Challenges in Freeze–Thaw Processing of Bulk Protein Solutions
7.2 General Aspects
Proteins themselves do not represent an administrable dosage form. To constitute proteins in a stable dosage form, various excipients are added to impart protection to proteins and bulkiness to the dosage form. In many instances, aqueous phase is the primary medium for forming a homogeneous mixture of proteins and excipients.
It also determines the dynamic and kinetic behavior of proteins and solutes in solu-tion, frozen, and dry states. As a fi rst step in becoming familiar with cryopreserva-tion of proteins, it is advisable to consider the properties of proteins and physical behavior of water as a function of temperature.
7.2.1 Protein Structure and Stability
Proteins, made of sequence of amino acids, are characterized by a unique three- dimensional structure. The native structure of proteins is the result of a balance of interactions including covalent and non-covalent interactions (electrostatic interac-tions, hydrogen bonding, hydrophobicity, and van der Waals forces). Hydrogen bonding is important for formation of secondary structures, while electrostatic and hydrophobic interactions are needed for stabilizing tertiary structure of proteins.
Both intramolecular and external environment interactions determine the stability of 3D-structure of proteins. For large multi-domain proteins, gentle conditions are suffi cient to initiate protein unfolding, whose free energy is reported to be quite small (21–63 kJ/mole) (Mozhaev and Martinek 1990 ). Since the folded state of
protein is marginally stable than the unfolded state, changes in external conditions (e.g., temperature, pH, additives) trigger conformational changes (e.g., unfolding, aggregation, oligomerization, and fi brillation) (Fig. 7.1 ). Moreover, partially unfolded states are more susceptible to aggregation than the native or unfolded state, due to exposure of contiguous hydrophobic regions that are buried deep in the native state or inactivated in the denatured state.
Protein aggregation can be physical or chemical or both, resulting in the forma-tion of high order oligomers (e.g., trimers, tetramers, or hexamers). Physical aggre-gation involves non-covalent interactions arising from the hydrophobic regions of the protein unfolded state, while chemical aggregation arises from covalent inter-actions, such as disulfi de bond formation. Depending on the spacial- and time- scale of exposure to extreme conditions, aggregation phenomenon can be either reversible or irreversible. The high energy of activation of irreversible aggregation makes the process slow, but can have direct impact on manufacturing processes (e.g., sterile infi ltration), drug potency, and immunogenicity. These aggregates (physical or chemical) can be either soluble or insoluble, and can form at the same
Fig. 7.1 The fi gure illustrates many conformational changes that proteins undergo in the presence of environmental conditions including excipients and temperature. During reversible equilibrium, dark arrow represents a thermodynamically favored equilibrium towards a particular state. Dotted brown arrow represents the least favored thermodynamic equilibrium. Modifi ed and adapted from Dobson ( 2003 )
time during processing and storage. Other degradation reactions that inactivate proteins include protein fragmentation, aspartate isomerization, oxidation, deami-dation, and hydrolysis.
7.2.2 Water: States of Matter
Frozen storage and freeze-drying are used as preservation technologies for food and pharmaceuticals. The product shelf-lives can be closely correlated to water and solution transitions that occur during freezing and freeze-drying processes (Franks 2003 ). Figure 7.2a shows different phases and states of water as a function of tem-perature. The phase and state behavior of water becomes even more complex in the presence of solutes. This has led to the construction of state diagrams, combining conventional solid–liquid and solid–solid phase coexistence data with temperature-compositions relating glass transition profi les. Figure 7.2b illustrates a popular state diagram for the sucrose–water system.
