Prospects in Lyophilization

The development of Lyophilization/Freeze-Drying is almost a century old venture since the 1906 publication of Bordas and d’Arsonval¹. In the first part of the 20th century, pioneering work in the field has been carried out by many remarkable scientists, such as Altman, Gersh, Greaves, Flosdorf², Henaff and others, but it is mainly under the pressure of servicing the second world war battle fields that Lyophilization took its marks with freeze-dried plasma and later under the impetus of the Nobel Laureate Ernst Chain with freeze-dried Penicillin. As any new technology, the field of freeze-drying opened more and more and became strongly rooted in the biological, pharmaceutical, food and cosmetic industries. In the early sixties, many products in the chemical field and even radioactive wastes were dried from the frozen state. Thus, the new technology diversified and, as it is often the case, some cross-fertilization took place between the different applications bringing new opportunities in already “classical” areas. The purpose of this article is precisely to give an overall survey of this evolution and attempt to identify the mainstreams of further diversification of Lyophilization. As such, it was compulsory to review a certain number of scientific and industrial areas that lie outside of the main focus of pharmacy.

As this is a most general case in science, Lyophilization has been propelled in the last decades by a push-pull phenomenon between products and technology. New products fulfill new needs; open new markets but, in turn, request new developments in process and industrialization. Sometimes they are purely genuine; sometimes they are just a consequence of the spin-off of other technologies. This article highlights various topics from basic science to engineering and from technology to consumer acceptance.

I Biological Products and Pharmaceuticals

1. Some Basic Issues

For a very long time, Freeze-Drying has been essentially concerned with the preservation of unstable biochemicals and, more particularly, of injectables. The very first issue has been, then, to secure their sterility and safeguard their potency during processing, storage and reconstitution. At the onstart most products were freeze-dried as such, either as a natural substance, such are blood plasma, biosynthetic isolates or antibiotics or else, for vaccines, as controlled concentrates of inactivated and/or living cells resulting from selected culture fermentation broths.

Often the product was bulky and resistant (like blood plasma) and could be processed directly. However, as more and more new advances were made, many products became difficult to treat and required the incorporation of a whole set of additives: cryoprotectors, free-radical scavengers, stabilizers and so on. Today, and with the rocketing development of biotechnology, more and more potent and fragile compounds are produced and, most often, their activity is such that they represent only a few milligrams in the basic formulation. There is, thus, an obvious need to incorporate the active substance into a solid matrix which will prevent it to fly away during drying and keep it secluded in a confined stable environment during storage. This means that, on top of the already quoted cryoprotectors, stabilizers and the like, different bulking agents have to be added like sucrose, mannitol, lactose …, the behavior of which might have a major impact on the properties of the active substance. This is particularly true for proteins where a given steric configuration should be preserved during the whole process and withstand the stress of freezing and drying. Carpenter, Pikal³ and others have shown that developing a glassy matrix during freezing could substantially reduce the osmotic and mechanical strains to the molecules. This, unfortunately, might also be contrary to the requirements of the drying process itself, which is easier to carry from a non amorphous state. Thus, it might be necessary to mitigate between the conventional freezing and what we called in 1960 a "thermal treatment," most often referred to today as an annealing process: double freezing with intermediate rewarming.

Depending upon the products, there might be very narrow margins to that exercise and the end-points’ temperatures. Velocities of cooling and rewarming need to be known very accurately by previous laboratory determinations such as Differential Thermal Analysis (DTA) or Differential Scanning Calorimetry (DSC), Low Temperature Electric Impedance measurements, velocity of crystallization in the supercooled state …

Moreover, in this context of highly diluted active substances of powerful potency (like for instance Botulinum toxin) there might be a strong interference between the product and the container-closure system. Glass manufacturers have pioneered in the field showing that a Type I tubing vial might interact with the solution prior to freezing, and this is often in an irreversible way. For instance, it might adsorb more than 50 percent of an active protein⁴ (tests have been made with a nicotinic acetylcholine receptor) and equally leach substantial amounts of undesirable glass components (sodium, calcium, boron, aluminium …) into the solution which may also dissolve the inner surface of the container. To obviate those problems, a glass manufacturer developed the Type I Plus vials in which, thanks to a special high temperature plasma process (PIVD: Plasma Impulse Chemical Vapor Deposition), a very thin coating (100-200 nm) of pure silica is deposited and strongly bonded to the glass surface (something like a quartz glazing of the inner wall of the vial) which becomes then totally neutral. Much interest is given today to this new process, at a time where Regulating Agencies, and especially FDA, are deeply concerned by the effect on brain and liver of aluminium released from glass in very young infant vaccines. Silica coating affords then an excellent protection, still better than some new cyclo olefin polymers (such as Topas 6013) that could be also used to manufacture the entire vial. Novel design of vial⁵ have been developed which have compartments so that drugs are kept separate from filling until just before administration. By this drugs which react together can be stored in the same vial.

