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Large-Scale Freezing of Biologics A Practitioner's Review, Part One: Fundamental Aspects

Satish K. Singh, Parag Kolhe, Wei Wang, and Sandeep Nema

Reprinted with permission from BioProcess International 7(9) (October 2009)

roduction of biologics is an large-scale processes (part one) and expensive process, and to critically examines some technologies optimize capacity use, bulk and systems available to provide P protein solution is often guidance on rational development of produced in manufacturing this unit operation (part two). An campaigns. It is converted into drug abridged version of these two articles product based on market demand and has been published previously (2). For

therefore may have to be stored for ehse (www.sxc.hu) our purposes, we are limiting relatively long periods. To decouple R consideration of biologics to protein the bulk solution production from that biotherapeutics only. of the final drug product, bulk is often stored frozen. ann-kathrin Fu n d a m e n t a l As p e c t s o f Fr e e z i n g Transport of frozen bulk product Proteins can undergo degradation by between sites offers several practical many mechanisms. However, the advantages over its transport in the primary mechanism of concern with state (2–8 °C). Maintaining frozen storage is aggregation, 2–8 °C requires accurate control although some chemical-reaction– systems to ensure that a product does Publicly available information based mechanisms could arise with not get too cold and (partially) freeze. suggests that nearly half of certain susceptible proteins. A liquid shipment also subjects commercial biotherapeutics are stored Knowledge of the fundamentals of protein to greater degrees of agitation frozen (1). Given the high value of freezing and thawing is geared toward stress at air–liquid interfaces. So a product being processed in the understanding their impact on and the successful bulk storage program will freezing operation, it is surprising that prevention of (permanent) structure enhance bioprocess capacity use and there is little scientific guidance loss and concomitant formation and reduce overall cost of production. available for practitioners. Literature growth of aggregates. However, success requires careful on the impact of protein freezing is A discussion of the low- consideration of biophysical and limited to very small-scale behavior of proteins must engineering principles in development experiments that, although useful, do distinguish between the effects of the of a frozen-storage operation and its not address complications created by low temperature or cold per se from impact on the product to be frozen. the relatively large heat and mass that of the freezing process itself. transfer dimensions of practical large- Franks distinguishes the former as Pr o d u c t Fo c u s : Pr o t e i n s scale systems. The subject is expensive “impact of chill,” which involves for academic research and not biological changes produced by Pr o c e s s Fo c u s : Formulations a n d interesting enough from a results changes in the property of liquid d o w n s t r e a m p r o c e s s i n g perspective because real-time, long- with temperature (e.g., term stability studies are required. constant, dielectric Wh o Sh o u l d Re a d : Pr o c e s s /p r o d u c t Unlike liquid-state stability studies, constant, hydrogen bond energies, and d e v e l o p m e n t , f o r m u l a t o r s these cannot be meaningfully hydrophobic interactions) leading to 3, Ke y w o r d s : Stability, aggregation , accelerated. A lot of knowledge resides hydration of the hydrophobic core ( 4 b u l k intermediates , e u t e c t i c , s t o r a g e , in the industry, and freezing practices ). Generally reversible protein T , t r a n s p o r t , denaturation are often empirical. structure changes that occur as a g This two-part review discusses the consequence of low temperature are Le v e l : Intermediate basics of freezing biologics relevant to termed cold denaturation.

32 BioProcess International Oc t o b e r 2009 Figure 1: Schematic representation of a freezing process shows different stages and nature of The freezing process, however, frozen material. Cooling starts at a, and the solution supercools to b. occurs at b, followed by freezing of the whole mass between c and d, representing the freezing time or subjects proteins to other stresses as a duration. Freezing temperature drops during this process as the unfrozen fraction becomes consequence of the removal of water progressively cryoconcentrated. The rate of cooling (heat removal) determines the number of as . The resulting cryoconcentration nuclei formed and the ice size, with slow cooling leading to fewer and bigger . At d, and desiccation of protein can be the unfrozen fraction either crystallizes (eutectic solidification) or converts to in a maximally classified as osmotic stresses. Other cryoconcentrated matrix. After further removal of sensible heat and cooling to point e (the target temperature), the process is considered complete. freezing-process–induced stresses include ice interface formation, pH Solution Ice nuclei changes, and separation. Protein AB structure changes that occur as a B nucleation C freezing point depression to Tf consequence of such stresses have a CD freeze-concentration

