Challenges in Freeze–Thaw Processing of Bulk Protein Solutions
7.3 Freezing-Induced Stresses
the formation of more nuclei of small size. Post-nucleation, a number of factors infl u-ence the number, size, and shape of ice-crystals. Not all the crystal dimensions pose challenges to the stability of proteins. For example, dendritic ice- crystals produce uni-form distribution of solutes and prevent cryoconcentration of solutes.
Although, the exact mechanism of cold denaturation is still not clear, hydration of hydrophobic nonpolar residues of proteins is largely believed to be a causative phenomenon (Privalov 1990 ; Dias et al. 2010 ; Lopez et al. 2008 ). As discussed earlier, the folded state or self-aggregated state (native state) of proteins can be imparted to hydrophobic interactions among nonpolar amino acids. The hydropho-bic effects can be measured by the heat capacity of transfer from bulk liquid to sur-rounding water via Kirchhoff equation, ΔCp =Cpwater−Cpbulk . For globular proteins, Δ C p is positive, which is due to the hydration of nonpolar amino acids. Frank and Evans computations revealed that hydration of nonpolar solutes results in an increase of order of water molecules (shell-water) around the nonpolar solute (e.g. protein), i.e., increase in the number of hydrogen bonds between ordered water molecules (Franks and Evans 1945 ). In most of the orientations around protein, shell-water has at least one free (unsaturated) hydrogen bond towards the protein, which facilitates free-water molecules to infi ltrate folded protein and passivate unsaturated hydrogen bonds of shell-water. At the same time, hydrophobic association between nonpolar solutes destabilizes 3D-structure of proteins (Dias et al. 2010 ).
Cold denaturation temperature may be dependent on solution pH, protein con-centration, and presence of additives (Tang and Pikal 2005 ; Lazar et al. 2010 ).
Protein formulations may suffer pH changes during freezing due to crystallization of buffer components. As a result, cold denaturation temperature might increase, causing protein cold denaturation at higher temperatures. On the other hand, Tang and Pikal reported a lower cold denaturation temperature in the presence of addi-tives (e.g., sucrose and trehalose) and high protein (β-lactoglobulin) concentration in protein formulations. Therefore, cold denaturation temperatures of protein formu-lations must be evaluated before considering the storage temperature of biologics.
It is believed that storage temperatures in the range −70 to −80 °C would provide a low risk towards cold denaturation, and subsequently cause a slow aggregation.
While, storage temperature in the vicinity of −20 °C could present a risk of aggrega-tion, since glass transition ( Tg′ ) and crystallization phenomena for many solutes occur at −20 °C, which could increase the probability of protein aggregation.
7.3.2 Ice–Liquid Interfacial Denaturation
A liquid is said to be super-cooled, if the temperature drops below the equilibrium freezing point ( T f ) of water, and remains unfrozen. Unless seeded with crystalline ice, pure aqueous solutions can be super-cooled to −40 °C (Franks 1993a ). Below
−20 °C, the specifi c heat of aqueous solutions decreases rapidly leading to ice- crystallization with decreasing temperature. Factors, such as cooling rate, tempera-ture, nucleation density, and heterogeneity of the substrate/solute, determine the dynamics of ice-formation, such as the number, size, and shape of ice-crystals. In general, faster cooling rates result in a high degree of super-cooling and high nucle-ation rates. Freezing produced from fast cooling results in the formnucle-ation of a large
number of small ice-crystals, leading to large planar ice–liquid interfacial areas, which are considered to be detrimental to the stability of proteins.
Protein unfolding at ice–liquid interface has been demonstrated for a number of proteins including lactate dehydrogenase (LDH), IgG, and azurin. Schwegman et al. compared the infrared (IR) spectra of IgG and LDH in initial solution (prior to freezing), interstitial space (unfrozen liquid), and ice-crystal (Schwegman et al.
2009 ). IgG infrared spectral data from initial solution and interstitial space over-lapped each other, indicating that the native state of IgG has been retained in inter-stitial space solution. In contrast, there are qualitative and quantitative differences between the spectra recorded through the ice-crystals relative to the spectra of solu-tions prior to freezing or in the interstitial space. An increase in the intensity of bands (1,668, 1,690, and 1,625 cm −1 ), characteristic of intermolecular β-sheet struc-tures (main component of aggregates) was observed. For LDH, IR spectra collected from ice-crystal showed a decrease in the intensity of bands characteristic of α-helix (1,654 cm −1 ) and intramolecular β-sheet (1,638 cm −1 ) structures, and an increase in the intensity of band corresponding to intermolecular β-sheet structure. The inter-molecular β-sheet structural changes associated with ice-crystals of LDH and IgG indicate protein aggregation at the ice–liquid interface, while the changes in α-helix and intramolecular β-sheet structure refl ect the loss of native protein structure.
