid anhydrous,Fisher Scientific) at pH 5.7. BSA was
Freeze Ice FrontContainer Conditions Velocity (mm/h)
Wedgea normal (4.3 h) 3013-h extended 4
1-L bottles fastest (12 h) 7slowest (34 h) 3
20-L carboys fastest (42 h) 5slowest (70 h) 3
aCryoWedge 20
Table 5. Average ice front velocities for threecontainer types
Left Middle Right
Front 17.5 22.0 17.7Middle 23.4 27.9 23.6Back 15.7 20.2 15.8
aEach data point is an average of threebottles in the array stacked on top of eachother (similar to viewing the freezer from thetop down); statistical significance wasdetermined by difference in means (0.05,pooled standard deviation is 1.7 hours).
Table 4. Average time to freeze (in hours)based on horizontal position of 1-L bottlesin the 20 °C freezera
Figure 3. Contour plots of (a) BSAconcentrations (mg/mL) and (b) sodiumcitrate concentrations (mM) measured inthe 1-L bottle that was the last to freeze inthe 333 array in the 80 °C freezer.Each intersection of the grid in the plotrepresents a data point (a concentrationmeasurement).
0.5 1.5 2.5 3.5 4.5
Bottle Diameter (cm; left to right)
Bot
tle H
eigh
t (cm
)B
ottle
Hei
ght (
cm)
5.5 6.5 7.5 8.5
0.5 1.5 2.5 3.5 4.5
Bottle Diameter (cm; left to right)
5.5 6.5 7.5 8.5
7.70-8.407.00-7.706.30-7.005.60-6.30
2.10-2.801.40-2.100.70-1.400.00-0.70
117-126108-11799-10890-9981-90
27-3618-279-180-9
15.0
13.5
12.0
10.4
8.8
7.2
5.7
4.0
2.5
1.1
0.5
15.0
13.5
12.0
10.4
8.8
7.2
5.7
4.0
2.5
1.1
0.5
(a)
(b)
4.90-5.604.20-4.903.50-4.202.80-3.50
72-8163-7254-6345-5436-45
Containers Time to Freeze (h) Time to Thaw (h)for Freezing 20 °C 50 °C 80 °C 22 °C 25 °C 2–8 °C
1-L bottles 13–34a —b 12–25a 12–24a —b 48–120a
Wedge —b 4.3 —b —b 1.5 —b
aTime is the range measured for 1-L bottles. bNot determined cError 0.04 h for tfreeze and 0.02 h for tthaw (one standard deviation).
Table 3. Time required for freezing and thawing 1-L bottles and CryoWedge 20
26 BioPharm MAY 2002
28 BioPharm MAY 2002
Process Development
(Beverage-Air, www.beverage-air.com) oran 80 °C upright freezer (RevcoTechnologies, www.revco-sci.com).
The freezing. Bottles were arranged in thefreezers in either a 333 array (27 bottles)or as part of a full load (a freezer completelyfilled with solution containers). Carboyswere loaded into freezers individually or aspart of a full load. Protein solution (350 mL)was added to the CryoWedge, and thestandard freeze–thaw profile was initiated tocomplete the freeze within 4.3 0.04 hours,one standard deviation.
The sampling. Three additional wedge runswere completed with sampling limited to alongitudinal slice through the center point. Totest our hypothesis that ice front velocityinfluences the extent of bulk-scale freeze-concentration, another run analyzed the entirewedge after an extended freeze (13 hours).Temperatures in the wedges, bottles, carboys,and freezers were recorded using type-Tthermocouples. Samples (about one mL each)were cored or cut from the frozen material,melted, and diluted before testing. The entirefrozen mass was cut, melted, and analyzedfrom containers 50 mL or smaller.
Protein concentrations were analyzed usingan ultraviolet (UV) Genesys 5 spectrometer(Thermo Spectronic, www.thermo.com) at280 nm with correction for light scattering at320 nm. Sodium citrate concentrations weremeasured by conductivity using a Model 20Accumet pH/conductivity meter (DenverInstrument Company, www.denverinstrument.com).
selected for this study because it is amedium-sized protein (66 kD) that shouldexperience close-to-average mass transferbehavior (molecular weight is inverselyproportional to the diffusion coefficient).Sodium citrate was used because itmaintains a relatively constant pH duringfreezing (10,22).