c) A Ice Crystals greater probability of being o irreversible, and are classified as freeze 0 denaturation (3, 4). T f B C Rate of Cooling: Freeze–thaw behavior of proteins has been studied Temperature ( Temperature Unfrozen fraction extensively but primarily in D microscopic or small volumes (a few E milliliters or less), often in conjunction Freezing Time with lyophilization. Among the Target Temperature Time parameters considered important are Slow Cooling Fast Cooling the rate of processing (cooling and heating) and composition. Effects as Eutectic or presented in the literature provide no Maximally cryoconcentrated matrix clear guidance because the terms fast and slow are specific to each study, and rates (if) reported vary widely (5). unfrozen mixture of solutes (including diagram (if available). For Further, the use of minimal volumes the protein) because growing ice noncrystallizable excipients, makes the “process” aspect of crystals will exclude solutes. The cryoconcentration behavior is better literature studies difficult to increasing concentration of solutes in described by state diagrams instead extrapolate to freezing large-volume the unfrozen fraction results in a because practical freezing processes bulk protein solutions. continuously decreasing freezing point are seldom carried out under The “rate” of cooling in practical (Figure 1, cd). Progressive freeze- equilibrium conditions. Combinations systems comes into play indirectly concentration of the unfrozen mixture of crystallizable and noncrystallizable through its effect on the nature of the leads to an increase in viscosity, excipients will give intermediate ice interface created. Moreover, rate as reduction in diffusion coefficients, and behavior depending on the ratio of the often used in the literature refers to ultimately a glassy matrix when no two (9). Figure 1 shows a freezing the drop in temperature over time. more water can crystallize. If all the curve with schematics showing the But the more important rate relates to freezable water is converted to ice, nature of frozen materials in solution the time required for a solution to then the concentration of solutes can at various studies. actually freeze and transition from become very high. In practical systems, a small liquid to a solid or glass phase In the case of crystallizable amount of supercooling is followed by (indicated as freezing time or duration excipients (e.g., NaCl), reaching a rapid ice nucleation throughout the in Figure 1). Any practical system solubility limit during bulk solution, so not all the solutes are larger than a few hundred milliliters cyoconcentration could cause them to pushed toward the geometrical center. will freeze over a period as the process crystallize out of solution, forming a However, because of the heat transfer progresses, thus producing much more eutectic. For a 150-mM (~0.9% w/w) distances involved, ice growth is faster complex behavior than can be NaCl solution, the eutectic at –21.2 °C at the walls than in the center, so described by a single rate parameter. has a concentration of 23.3% w/w, a some diffusion of solutes toward later- Although beyond our scope here, it is ~25-fold increase (Figure 2) (6). Most freezing regions occurs. Excluded clear that geometry (heat and mass carbohydrates (e.g., sucrose) tend to be solute at the interface depresses the transfer dimensions) is important to noncrystallizable, so their maximal freezing point of solution just in front the outcome of the process as well. freeze-concentrated (equilibrium) of it to below the freezing point of the Osmotic Stresses — Dessication concentration is around 80% w/w remaining nominal bulk solution, and Cryoconcentration: As an aqueous (Figure 3) (7, 8). General trends in the creating a situation in which solution solution freezes, conversion of water cryoconcentration behavior of easily further from the ice front is in a into ice causes progressive freeze- crystallizable excipients can be supercooled state (constitutional concentration (cryoconcentration) of the followed using an equilibrium phase supercooling). This leads to instability

