Short Cycle Times for Cost-Efficient Processing in Lyophilized Formulations

• Aston University

This review will highlight rational approaches and considerations for cost-efficient, short-cycle development with emphasis on bulk freeze drying. It must be noted that published work on pharmaceutical freeze drying can be drawn from a variety of backgrounds. This may include food, vial or spray systems and we aim to draw information from these studies towards these aims.

Material quality is affected across all stages of lyophilization and has been reviewed extensively; (I) stabilization [1] (II) freezing [2] (III) primary drying [3,4] (IV) secondary drying [5,6] (V) back fill and (VI) product sealing [7-9].

We characterise quality factors as the term given to the individual priorities developers place on distinct aspects of a formulation to define its final acceptance. We intend to discuss some of the most common quality factors assigned to lyophilized products examining each for its implications and limitations. It is never the case that all factors can be maximized, a rational selection based on the formulation must be achieved. The economics of freeze drying do not allow for both the cost-efficient production and ability to obtain the highest quality score across all descriptors, with a mixture of factors being the most common outcome.

Morphology

Physical appearance is the most common quality factor for any product. However, its priority with respect to other descriptors is important. It would be important to describe any particular appearance in color, shape, texture or porosity that is a priority, as during cycle design color may be lost without affecting taste.

The morphology of a freeze-dried cake is often seen to provide reassurance of quality to an end user. In particular, physicians are known to reject materials with poor visual appearance. It is largely a subjective measure but a cake volume representative of the original frozen mass with consistent coloring and minimum surface abnormalities are typically considered ideal freeze-dried materials.

To alter morphology, annealing during the freezing step can be adopted. To anneal a formulation it is held frozen at an elevated temperature for an extended period often above the glass transition temperature of the maximally frozen concentrate (Tg’) or eutectic temperature. The crystal reorganization of ice and eutectic phases will often reduce product resistance during sublimation with a resultant decrease in primary drying time [10].

Processing of freeze-dried materials as dried products demands free flowing powders and is found in both food [11,12] and pharmaceutical industries [13,14], careful characterization of the morphology is needed to allow processing or packing. Primary drying temperatures above glass transition or collapse are known to significantly affect morphology producing shapes pyramidal in structure or high moisture content film residues. To maintain flow properties a consistent morphology must be demanded [15].

Figure 1. - Three-dimensional cross-sectional X-ray CT into packed powders. Left: Freeze-dried mannitol powder. Right: Fluid bed dried mannitol powder. Scale bar: 1.25 mm

X-ray micro-computed tomography (X-ray CT) is allowing the investigation of the micro-structure of lyophilized powders (Figure 1) providing data on surface area, particle orientation and particle size distribution of the freeze-dried powders. Previously, Parker et al. [16] quantitatively studied the micro-collapse of Bovine Serum Albumin lyophilized cakes, correlating porosity and connectivity with electron microscopy images in freeze-dried cakes.

It is suggested that the non-destructive analysis of powders with this technique may expand to allow characterization of characteristics as diverse as flow and dissolution through surface area and geometric modelling. Unlike scanning electron microscopy (SEM) it has the ability to examine the internals of a sample without disruption of the original sample-defining morphology within the highly heterogeneous freezedried cakes.

Activity

Intrinsic activity of an Active Pharmaceutical Ingredient (API) will affect the priority of any activity quality factor during cycle design. Although bulk freeze drying is commonly used for small molecules [17] such as antibacterials (e.g., Azithromycin [18], Clarithromycin [19]) and anticancer agents (e.g., Doxorubicin, Cisplatin [19]) there have been clear trends to explore larger sensitive bio-molecules.

Small drug molecules are intrinsically robust through primary and secondary drying. Extensive reviews of small molecules [17], biopharmaceuticals [20] and their excipients approved over the decades by the Food and Drug Administration (FDA) have been made. Common concerns for small molecules during lyophilization include avoidance of melt back as a result of change from solid to a liquid state [21] during primary drying.

Protein API’s have particular stability issues and require careful control of sublimation below critical processing temperatures (T_c or T_g’) to avoid activity loss [38]. The three-dimensional structure of proteins make them particularly sensitive to over drying or under drying, requiring carefully titrated levels of moisture [6] within the powdered state.