The containers. One-liter HDPE bottles and20-liter HPDE carboys (Fisher Scientific)and a variety of smaller containers(centrifuge tubes from one to 50 mL) werefilled with the buffered protein solution.Bottles and carboys were loaded into eithera 20 °C upright, E-series freezer
We extensively sampled two bottlesfrozen in the 20 °C freezer (n73 andn144) along vertical planes through thecenter of each and on a single bottle(n154) from the 80 °C freezer. Sampleswere also removed at locations 90° from theplanar section (isotropic determination) andfrom bottle tops (just beneath the screwcap). The wedge was extensively sampled(n90, sample size about 1 mL) after thestandard freezing protocol was completed.Each hole shown in Figure 2 was sampled atmultiple depths (1 cm, 2 cm, 3 cm, and soon) so that the concentrations of salt andprotein could be mapped from the bottom tothe top of the wedge.
The calculations. Mass balances werecalculated (for both protein and salt) withthe data from the bottles and wedges toverify the accuracy of the methods. Resultsfrom extensively sampled bottles showedthat solute concentrations were greatest nearbottom-center in all three bottles, so eachremaining bottle (n32) was sampled at 13 locations near bottom-center.
Only samples removed from solid phaseafter freezing were analyzed. Sampling fromthe liquid phase during the freezing processwas not performed because that couldintroduce bias by measuring solutes thatconcentrate in the amorphous phase (if icelocalized in the sampling zone wasexcluded). For instance, sampling through anarrow tip (such as with a syringe or pipette)can filter out ice crystals.
Figure 4. Contour plots of (a) BSAconcentrations (mg/mL) and (b) sodiumcitrate concentrations (mM) measured inthe 1-L bottle that was the last to freeze inthe 333 array in the 20 °C freezer.
Our study suggests that controlling theimpact of cold denaturation, ice surfacearea, and progressive freeze-concentration in bulk processing canminimize the stresses that proteinsexperience during freezing and thawing.
Cold DenaturationLow temperature can cause spontaneousunfolding of proteins (7). Refolding andaggregation pathways compete forunfolded protein molecules. Slow freezingof solutions can lead to elevatedconcentrations of macromoleculesinteracting over extended periods of time,increasing the likelihood that an unfoldedmolecule will aggregate.
Ice Surface AreaProteins can denature near ice surfaces(6). Limiting nucleation and maximizingice crystal growth during freezing shouldminimize the ice surface area per volumeof ice formed. Submersion methodsgenerate large amounts of ice surfacearea and should be avoided (23,25).
Progressive Freeze-ConcentrationsBulk-scale freeze-concentration ofsolutes has been identified as a serious,yet avoidable, problem associated withfreezing. Bulk-scale freeze-concentrationis minimized by increasing heat transfersurface area, shortening characteristicfreeze distances, and increasing the rateof the ice front’s advance.
Minimizing Protein Stress
0.5 2.5 4.5Bottle Diameter (cm; left to right)
Bot
tle H
eigh
t (cm
)B
ottle
Hei
ght (
cm)
6.5 8.5
0.5 2.5 4.5
Bottle Diameter (cm; left to right)
6.5 8.5
4.50-4.804.20-4.503.90-4.203.60-3.903.30-3.603.00-3.30
0.90-1.200.60-0.900.30-0.600.00-0.30
54.00-60.0048.00-54.0042.00-48.00
18.00-24.0012.00-18.006.00-12.00
15.0
13.5
12.0
10.4
8.8
7.2
5.7
4.0
2.5
1.1
15.0
13.5
12.0
10.4
8.8
7.2
5.7
4.0
2.5
1.1
(a)
(b)
2.70-3.002.40-2.702.10-2.401.80-2.101.50-1.801.20-1.50
36.00-42.0030.00-36.0024.00-30.00
30 BioPharm MAY 2002
Process Development
Bulk-Scale ResultsFreeze-concentration results from bulk-scalesampling were characterized for one-literbottles, for wedges, and for other containers.