34 BioProcess International Oc t o b e r 2009 in the ice front, increasing the overall effect depends on the cryoconcentration affect a frozen probability of dendritic ice formation container’s depth and geometry. biologic in a number of ways. because protuberances can better shed The flip side of cryoconcentration Increasing ionic strength during into larger and supercooled is desiccation. As freezing proceeds, freezing can reduce its solubility and volumes of liquid (10). removal of water (as ice) dries out of potentially also destabilize protein Dendritic growth of ice crystals the protein in the amorphous (glassy) structure through disruption of salt- enables entrapment of solute in phase. Each protein molecule bridges. Buffer salts can crystallize if unfrozen channels between the structures the water molecules around their concentration limit is reached, dendrites. However, solutes trapped it, with a first hydration shell of water which changes pH. Among common between those ice crystals still molecules (~0.3 g/g) directly attached buffers used for biologics, the sodium experience a localized to the protein. The water molecules phosphate buffer mixture is cryoconcentration effect as water is aid in proper folding and lubricate particularly susceptible: pH can change extracted and the channel narrows by movement of a protein’s amino-acid from 7 to ~4 on precipitation of the growth of ice. Thus, concentration backbone and side groups by rapid dibasic salt below 0 °C. Even if the gradients are generated and “frozen- formation and exchange of hydrogen salts do not reach their solubility in.” Figure 4a shows an example of bonds (11). Hydration is thus limits, their pKa value is sensitive to concentration gradients in a bottle important for maintaining the three- temperature, so pH shifts will occur containing a monoclonal antibody dimensional structure of a protein. during freezing and in the frozen state. (MAb) solution. Such gradients can The entire hydration shell of the Data are available for pKa/temperature persevere if thawing is carried out protein is unlikely to be completely in the liquid state (13). In general, without mixing (Figure 4b). removed by freezing: Part of it is dissociation constants for carboxylic Our in-house observations show unfreezable or bound water, and outer and inorganic acids have dpKa/dT that proteins and other excipients shells or loosely bound water can be values close to zero. They are slightly cryoconcentrate to nearly the same lost. Structural changes have been negative (~0.01) for secondary amines extent: The impact of differences in recorded for lysozyme (amide I band- and more negative (~0.015–0.02) for diffusion coefficients is overridden by narrowing implying protein–protein primary amines. Changes in pH in the convective effects. Convective effects interaction) as water is removed during frozen state are not predictable, but also become important in large-scale freezing in the absence of some data are available (14, 15). systems, where temperature such as sucrose (12). Other excipients (e.g., mannitol, differences create density gradients in The steepest changes were recorded at glycine, and polyethylene glycol) in a the unfrozen solution. The excluded low hydration levels under ~15%. formulation can crystallize and/or solute just ahead of the ice front has a Structural change was prevented with phase separate during freezing. As in high concentration of solutes (and thus 10% sucrose, which satisfied the all situations involving equilibrium in a higher density) than the bulk. protein’s hydrogen bonding practical processes, the actual extent Density gradients create a convective requirements. of of buffer or other flow that carries solutes down toward Cryoconcentration Consequences: solution components depends on the bottom of the container. The Ice formation and consequent volume, cooling rate, the presence of

Figure 2: of an NaCl–water system Figure 3: State diagram for a noncrystallizing solute (e.g., sucrose) shows the different states of a frozen matrix. Nonequilibrium cooling 40 leads to formation of less-than-maximally freeze-concentrated glass with an effective glass-transition temperature

Temperature (°C)Temperature −20 concentrated solute las G −40 Tg −30 Ice + NaClt2H2O Ice and freeze- concentrated glass −60 e rv −40 (°C)Temperature u C 0 10 20 30 40 50 60 70 80 90 100 on Maximally −80 iti ns freeze- Tra concentrated NaCl (% W/W ) ss −100 Gla glass s las −120 G 1011Pas −140 0 0.2 0.4 0.6 0.8 1 Weight Fraction of Solutes