Fig. 7.2 ( a ) Hypothetical phase diagram of liquid water including super-heated and cryo- quenched states. Phase and state transition temperatures at atmospheric pressure are indicated: T B , boiling point; T m , melting point; T hom , homogeneous nucleation; T x , devitrifi cation temperature; T g , glass transition temperature. Modifi ed and adapted from Mishima and Stanley ( 1998 ). ( b ) A schematic temperature–concentration state diagram for an aqueous carbohydrate solution, showing the glass transition curve, which extends from T g (glass transition temperature) of pure water (−134 °C) to T g of pure solute (52 °C), the equilibrium freezing (liquidus curve), which extends from T m (melt-ing temperature) of pure water (0 °C) to eutectic temperature ( T e ) of the solute. The liquidus curve extends below T e in a nonequilibrium state to intersect the glass transition line at Tg′ , which repre-sents the glass transition temperature of the maximally freeze- concentrated solution. Wg′ repre-sents the amount of unfrozen water (100 % solute, C g ) entrapped in the glass. Point Tm′ represents collapse temperature of the glass during warming. T g and W g represent a temperature–concentra-tion relatemperature–concentra-tionship in a glass formed as a result of less than maximal ice-formatemperature–concentra-tion. Adapted from Goff et al. ( 2003 )
Under equilibrium conditions, cooling of an unsaturated solution leads to eutec-tic point ( T e ), where liquidus and solidus curves meet and anhydrous sucrose pre-cipitates. This temperature is termed as eutectic temperature. At ordinary cooling rates, probability of sucrose nucleation at T e or below is low. Further, continued cooling produces supersaturated solution undergoing vitrifi cation (solidifi cation of super-cooled solution without ice or solute crystallization) at glass transition tem-perature ( Tg′ ; −32 °C). At this point, solution consists of 80 % w/w sucrose and represents amorphous, super-cooled, and high viscous system, referred to as maxi-mum freeze-concentrated phase. In general, biologics are stored frozen below Tg′ to avoid crystallization of solutes and associated pH changes which can cause protein denaturation.
However, the challenge of cryopreservation by vitrifi cation is avoidance of ice- formation. Ice-formation consists of nucleation followed by growth. For nucleation to occur, molecules in the liquid water are subjected to small transient energy and density fl uctuations during Brownian diffusion. Occasionally, these fl uctuations lead to formation of clusters similar in dimensions to ice-crystal. The temperature at which crystallization is favored is approximately −40 °C in pure water, termed as homogeneous nucleation temperature ( T hom ). The value of T hom decreases by approx-imately 2 °C for every 1 °C decrease in T m as solute concentration is increased (Mehl 1996 ). Below T hom , solution is unstable against ice-formation because homo-geneous nucleation occurs quickly. Between T m and T hom , solution is metastable against ice-formation. Ice can form, if nucleation is assisted by particles or surfaces that lower free energy barriers to nucleation. This is termed as heterogeneous nucle-ation, which can occur at higher temperatures ( T het ) than homogeneous nucleation temperature. Heterogeneous nucleation is the predominant mechanism of ice-for-mation in biologics.
The solute concentration at which T hom crosses Tg′ is the lowest concentration at which it is possible to avoid homogeneous nucleation. The presence of solutes (e.g., cryoprotectants) in water depresses T hom , and thereby inhibits homogeneous nucle-ation. The lower the solute concentration, the faster cooling must proceed to avoid ice-formation (Fahy and Rall 2007 ). The minimum cooling rate necessary to avoid signifi cant ice-formation during cooling is the “critical cooling rate” of a cryopro-tectant solution. The minimum warming rate to avoid signifi cant ice-formation dur-ing warmdur-ing from a super-cooled/vitrifi ed state is the “critical warmdur-ing rate.”
Critical warming rates are typically 2–3 orders of magnitude greater than critical cooling rates. Ice-formation during warming is termed as “devitrifi cation,” which happens faster than cooling because ice nucleation occurs at lower temperatures than ice growth. Nucleation at very low temperatures primes the solution for exten-sive ice growth at relatively warmer subzero temperatures. Nucleation rate increases by a factor of nearly 50 for each 1 °C lowering of temperature.
Nucleation and growth are kinetic processes, and therefore are determined by rate of cooling. Cooling rate determines the degree of cooling, but could not directly con-trol. In the absence of nucleation, rapid cooling rates produce greater degree of cool-ing. Nucleation in a low temperature super-cooled solution will be rapid and leads to
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.