What is true for the container in the "container closure system" is equally true for the closure alone and, essentially, for the elastomeric stoppers, generally butyl-rubber, which cap the vial. Fran DeGrazio⁶ has studied many alternatives to their formulation trying to keep down the extractables (essentially volatile) and leachables and prevent adsorption of oils, waxes, polymers and others on the freeze-dried plug. Innovations in the stopper design like presence of ventilation holes and sterilized filter⁷ prevent contamination of the lyophilized product with bacteria, dust etc.

Another critical issue in the same field is the determination of the optimum residual moisture, understanding that it could result from the drying process itself obviously, but also from release by the stoppers of water picked up during the sterilization process⁸. Fran DeGrazio, Maninder Hora, and others did show that this phenomenon definitely influences the storage ability of the freeze-dried products, which also depends, among other issues, upon the quality of the fit between the vial and the stopper which, in turn, depends upon how tight the manufacturers can guarantee the dimensional tolerance of their products⁹.

Water is, indeed, a recurrent and unavoidable issue in the whole Lyophilization process. Formally regulated by the different responsible agencies, the residual moisture is, indeed, a floating, nebulous concept which is quite difficult to grasp with exactitude. Neither Karl Fischer titration¹⁰, neither Thermogravimetry nor Equilibrium Water Vapor Pressure Determination could bring the right answer. Moreover, the first two techniques being destructive, they prevent any follow-up of the "fate" of the water in the product - with time. They titrate the "total water," as a chemical, without discriminating between the part which is free to move and exchange between the stopper and the cake and the one, bound to the cake, which is often essential to maintain within the freeze-dried active substance, the tridimensional structure which is at the root of its potency. The Equilibrium Water Vapor Pressure method solves the follow-up issue since it is a non-intrusive, non destructive technique but it gives an "indirect" reading of the water content. Finally, in the dry product itself and without any interference from the container-closure system, there are movements of water with time and, that, during storage, the ratio between "free" and the different types of "bound" water changes and might impact the final potency.

These are only limited glimpses at the basic issues that any professional has to challenge when performing freeze-drying of biological products and pharmaceuticals.

2. A Few New Original Technologies

There are several interesting break-throughs that could be applied to Lyophilization of Biological Products and Pharmaceuticals.

2.a. Inert Carriers

When dealing with a highly diluted active substance, try and get rid of the bulking agent. One possibility is to enclose the liquid to be dried into a porous matrix where it will be kept, in the course of drying, and thus, prevented to fly away with the water vapor stream. Porous polymers, sintered metals, ceramics, porous glass, inorganic textiles, multilamellar pads … could, at first glance, bring a solution provided they demonstrate simultaneously, a certain number of properties:

• They can adsorb the liquid solution easily as a hydrophilic material and withstand freezing and drying without mechanical rupture.
• They do not interact with any element of the formulation.
• They are clean and deprived of residues, particles or contaminants.
• They hold enough liquid per unit volume, which means that they present at least 30 to 50 percent porosity.
• They can be shaped as well defined geometrical units: discs, rods, spheres … to be incorporated into the vial.
• Their pore structure is thin enough to hold the solution but wide enough to let the water vapor escape from the frozen liquid which means an open-pore structure, a "sponge" with interconnecting interstitial channels.
• They release the key enclosed adsorbed product when they are flooded with the reconstitution fluid.

Fulfilling these requirements is not an easy task, and actually very few products are susceptible to provide this complete set of properties. Whilst most of the structural issues can be solved, the most difficult, by far, remains the latter one: The "carrier" should release the totality or at least a major known amount of the active substance at the time of reconstitution. To that end, developments have been made to "exhaust" the carrier by percolating through it the dissolution fluid under pressure, as would be done with a conventional on-line filter. Special syringes have been manufactured where the original solution to be dried is pumped through the carrier, then allowed to dry there and finally extracted by the reconstitution fluid using the same type of mechanism.