Addition of polysorbate 80 (protein stabilizer against surface-induced denaturation) to LDH prior to freezing resulted in the decrease of nonnative intermolecular β-sheet signal in the spectra of LDH protein adsorbed to ice interface, indicating that ice–
liquid interface contributes to denaturation of proteins.
As discussed earlier, nucleation, cooling rate, and temperature infl uence the nature of ice-formation. In a controlled ice-formation study via seeding, catalase enzyme was frozen after prior equilibration at −2 or −10 °C by seeding with ice (Fishbein and Winkert 1977 ). Lower ice nucleation temperature (−10 °C) resulted in greater denaturation (60 % loss) than that observed at higher nucleation temperature (−1 °C/25 % loss). For samples frozen by seeding at −2 °C and cooled to −25 °C at different cooling rates, the extent of damage was greater for samples subjected to faster cooling post-freezing. Irrespective of the approach used to produce freezing (seeded or non-seeded) in protein solutions, a greater degree of super- cooling results in irreversible protein degradation (Cao et al. 2003 ). Jiang and Nail investigated the effect of different freezing methods on the activity of LDH using liquid nitrogen-induced freezing, precooling, and ramp cooling of shelves (Jiang and Nail 1998 ) . Higher degree of super-cooling and low stability was observed with liquid nitrogen-induced freezing; whereas low super-cooling and high stability were observed with precooled shelf method; and ramp cooling method resulted in intermediate stability values. The authors hypothesized that even though ramp method is the slowest freezing method, it resulted in a greater degree of super- cooling and a better thermal equilibration than precooled shelf method. When ice nucleation occurred, the freez-ing rate in ramped samples was faster than the rate in samples placed on precooled shelf. It is therefore important to separate effects of cooling rate from those of freez-ing rate in the study of freeze–thaw stability of proteins.
7.3.3 Cryo (Freeze)-Concentration
Cryoconcentration involves partitioning of solution into ice phase and freeze- concentrated liquid phase (unfrozen liquid containing proteins and excipients) of solutes. As freezing continues, growing ice-front preferentially takes up water mol-ecules from the ice–liquid interface and excludes other solutes due to crystallo-graphic dissimilarities. As shown in Fig. 7.3 , exclusion of solutes by the moving ice-front leads to an increase in concentration of solutes in the residual unfrozen water (Franks 1993a ; Schneider et al. 1973 ). The excluded solute, which is concen-trated near the ice–liquid interface, is moved away from ice phase by diffusion and convection (Butler 2002a ). Due to segregation of ice phase, an increase in the con-centration of solutes, such as salts and buffers in the unfrozen liquid phase leads to changes in pH, ionic strength, osmolarity, and viscosity. Together, these physico- chemical changes could cause protein denaturation.
Inside the freeze-concentrated liquid phase, solution viscosity increases with increasing solute concentration. When ice-crystallization is complete, the freeze- concentrated liquid phase reaches a maximum in its concentration. Now, the viscos-ity of freeze-concentrate increases several orders of magnitude than that of the initial solution over a narrow temperature interval, during which the physical state of freeze-concentrate changes from unfrozen liquid state through visco-elastic rub-bery state to “glass-like” solid state. The temperature at which the freeze- concentrate liquid phase undergoes this liquid/solid transition is termed as glass transition tem-perature ( Tg′ ) (Carpenter and Crowe 1988 ; Franks 1993b ).