One-liter bottle characterizations. Contour plotsin Figures 3 and 4 show significantconcentration gradients from bottles frozenin 20 °C and 80 °C freezers. The figuresshow measured protein or saltconcentrations from the central planarsection of a one-liter bottle. Mass balanceswere accurate to within four percentagepoints for one-liter bottles, which is alsowithin the acceptable error rate for theanalytical methods.
Figure 3a shows that the range of proteinconcentration in the bottle from the 80 °Cfreezer varied more than 20-fold from lessthan 0.4 mg/mL to an excess of 8.3 mg/mL(more than eight times the initialconcentration). The most concentrated
sample was taken from the bottom center ofthe bottle (1–3 cm from the bottom). Sodiumcitrate concentration varied 0.3–12 timesinitial salt concentration (Figure 3b), andboth protein and salt solute distributionsfollowed similar trends. BSA concentrationvaried between 0.3 and 4.6 times the initialconcentrations in the bottle frozen in the20 °C freezer (Figure 4a) and sodiumcitrate varied 0.2–6 times its originalconcentration (Figure 4b).
Additional sampling plans (90° from thecenter plane) confirmed that the results forthe planar section (Figures 3 and 4) wererepresentative of the entire bottle, so theywere not reported. Ice samples from frozenmaterial near the bottle tops (not shown)exhibited increased concentration, rangingfrom 1.2 to 3.8 times the initial BSAconcentration. No clear correlation existsbetween concentration and position near thetop of the ice matrix in bottles.
Highest concentrations. Table 1 lists themaximum observed concentrations(individual samples) of BSA and sodiumcitrate from all one-liter bottles (n35), andfrom the wedge. Some regions in the bottlesapproached a 12-magnitude increase of BSAand a 16-magnitude increase of sodiumcitrate concentrations. Sodium citrateconcentrations consistently increased morethan the larger BSA molecule. Mass transfergoverns partitioning of solute moleculesbetween the liquid and solid phases duringfreezing (18,29), and steeper concentrationgradients for salts (relative to proteins) resultfrom higher diffusion coefficients forsmaller salt molecules.
Wedge characterization (standard freeze time 4.3 h).BSA and sodium citrate concentrations in the
wedge were uniform in all sampling locationsexcept for a small volume near the center inwhich concentrations were slightly elevated(see the red section in Figure 2). The BSAand sodium citrate concentrations found atmultiple depths in this center location arelisted in Table 2. Maximum concentrationsmeasured in the wedge were much loweroverall when compared with bottles. A massbalance performed on the wedge data wasaccurate to within four percentage points forboth BSA and sodium citrate.
Wedge characterization (extended freeze time 13 h).BSA and sodium citrate concentrations wereelevated near the center of the wedge(Figures 5a and 5b) after extended freezing(8.7 h longer than protocol). Table 1 listspeak concentrations.
Other containers. In smaller containers, suchas centrifuge tubes or sample vialscontaining from one to 50 mL of solution,freeze-concentration was minimal, so nocontour plots are shown. Maximum BSAconcentrations measured are reported inTable 1.
Time and Velocity FactorsFreezing and thawing times in bottles andthe wedge are reported in Table 3.
Freeze times. The bottles required three toeight times longer to freeze than the wedge.Variability in the time to freeze wasdependent on bottle position within thearray. Table 4 shows bottle average freezingtimes in the 20 °C freezer based onhorizontal position in the array. Bottles inthe middle row of each freezer took longerto freeze than those in the front or backrows.
The difference in freezing times for thebottles was significant (difference in meanstest, 0.05; pooled standard deviation, 1.7 h) even for small loads (5–10% ofcapacity). Differences in freezing timesbetween containers was exacerbated when afreezer was filled to capacity. Thatdifference can be seen in the freeze time fora single 20-L carboy in the 20 °C freezer(42 h) compared with the freeze time whenthe freezer was completely loaded withbottles and carboys (70 h).
Direction of freezing. The bottles froze in the20 °C freezer from the bottom upthroughout the array, whereas in the 80 °Cfreezer, the array froze from the top downbecause of differences in air circulationpatterns between the two freezers.
