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 FUNDAMENTAL AS P ECTS O F FREEZING 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 liquid 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 temperature 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 PRODUCT FOCUS : PROTEINS scale systems. The subject is expensive “impact of chill,” which involves for academic research and not biological changes produced by PROCESS FOCUS : FORMULATIONS AND interesting enough from a results changes in the property of liquid DOWNSTREAM P ROCESSING perspective because real-time, long- water with temperature (e.g., term stability studies are required. ionization constant, dielectric WHO SHOULD READ : PROCESS /P RODUCT Unlike liquid-state stability studies, constant, hydrogen bond energies, and DEVELO P MENT , F ORMULATORS these cannot be meaningfully hydrophobic interactions) leading to 3, KEY W ORDS : STABILITY, AGGREGATION , accelerated. A lot of knowledge resides hydration of the hydrophobic core ( 4 BULK INTERMEDIATES , EUTECTIC , STORAGE , in the industry, and freezing practices ). Generally reversible protein T , TRANS P ORT , DENATURATION are often empirical. structure changes that occur as a G This two-part review discusses the consequence of low temperature are LEVEL : INTERMEDIATE basics of freezing biologics relevant to termed cold denaturation. 32 BioProcess International OCT O BER 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. Nucleation 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 ice. The resulting cryoconcentration nuclei formed and the ice crystal size, with slow cooling leading to fewer and bigger crystals. At D, and desiccation of protein can be the unfrozen fraction either crystallizes (eutectic solidification) or converts to glass 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 phase separation. Protein AB supercooling 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 solid 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 OCT O BER 2009 in the ice front, increasing the overall effect depends on the cryoconcentration affect a frozen probability of dendritic