36 BioProcess International Oc t o b e r 2009 other solutes, initial concentrations, mechanisms stabilized against are strictly limited to cold denaturation, and nucleation rates. Recently, liquid– surface-induced denaturation and cold then stability in the frozen state liquid phase separation of a protein- denaturation. probably would not be an issue. rich from a protein-poor phase has Cold Denaturation: As the However, chill-induced unfolding been reported in a high-concentration temperature of a solution drops, probably makes a molecule more MAb solutions in high ionic strength properties of the aqueous solvent susceptible to freeze-induced stresses, buffer when cooled to 0 °C (16, 17). A medium change including its dielectric leading to aggregate formation and/or number of proteins display this effect constant, acid/base ionization loss of structure. (18–20). High-salt and/or high- constants, diffusion rates and mobility, Storage, Shipping, and the Glass sequence hydrophobicity (21) solubility of hydrophobic residues, and Transition Temperature: Once contributes to the accessibility of this hydrogen bond energies. Those formulated and frozen, a protein must transition. Cryoconcentration could changes in themselves, without the be stored and remain stable over long trigger this in susceptible protein complicating effects of freezing and periods — extending into years. When systems with low salt content as well. phase changes, cause reversible freezing is complete, the protein and Cryoconcentration can also affect changes in protein structure and cold other solutes are concentrated into a reaction rates. Reduction in denaturation. This low-temperature highly viscous amorphous matrix with temperature lowers the rate of effect (“chill”) is distinct from the a characteristic temperature called the degradation reactions (the Arrhenius effect on protein structure that comes temperature (Tg), above effect), but cryoconcentration can from the actual freezing (e.g., which the matrix is regarded as counteract that through an increase in cryoconcentration, phase changes, and “rubbery” and below which it is “glass” the concentration of reactants. So ice surface denaturation) discussed (Figure 3). The actual transitions are reactions such as oxidation can be above and elsewhere (3, 25, 26). not so distinct, but molecular enhanced, especially when the A more precise thermodynamic relaxation and related viscosity and solubility of oxygen increases as explanation for cold denaturation mobility phenomena show a temperature drops while ice formation comes from considering the free Williams–Landel–Ferry (WLF) type also excludes . Dissolved oxygen energy of protein unfolding. Cold- of dependence just above the Tg, which at high concentration can be trapped induced unfolding (cold denaturation) has a much greater sensitivity to along with proteins in the final glassy is a physical consequence of the temperature than in an Arrhenius matrix. Other potential temperature sensitivity of noncovalent relation (36). Viscosity decreases incompatibilities among solutes and electrostatic and hydrophobic dramatically in the vicinity of and impurities (e.g., peroxides, trace interactions, which become weaker at above Tg. Thus, even if the matrix metals) could also be exacerbated. lower (3, 27, 28). It is a appears nominally frozen in this At the Ice Interface: Proteins also thermodynamic consequence of the temperature region, diffusive processes interact with the ice surface, resulting large and positive ΔCp of proteins can occur over long time-scales. in perturbation of their native unfolding (and which, within The storage temperature for a frozen structure. In effect, they can (partially) experimental error, can be considered matrix must be set based on Tg with denature at the ice interface through a constant for each protein) (29). Cold- some margin because even reduced weakening of their hydrophobic bonds denaturation temperature has never molecular mobility can affect the as well as adsorption onto the ice been experimentally observed because stability of protein over long time surface (22, 23). The extent of it generally lies below 0 °C, at which scales. The US Pharmacopeia defines structural perturbation depends on the point the chill-induced effect is the –20 °C condition as a –10 to –20 °C rate of cooling or heat removal, which difficult to separate from that of range, whereas the Ph. Eur. defines it determines the number of nuclei, the freezing itself (30). However, changes as –20 ± 5 °C. Periodic fluctuations on size of ice crystals formed, and the in solvent conditions such as pH, the high side of the storage temperature interfacial area generated (Figure 1). addition of chaotropic agents, and can speed up diffusive processes, and Protein–ice interaction lowers the other perturbants have been used to large deviations will negate the effect free energy of the denatured state make the cold-denaturation of a low storage temperature. Storage more than that of the native state. temperature more accessible (31–34). temperature must be chosen such that Interactions are therefore stronger for From a pharmaceutical perspective, the high-temperature part of the cycle an expanded protein with a larger commonly added sugars and polyols and the time spent above the set-point solvent-accessible surface area, which move the cold-denaturation will still provide an acceptable product. aids in adsorption (24). A strong temperature lower, thus stabilizing a If data are available, then mean kinetic positive correlation has been shown protein molecule (35). temperature estimations can be applied between freeze denaturation and In practical terms, the reversible to determine the acceptable range. surface denaturation (23). When nature of unfolding due to chill- In a well-designed formulation, freeze–thaw cycling studies are induced cold denaturation is not in freeze-induced structural perturbations performed to identify cryoprotectants itself detrimental to the storage will be largely reversible upon thawing, (including surfactants), the primary stability of a protein. If the stress were although some fraction may become