Today, the inert carrier issue is still under development and it is more than likely that the enormous amount of research which is currently devoted to new materials, whether glass and glass derivatives, polymers, fibers … will bring some challenging contributions in this area.

For some dermatological and surgical uses, and also in the vast field of cosmetics, freeze-dried carriers have been in use for several years. They, generally, consist of natural products like collagen, agar-agar, vegetal extracts which are doped with very small amounts of active substances freeze-dried and used as dressings on burns, scars, bleeding areas or as beauty masks on the face and/or eye-lids. Depending upon their formulation, they can either be stripped off after release of the active compound or else remain in situ where they dissolve away in a rather short time in the case of dressings or are easily washed out for beauty masks.

Whilst carrier technology appears as rather specific to the pharmaceutical and cosmetic industries, the next developments that are described herewith are actually the results of technology transfers from the food industry, namely soft ice, continuous freeze-drying, and Radiation Treatment.

2.b. Soft-ice

Everybody is familiar with the concept of sherbets and soft ice creams. They are basically frozen plastic pastes which present a high viscosity at relatively moderate negative temperatures. They are not free-flowing but can be stirred, mixed under moderate mechanical strength and can incorporate solid particles like chocolate crumbs, fruit, nuts and others without losing their structure. Their rheological properties are rather complex and highly dependent upon temperature.

In the preparation of many pharmaceuticals some components in the formulation are not compatible together. For instance, if acetyl salicylic acid is mixed with sodium bicarbonate in solution, there is an immediate reaction and a vigorous release of carbon dioxide. Nevertheless, it might be of interest to freeze-dry them together in order to have instant sparkling aspirin, but this is not possible and the only way around this is to compact the products together in the dry state. The resulting tablet is not very stable and it takes a relatively long time to get back into solution.

Conversely, if such reactive substances are mixed together at a low temperature after incorporation in a soft ice, they will not react and the resulting paste can be molded in appropriate shapes and hardened by further cooling. The material can then be freeze-dried without difficulties. In that state, lyophilized aspirin, for instance, is perfectly stable and, when water is added back, it reconstitutes as a sparkling fluid in a few seconds because of its high porosity.

The "soft ice" technology is thus a very precious tool to prepare complex products, most often for oral route. Pure chemicals, drugs, vitamins, and mineral salts, can be successfully freeze-dried in that way in rather elaborate formulations since it is possible to mix together "sherbets-lines" issued from different solutions and even add to the whole other solid ingredients as finely dispersed powders. The key to success in that process is a good control of the temperature of the icy paste, which is generally prepared in a cylindrical double wall heat exchanger with a continuous scraped surface maintained at temperatures between –4°C and –20°C, depending upon the nature of the treated products. When the soft ice mixture is duly completed, it can be molded by conventional equipment in plastic blisters and frozen hard in a blast tunnel. Then, this material can enter the freeze-dryer. Altogether, it is a rather simple on-line galenic process.

2.c. Continuous Freeze-Drying

Any chemical engineer dreams of a continuous process because it is easy to control and gives manufactured products a consistent quality. Freeze-Drying has not escaped this trend and, as early as in the 60's, semi-continuous to continuous equipments have been designed and built for the food industry. LEYBOLD was among the very first to do it and G.W. Oetjen developed the C.Q.C. process into an industrial reality for milk products. The operation was, indeed, sequenced into several phases. The frozen products, most often under granular form, were loaded on trays placed on a special carrier, hung to a monorail which traveled all along the freeze-drying tunnel between heating plates in successive steps through vacuum locks closed by sliding gates. From the entrance lock to the outlet, the total cycle time was of the order of several hours.

An alternative to this system was introduced by ATLAS who pioneered the so-called "Conrad" System in which the loading of the frozen goods was done tray by tray through a small side lock. Coffee, milk but also vegetables, fish fillets, meat have been successfully treated in that way, essentially by radiant heat.