Fig. 7.3 Representation of cryoconcentration of solutes (proteins and excipients). Freezing-front moves from the storage container wall surface (heat transfer surface) towards the center of the container. Slow freezing rates cause solute exclusion from ice-liquid interface, and result in solute cryoconcentration towards the middle and center of cryovessels (Kolhe and Badkar 2011 )
Formation of “glassy” homogeneous protein–solute matrix is dependent on the formulation composition and cooling rate. At slow cooling rates, crystalline solute with least solubility in unfrozen solution may fi rst crystallize out at a temperature, referred as crystalline temperature ( T c ). A further decrease of solution temperature may crystallize together the least soluble solute and ice as a mixture. This tempera-ture is referred to as eutectic crystallization temperatempera-ture. Crystalline and eutectic crystallization temperatures occur in between melting ( T m ) and Tg′ of frozen matri-ces. As a result of crystallization, the freeze-concentrated liquid solute phase may further evolve into eutectic and/or amorphous phases, refl ecting the separation of crystalline and noncrystalline solute phases, respectively. As described above, non-crystalline solutes transform into a glassy state with decrease of temperature. Both crystallization and phase separation of excipients from the homogeneous mixture deprive the proteins of their stabilizing effects.
7.3.3.1 Crystallization of Solutes
Crystallization phenomenon excludes protective excipients from the vicinity of pro-teins and imposes stress on the stability of propro-teins. Particularly, crystallization of buffer salts causes large shifts (3–4 pH units) in the pH of frozen phosphate and carboxylate buffer systems (Pikal-Cleland and Rodriguez-Hornedo 2000 ; Van den Berg and Rose 1959 ; Lam and Constantino 1996 ). The pH changes during freezing are a function of buffer salt, concentration, and freezing protocol (ice seeding vs.
non-seeding). In sodium phosphate buffer, the pH changes could be due to solubility differences of the mono- and disodium salts, whose eutectic temperatures are
−9.5 °C and −0.5 °C, respectively. Therefore, disodium salt is less soluble than the monobasic salt, leading to its precipitation during freezing. Exclusion (crystalliza-tion) of disodium salt from the solution alters the ratio of basic to acidic salt species in the buffer solution causing a pH decrease. At high buffer concentration, the pH changes of the frozen solutions are even more dramatic due to crystallization of large amount of salts, which hampers buffering capacity. Also, the presence of salts, such as sodium chloride in sodium phosphate buffer increases the ionic product of the dibasic salt of sodium phosphate buffer, and thus exacerbates pH shifts.
Polyols are used as stabilizers in protein formulations. Sorbitol, a polyol, is an effec-tive protein stabilizer in formulations, Neulasta ® (pegfi lgrastim) and Neupogen (fi lgras-tim) in liquid state. In frozen state, sorbitol can exist as an amorphous solute ( Tg′ is
−44 °C) (Levine and Slade 1988 ). Piedmonte et al. reported that aggregation of Fc-fusion protein occurred in sorbitol containing formulations stored at −30 °C. Aggregation of Fc-fusion protein was attributed to the crystallization of sorbitol in frozen solutions stored above its Tg′ . Due to depletion of sorbitol from the vicinity of protein, stabilizing interactions between the amorphous sorbitol and protein were removed, leading to pro-tein aggregation. Unlike mannitol, sorbitol crystallizes at a slower rate, which should be considered during freezing of formulations and process intermediates (Piedmonte et al.
2006 ). In addition, these protein stabilizers (e.g., mannitol, sorbitol, and trehalose) undergo polymorphic transformations, which may exhibit different degrees of stabiliza-tion effects during freezing and varied solubilities upon thawing.
7.3.3.2 Phase Separation
Solutes in a freeze-concentrated unfrozen liquid phase are either miscible or immis-cible (Izutsu et al. 1996 ). During freezing, certain combination of solutes with steric hindrance or repulsive interactions separates into eutectic and/or amorphous phases.
The phase separating polymer excipients may stabilize or destabilize proteins.
Phase separating excipients may protect the multimeric proteins (e.g., LDH) by stabilizing subunit interactions. The repulsive interactions between protein and polymer co-solutes shift the equilibrium between the subunit association (e.g., monomers, dimers, and tetramers) towards a stable multimeric protein. In contrast, phase separating excipients can deprive proteins of their stabilizing interactions. It is not uncommon that a solution composed of two polymers with different miscibili-ties separates into more than one amorphous phase. And preferential partition of protein into one of the phases may deprive protein of the protective effects of the other stabilizers.
Relative contribution of each individual stress, i.e., cold temperature, cryocon-centration, and ice-formation, on the overall freezing-induced denaturation of pro-teins is still unknown. From the literature evidence on freeze-thawing-induced stresses, it can be inferred that the contribution of cold denaturation to the overall protein denaturation can be considered negligible, which implies that ice- formation- and cryoconcentration-associated changes are the potential contributing factors to the destabilization of proteins.