38 BioProcess International Oc t o b e r 2009 irreversibly damaged. More important, Th a w i n g o f Bi o l o g i c s 1 w1 w2 depending on the storage temperature = ` + ` Although freezing is the principal Tg Tg1 Tg2 in relation to Tg, some minor loss of factor that determines the stability of a structure in the frozen state may cause biologic when it is stored frozen, the aggregate formation over time when where Tg is the glass transition process of thawing deserves some (partially) unfolded molecules serve as temperature of the solution, and wi consideration. The major energy ′ nuclei and interact with their neighbors and Tg i are the weight fractions and requirement goes to provide the latent if mobility is sufficient. Thus, storage glass transition temperatures of the heat of melting. Although it is simple near and above the Tg of the matrix solution components. in principle, the process must be will allow denaturation and/or Commonly used stabilizers in controlled properly to ensure that wall aggregation to progress, albeit slowly. protein formulations include sugars, temperatures at the heat-transfer Ice crystal size and morphology can polyhydric alcohols, certain amino surfaces do not exceed allowable limits also change if mobility is sufficient. acids, and higher oligosaccharides. for a product. To ensure that the That leads to through Their utility in the frozen state comes thawed material does not overheat Ostwald ripening and to a from their inability to crystallize and a while a remainder is still frozen, the redistribution of solutes. Crystallizable lack of eutectic phase separation. Most mass should be agitated during excipients (e.g., NaCl, mannitol, such excipients are subject to processing, which ensures efficient heat glycine) trapped in a nonequilibrium or glass formation as transfer and prevents hot spots (43). An state during freezing can crystallize freezing proceeds, crystallizing agitation rate should be chosen to over time given the mobility allowed by excipients being generally unsuitable. provide adequate mixing without storage above Tg. Phase transitions over A comprehensive compilation of Tg is causing foaming or shaking-induced time have been shown in the frozen available (39, 40) although some values degradation. An air–water interface is state, recently reported for sorbitol, are disputed (41). Tg values generally often a site where proteins will unfold. leading to protein aggregation as the increase with molecular weight (7, 8). Apart from the above “macro” cryoprotective effect of excipients is lost Proteins in solution do not crystallize phenomenon, thawing can lead to ice because of crystallization (37). on cooling. Being polymeric, they also recrystallization and annealing. The type of container can exhibit a glass transition temperature, Because of higher , potentially also affect frozen-state which tends to range around –10 °C small ice crystals can melt and behavior through adsorption, regardless of protein size and preferentially refreeze onto larger depending on the hydrophobicity of its structure: –11 °C for ovalbumin, crystals, leading to Ostwald ripening interior surface, although other factors –13 °C for lysozyme, –15 °C for effects. In most situations, however, could be confounding the reported myoglobin, –11 °C for BSA, and this should not be of any practical results (38). However, most practical –9 °C for lactic dehydrogenase (42). concern if the process is carried out systems will have low surface areas in The Tg for pure water is generally reasonably rapidly. Similarly, relation to their volume, which reduces accepted to be ~135–140 K (about crystallizable excipients frozen into a the relative impact of the container –135 °C), at the lower end of the metastable state (e.g., mannitol, NaCl) interface compared with other factors glass-transition curve (Figure 3). can recrystallize during thawing and discussed here. Similar to storage Knowledge about Tg provides a have been indirectly implicated in vial temperature, a shipping temperature strategy to adjust it by changing the breakage during the process (44, 45). must be chosen with regard to the ratios of the major components in a properties of the glassy matrix. formulation, namely the stabilizer and St a b i l i z i n g Pr o t e i n s Ag a i n s t t h e Eff e c t s o f Fr e e z i n g Shipping below Tg is desirable. the protein. For common stabilizers Formulation Composition and T : g such as disaccharides, an increase in It is apparent from the above It is clear that Tg is a key bulk protein concentration with a discussion that, except for pH changes formulation parameter. The ability to concomitant decrease in that of the — and to some extent, phase manipulate it can thus be useful in stabilizer will raise Tg. It is unlikely separation — elimination of stresses is enhancing the stability of a protein in that the stabilizer can be completely not an option. Cold-denaturation, a frozen matrix. A solution’s Tg eliminated and still retain adequate osmotic stress (cryoconcentration, depends on the nature of the glass protein stability in the frozen state. dessication), and ice-interfacial stress formers involved and their But careful adjustment of their ratio are inevitable consequences of concentrations in the glass. The main can be made such that a Tg 5–10 °C freezing. Formulations and processes components of protein formulations above the (intended) storage therefore must be designed to be are buffer salts, stabilizers and/or temperature is obtained while cryoprotective against all these factors. cryoprotectants, and the protein itself. maintaining the stabilizer’s Formulation additives that have Assuming the additivity of their cryoprotective effects. More discussion been empirically found to be useful as properties (free volume), Tg is given by about practical formulation cryoprotectants include sugars, the Gordon–Taylor equation or its development is provided in part two of polyhydric alcohols, higher simplified version, the Fox equation: this review. oligosaccharides, amino acids, and