Figure 5. Contour plots of (a) BSAconcentrations (mg/mL) and (b) sodiumcitrate concentrations (mM) measured inthe CryoWedge after an extended freezeprotocol (13 hours).
1.3 2.9 5.7Distance Across the Wedge (cm)
Dep
th o
f Wed
ge,
from
the
Bot
tom
(cm
)D
epth
of W
edge
, fr
om th
e B
otto
m (
cm)
6.8 8.6 10.5
1.3 2.9 5.7Distance Across the Wedge (cm)
6.8 8.6 10.5
0.00-1.001.00-2.002.00-3.00
3.00-4.004.00-5.00
6.00-12.0012.00-18.0018.00-24.0024.00-30.0030.00-36.00
36.00-42.0042.00-48.0048.00-54.0054.00-60.00
5.0
4.0
3.0
2.0
1.0
5.0
4.0
3.0
2.0
1.0
(a)
(b)
Figure 6. Total protein reported as thepercentage exposed to different levels offreeze-concentration in a representative 1-L bottle and in the wedge.
1.17.3
41.2 50.499.5
0.5
Representative1-L Bottle
CryoWedge 20
Less than 1.2 1.2 - 2 2 - 4 > 4
32 BioPharm MAY 2002
Process Development
refrigeration required up to five days to thawcompletely; the wedge thawed in less thantwo hours.
Load FactorsTwenty-seven bottles in the 20 °C freezeroccupied five percent of its capacity, and theunit reached temperatures within a fewdegrees of the set point in less than two hours.The 80 °C freezer, however, was challengedwhen loaded with 27 bottles (about 20% full).More than 12 hours after loading, itstemperature was still only 40 °C . The 80 °C freezer failed to reach the set pointbefore all one-liter bottles were frozen.
Time required to freeze and thaw 1–50 mLcontainers was minimal compared to bottles.Once nucleation occurred, the smallercontainers froze within minutes, similar tosupercooled lyophilization vials (flashfreezing). Ice front velocities were rapid,estimated at approximately 600 mm/h orgreater. Thawing times were generally lessthan a few hours on the lab bench.
Larger containers required extended timeto freeze in the 20 °C freezer; a single 20-L carboy required 42 hours, and carboysin a fully loaded freezer required up to 70 hours. Ice front velocities based on thosefreeze times are included in Table 5. The 20-L carboys required three to four days tothaw at 22 °C. Carboy users report thatthawing 20-L containers requires up to 30 days or longer at 2–8 °C.
Why It Is ImportantThe total protein present in highlyconcentrated environments is importantbecause aggregation rates depend on proteinconcentration. Figure 6 compares proteinconcentration increases in a one-liter bottlefrozen in the 20 °C freezer with that of thewedge. About 50% of the total protein in thebottle was exposed to a 1.2 or moremagnitude concentration increase, and about8% of the protein was exposed to a twomagnitude increase.
The wedge exposed only 0.5% of theprotein to more than a 1.2 magnitudeincrease in concentration (maximumobserved local concentration increased 1.3 magnitude). Similar results are reportedfor the CryoWedge 100, in which noticeableincreases in concentration were reportedonly for the center point or last point tofreeze (20). Another analysis showed that
only about 1% of the total protein is at thatcenter point location and directly affected bythe freeze-concentration, which agrees withour results (30).
For aggregating systems, protein lossincreases linearly with time, typically by atleast the square of the protein concentration(31,32), and is generally increased by highersalt concentrations (33). Differences infreezing and thawing times can increaseaggregation by an order of magnitude. Highlocal concentrations of protein and saltexacerbate protein loss. In unagitatedcontainers, high local concentrations canremain after thawing. Protein loss increasesdramatically under such conditions asproduct temperature approaches ambientbecause the aggregation rate is exponentialwith temperature. Even for volumes as smallas one liter, a freeze–thaw operation in acontainer not specifically designed for it willresult in conditions that greatly favor proteinaggregation.