Obtaining a frozen matrix of a solution can often determine final activity. During the freezing step high degrees of super cooling affect activity recovery for enzymes with an inverse relationship between the level of super cooling and activity [22]. The higher interfacial areas between ice and concentrated solute enhance adsorption and have been linked to reduced activity caused by aggregation and protein unfolding [22- 26]; however, for other typically smaller API’s the interfacial area is of less significance. Adopting a reproducible ice nucleation process is desirable. Simple and more advanced ice nucleation techniques have been studied using; nucleating agents [22,27,28], ultrasound [2,29], ice fog [30,31] and electric filed [32] which are discussed by Bursac et. al. [33]. A more recent approach involves pressurization of the product chamber with argon gas to 26-28 psig, cooling to a desired nucleation temperature and depressurizing the chamber to 1 psig triggering a precise nucleation point [34]. The aim of all nucleation methodologies is to avoid high degrees of super cooling and induce controlled nucleation at high temperatures for production of large ice crystals with minimal batch variation [28]. The large ice crystals formed minimize the ice water interface preventing protein adsorption and the consequential denaturation while maximizing recovery [22, 29, 33].

Dissolution

High dissolution rates for freeze-dried materials are common. However, where further enhancement is required, choice of excipients and cycle design may affect a product significantly. Non-reducing sugars are commonly used excipients and the use or optimization of the differing physical states of a formulation can affect the dissolution as defined in the Noye’s Whitney equation [35] as adapted:

Where ‘D’ is the diffusion coefficient (of the active molecule), h is the thickness of the diffusion layer surrounding a surface (A). Cs is the saturation solubility of the drug in the diffusion layer. Ct is the concentration of the drug in the reconstitution medium at time t. dm is the mass change in solution over a period of time (dt).

Formulations with a high level of nucleation sites produce small ice crystals with a resultant large surface area and porosity. This change in surface area (A) provides a higher dissolution rate (dm/dt) for a short reconstitution time. In contrast, annealing produces large ice crystals with small surface area in the associated phase (A) this increases reconstitution time (dm/dt). This affect was seen as an 18-fold decrease in dissolution rate for recombinant human interferon-γ cakes [36].

Solutions with high masses of dissolved solute are seen to reduce the level of crystallinity (D) during freezing often forming amorphous disorganized states. Freeze-dried pegylated or high-level sucrose (>50mg/ml) systems have often been shown to have shortened reconstitution times [37]. Other approaches have included altering the reconstitution medium (C_s) or resorts to modify the actual API (D) [20].

Long-term Storage

Storage can be considered effective in even poor freeze-drying processes, this is due to degradation rates in powders often being in orders of magnitude less than those of the solution. For bulk freeze- dried materials a long-term storage of two years would be a common expectation.

The degradative processes common to lyophilization [38] are predominated by hydrolysis. Careful optimization of the secondary drying phase [6] can minimize the final water content while avoiding over drying or scorching of a powder.

Selection and control of excipients can extend the shelf life of products. Fully crystallized mannitol was shown to prevent escalation of moisture levels in Bacillus Calmette-Guérin (BCG) vaccine on long-term storage [39]. Conversely, many proteins have been reviewed [38] to show damage when exposed to crystallization during storage. It is of particular note that unstable excipients may release moisture to a system or change on extended storage. An unstable mannitol hydrate required extension of the secondary drying period by 13 hours to transform the powder state for long-term storage [40]. In these cases, it is important to have a clear understanding of the degradative stresses for the individual API and control crystallization through annealing and the regulation of moisture levels.

Exclusion of reducing agents is common; these slow reduction reactions are able to damage API’s over extended periods of storage. The Maillard reaction describes when reducing sugars are oxidized by amino acids producing a browning of a formulation over long periods, moisture levels can retard or accelerate such a reaction [38,41].

Cost

Although not a direct factor for a formulation, the economics [42] of a freeze-drying process may override all other quality factors. This is particularly the case for low-value solutions; but may be particularly of concern for re-optimization of an existing freeze-drying cycle or development of a generic pharmaceutical copy. The cost of freeze-drying cycles is often dictated by how well optimization was performed to allow scale up. Scale up of freeze-drying cycles has been discussed in depth [43], with particular importance placed on the monitoring and feedback of this information to the stages of drying. Limitations of the final production machine should be considered, as fast cycles often include high rates of drying requiring equally high condensation rates not available on all systems.