40 BioProcess International Oc t o b e r 2009 Figure 4a: Concentration distribution of a MAb in 1-L bottles after freezing at different conditions; surfactants. Other additives include protein concentration was measured by taking slices from the frozen bottle at different levels, as indicated on the y-axis. At each level, two samples were taken, one at the center and one at a 46– methylamines and lyotropic salts ( distance of r/2 from the center. 49). A number of those are proposed to function through the preferential 5 to −70 ˚C 5 to −40 ˚C 5 to −20 ˚C A exclusion mechanism (against cold denaturation by lowering the cold Side Side Side 7 Center Center Center denaturation temperature and against 7 7 6 osmotic stresses by stabilizing the 6 6 5 native state) while surfactants interfere 5 5 4 with interactions at the ice interface. 4 4 3 Because all stresses are present 3 3 2 2 2 concurrently, more than one additive 1 1 1 Bottle Slice Number Bottle Slice Number is usually required for maximum Bottle Slice Number protection. 0 10 20 30 0 5 10 15 20 25 30 0 5 10 15 20 25 30 A molecular modeling analysis of Protein Concentration Protein Concentration Protein Concentration X-ray diffraction peaks of ice led (mg/mL) (mg/mL) (mg/mL) Varshney, et al. to propose the existence B of 2–3 kbars local hydrostatic pressure during freezing (50). Such pressures are Left Left Middle sufficient to cause destabilization of Middle 5 5 Right Right protein structures and add an 4 4 intriguing stress mechanism active After Mixing during protein freezing. 3 3 Position

From a process perspective, within Position 2 2 current design limitations the only 1 1 process parameter available to practitioners is the cooling (heat 0 5 10 15 20 25 30 0 5 10 15 20 25 30 removal) rate. Because proteins Concentration Concentration interact with the ice interface and are (mg/mL) (mg/mL) prone to surface denaturation, a Figure 4b: Concentration distribution of a MAb in 1-L bottles after thawing under static conditions smaller interfacial area with larger ice at room temperature — and after mixing once thawed; significant concentration gradients are crystals (a longer freezing time) would formed during freezing and maintained if thawing is performed without mixing. be preferable. It would also allow the cryoconcentrated matrix to get closer to its maximally frozen concentration protein, and thawing should generally process economics. Knowledge of the — or nearer to the theoretical Tg be as rapid as possible (with agitation). fundamental phenomena described (Figure 3). However, a slower rate It is likely that proteins with here is critical to the rational design of allows longer exposure to the multimeric or multidomain structures successful formulations and processes. cryoconcentrated and/or pH-altered are sensitive to stress induced by Part two of this review will provide medium during the transition between freezing and thawing than monomeric practical guidance for this purpose. liquid and glassy solid, when there is proteins would be. still enough mobility to damage Ultimately, real-time stability Re f e r e n c e s proteins. Furthermore, it is possible studies must be conducted to determine 1 Singh SK. Storage Consideration As that higher degrees of the viability of the formulation and Part of the Formulation Development Program for Biologics. Amer. Pharmaceut. Rev. 10(3) cryoconcentration could be more storage temperature selected for a given 2007: 26–33. damaging to a protein than more protein. Unlike in the liquid state, 2 Singh SK, et al. Best Practices for dilute matrices would be. viable accelerated conditions are not Formulation and Manufacturing of Biotech Slower thawing rates can lead to available. However, studies should be Drug Products. BioPharm Int. 22(6) 2009: 32–48. recrystallization, especially in frozen performed both below and above Tg to matrices created by rapid cooling, determine its impact. A limitation to 3 Franks F. Protein Destabilization at leading to additional perturbation at such studies is that the difficulty of Low Temperatures. Adv. Protein Chem. 46, 1995: 105–139. the ice-liquid interface. Slow thawing simulating full-scale process conditions 4 Franks F. Nucleation of Ice and Its also causes longer exposure to during development studies. A range of Management in Ecosystems. Phil. Trans. Royal cryoconcentrated or pH-altered process conditions must be explored Soc. London, A. 361, 2003: 557–574. medium in the transition from a glassy and is covered in more detail in part 5 Bhatnagar B, Bogner RH, Pikal MJ. solid to a liquid phase. The basic rule two of this review. Protein Stability During Freezing: Separation that emerges is that an optimum Storage of bulk protein solution in of Stresses and Mechanisms of Protein freezing rate could be defined for each the frozen state is necessary for Stabilization. Pharm. Dev. Technol. 12, 2007: 505–523.

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