Product Quality IssuesWe statistically analyzed data from thebottles to determine the significance offreezer temperature and bottle locationwithin a freezer on total freeze time andsolute concentrations. Faster freezing of the333 bottle arrays in the 80 °C freezerthan in the 20 °C freezer was statisticallysignificant, although the difference averagedonly about three hours. The small differencein average freeze times can be ascribed tofreezer load.
Small vials of material produced in earlybatches can be frozen quickly in a freezerthat remains near the set-point temperature.With larger batches, variation will increaseas the freezer becomes less capable ofmaintaining the set point. In larger batches,containers are not exposed to the sameprocessing conditions, which raises GMPissues about product consistency andquality.
Total freeze time can be modeled usingfreezer temperature and bottle positionwithin the freezer, but all statistical modelsfailed to predict the maximum concentrationin a bottle. Even when several samplinglocations near the maximum were averagedto dampen extremes, no clear fit wasachieved for the extent of increasedconcentration as a function of freezer andbottle position. The lack of fit for all models
Ice front velocity. Average overall ice frontvelocities are reported in Table 5 as the ratioof characteristic length for ice growth to thetotal fusion time (solution at about 0 °C).Characteristic lengths were defined as theshortest distance from an active heat transfersurface (cylindrical radii for both one-literbottles, 4.5 cm, and 20-L carboys, 13.5 cm)to a center point in each container.
The arrowed lines in Figure 2 representcharacteristic lengths (Lc5.75 cm) in thewedge. Only the rapid ice front velocity (greater than 30 mm/h) achieved with awedge eliminated extensive freeze-concentration in all but the center of thecontainers. The lengthened freeze protocolin the wedge resulted in an ice front velocityof 4 mm/h. That slower-than-normalvelocity approached the ice front velocitiesmeasured in bottles. Intermediate velocitiesyielded levels of freeze-concentrationbetween the levels measured for BSA andsodium citrate in bottles and the morequickly frozen wedge.
A wedge slice. Additional data collectedfrom a longitudinal slice of the wedge(including the center point corresponding toprevious concentration maximums) showsthat rapid ice front velocity (700 mm/h),using liquid nitrogen cooling, preventedfreeze-concentration with a maximumconcentration of 1.1 mg/mL for BSA and an11 mM maximum for sodium citrate.Repeated measurements on the longitudinalcenter slice of the wedge after standardfreeze protocol (4.3 h) resulted in minimalfreeze-concentration with a maximumconcentration of 1.10.05 mg/mL (onestandard deviation) for BSA.
All concentration data collected in our labto date suggest that ice front velocity is thecritical factor in bulk-scale freeze-concentration; ice front velocity ismaintained or increased in largerCryoWedges/CryoVessels compared withthe wedge tested in our study. The ability toremove large amounts of heat releasedduring the fusion process is enhanced by thethreefold increase in overall surface areabetween a regular jacketed tank and aCryoVessel (including a 20% increase inactive heat transfer surface area).
Thaw times. Bottles took eight to 16 timeslonger than the wedge to thaw at ambienttemperature. Bottles thawed with
34 BioPharm MAY 2002
Process Development
may arise from the variation in nucleationtemperature due to uncontrolledsupercooling, which leads to differences inice crystal formation for each bottle (34).
Validating Freeze–Thaw ProcessesIncreases in concentration measured insamples from the tops of bottles along withthe volume expansion of ice toward the lidat the late stages of freezing suggest that anuncontrolled eruption of concentrated liquidoccurs at the surface. The lack of uniformfreezing within each container and betweencontainers fails to meet lot or batchconsistency definitions, raising productquality issues. Splitting one large batch intoindividual containers creates sub-batches,and cGMP requires sub-batches to beconsidered separately when evaluatingproduct quality.
Scale-down and stability testing. Considering thelarge differences in freeze-concentrationbetween bottles and smaller containers, thesmaller containers do not provide validrepresentations of what happens in largercontainers. They take very little time to freezeand thaw and are subjected to minimalconcentration increases, therefore, theyrepresent “best case” findings, which areparticularly inappropriate for use in stabilitytests.
The “Minimizing Stress” box listsmethods to help prevent protein degradationduring freeze–thaw unit operations.
AcknowledgmentsWe wish to thank John Carpenter and theanonymous reviewers for many helpful comments.
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