The cost of maintaining high vacuums and providing both low temperatures for condensation and high temperatures for sublimation drives the economic cycle around the batch production of freeze-dried items. Labor provides additional expense when a cycle is unable to complete on a convenient daily cycle resulting in driers being active but not productive. Any process that can be adapted to reduce these running times or minimize staff will benefit the associated costs. Bhambhani et. al.[44] provided discussion of primary packing in detail, while it was clear bulk drying provided the most basic of container system its use can be economical. Bulk freeze-drying maximizes shelf area minimizing costs of thermal transfer. Avoiding fill depths in excess of 2cm [45] is common to prevent product resistance to vapor flow limiting the maximum primary drying rate. However, products of high solute content may require special attention to prevent significant extension of the primary and secondary drying periods [45]. High super-cooling rates are to be avoided as production of large numbers of small ice crystals increases final product surface area increasing product resistance to vapor flow during sublimation [29,46] and increase total running time. Nucleation at higher temperatures is of particular use in the production of large contiguous ice crystals suitable for higher sublimation rates [28,33].

The role of controlled ice nucleation on the total length of some newly developed cycles may be self limiting since any resulting decrease in sublimation time may be offset by an increase in desorption time [29]. However, practically it appears that controlled nucleation does provide benefit, cycle lengths reducing by between 14 -41% [29,34,28].

Membrane trays of polypropylene and polytetrafluoroethylene laminate have been proposed which are able to minimizes product loss or blow out from trays [47] during or at the conclusion of drying [7]. Gassler and Rey attributed the superior heat transfer observed in membrane trays to low thermal inertia. Membranes were associated with a 20-30% reduction in freezing time and have been linked with 20-30% reductions in drying time when compared with stainless tray systems. However, the advantages of stainless steel trays are lost and product transfer may become restricted with less protection from rupture of the container. It is also clear that membrane sealed trays may demand a lowering of the sublimation rate requiring increased cycle lengths [48] due to container resistance.

The rule of thumb that a 1°C rise in product temperature will decrease primary drying time by about 13% is commonly quoted [45] and governed by the sublimation rate defined by [10] :

Where ‘dm/dt’ is the sublimation rate or mass transfer for water vapor (kg/s), ‘P0’ is vapor pressure of ice at the product temperature (Pa) , ‘Pc’ is chamber pressure (Pa), Rp is product resistance (Pa.s/kg) and ‘Rs’ is stopper resistance (Pa.s/kg ). During sublimation an increasing thickness of dried layer forms. This layer resists the flow of water vapor through the solidifying cake with no direct proportionality to mass flow. It is unaffected below collapse temperatures but may show dependence to any resistance within the container used.

Defining critical formulation temperatures is key to cost reduction over the long term. By using temperatures close to those for formulation collapse or glass state transition energy transfer is maximized, maintaining the highest sublimation rates possible. These temperatures can be exploited in the process of micro-collapse when the lower resistance of the dry layer between T_g’ and T_c allows enhanced rates of sublimation to be achieved.

High protein or solute concentration may contribute to alterations in the T_g’ or collapse of a formulation. Increased Bovine serum albumin (BSA) concentration has been found to increase T_g’ and T_c allowing aggressive drying temperatures [16]. Despite a low -32°C T_g’ for sucrose, 5 mg/l and 50 mg/l BSA can be successfully dried with a 25°C shelf temperature in the presence of sucrose with cakes finally exhibiting 2-year stability profiles inline with those freeze-dried at -25°C [16] .

Crystallizing agents may provide scaffolds to allow micro-collapse while maintaining macro structure by taking advantage of the high eutectic temperatures of sugars or salts. Mannitol sucrose mixtures have been dried at -10°C while avoiding visual signs of macro-collapse, exploiting the high eutectic temperature of mannitol -1.5°C [49]. Care, however, must be taken to consider the consequences of incomplete crystallization for long-term behavior.

Summary

Pharmaceutical scientists who bulk freeze dry need to foremost identify what quality factors are of a priority during cycle development since the economics of freeze-drying do not allow for both the cost-efficient production and the ability to obtain the highest quality score across all quality factors. Consider; morphology, activity, dissolution, long-term storage, packaging and cost.

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Author Biographies

Edmond Ekenlebie is a postgraduate in the laboratories of Dr. Andrew Ingham. A Pharmacist since 2006, his interests include the storage of biological molecules in the powdered state for pharmaceutical delivery. His current work is focused on lyophilization of antibody formulations and the characterization of bulk freeze-dried powders. The group based at Aston University in Birmingham UK has knowledge spread across the area of drug delivery with specific interests in lyophilization, transdermal and inhaled delivery using both in vivo and in vitro models, with close support for technology from a team of dedicated engineers.

Dr. Ingham qualified as a pharmacist in 2000 gaining his doctorate from the London School of Pharmacy (University College London) in 2004 for his work in freeze-drying technology. His research led to a position in a consultancy directly tailored to freeze-drying (lyophilization) and its associated powder technology. At Aston University he continues his characterization of freeze-dried material, within EU and MOD funding.