Quite obviously this technology worked, but it was still a semi-continuous process and the Food Industry was eager to develop a fully automated continuous operation for one of its leading products on the international market: instant coffee. Conventional process involves the extraction of low temperatures that do not strip off the negative aromas as found in conventional spray dryers because of the high temperatures. Freezing of the concentrated extract is critical since density, colour and brittleness of the frozen foams had to be made just right. Other processes that had to be taken into consideration were grinding to a regular granular size, sublimation and desorption avoiding loss of volatile aromas.

Thus, it is not surprising that it was only in the mid 60's that a fully continuous line was introduced. In one of the most advanced designs, granulated frozen extract is fed continuously into the dryer through a rotating lock and deposited on a 20 to 30 meters long vibrating tray on which it travels as a fluidized bed. It moves, indeed, on its own water vapor cushion all along the heated surface which provides the energy for sublimation. During that long transport, the granules are guided by vertical ribs into parallel channels and great care is taken to prevent attrition from mechanical chocks between the granules themselves and with the surface and walls of the tray. Though limited, attrition nevertheless does occur and generates a lot of "dust," which has to be stopped by a long semi-cylindrical screen deployed all over the vibrating tray. In that way, the fines are not carried away with the vapor stream towards the condenser and the pumps.

Under those circumstances, it can be easily understood that a continuous freeze-drying plant is a highly sophisticated piece of equipment which is designed and put together by the Food Industries themselves who keep the whole development as strictly confidential and assemble different components purchased from multiple unconnected manufacturers.

The efficiency of a vibrated tray freeze-dryer is enormous, and the drying times drop by more than one order of magnitude. We are speaking in terms of minutes instead of hours, and throughputs of tens of tons per day are no longer unrealistic. Moreover, the process is "intellectually clean" and the end product constant in quality.

This technology has been introduced to the processing of biologicals and pharmaceuticals. Indeed, despite the elaborate design proposed by equipment manufacturers and the care that the drug companies take of their freeze-drying operations, recurrent problems of potential heterogeneity between vials and ampoules within a same single batch are faced . The spatial distribution of 10,000 to 100,000 vials in a multi-shelf freeze-drying cabinet remains a problem. Some sit close to the door, some right in the middle, others near the condenser, some in the upper shelves, others down and, seriously, they do not dry in the same way. Drug manufacturers claim that all the products that they release are within very strict standards and the Regulatory Agencies keep a close eye on that issue and that they are more and more stringent on the validation tests. It remains, anyhow, that the homogeneity of a single batch is still a matter of deep concern. Can this problem be solved ?

A very simple idea is then to switch to a semi-continuous or continuous process as this is done in the Food Industry. Let us forget about the holy paradigm that a vial, which has been initially loaded with a given volume of solution, has to remain so until it is capped with its freeze-dried cake inside and placed into the commercial box. Let us try to consider a process in which the initial solution is distributed as individual droplets frozen into spherical granules of given geometry, continuously fed into a vacuum chamber and spread on a heated conveyor. On that tray, most probably a vibrated tray, they glide self-suspended on their water vapor cushion as a thin, regular, fluidized cloud at a well controlled operating pressure fit for sublimation, say a hundred microbar. At the end of the tray, they reach a transfer lock which discharges them on another conveyor placed in a second chamber fit for desorption and secondary drying at pressures of some tens of microbar. Finally, they enter an ultimate lock and are discharged into a receiving bin under dry neutral atmosphere. The efficiency of such a process is tremendous, and, if we have granules of 1 to 3 mm size, the whole freeze-drying operation might take less than 30 minutes!!

Now, at the end of the road, we have a population of granules, all dried in the same conditions, all equal in quality and size that can be numbered and fed into dry sterile vials as so many distribution machines can do. The result is a batch of identical vials containing, say 100 ± 1 freeze dried granules instead of a more or less regular cake, painfully manufactured over several days. Moreover, the product "elegance" is maximized and, in that dispersed form, its reconstitution is a matter of seconds.

This is not wishful thinking since we do have the basic knowledge, the practical know-how and a multi-year experience of this type of operation. It is just a change of mind, a new approach which deviates from historical practice. Unfortunately, we know that, this is sometimes more difficult to pass.

Some people will claim that instant coffee manufacturers do not care about sterility, that individual freezing of small droplets is a difficult undertaking, that maybe mechanically the granules will not prove resistant to the process… We know quite well that nothing yet has been completely solved, but we do have industrial solutions to those specific issues and they can be successfully challenged if, at the same time, some developments are made on the formulation side.

2.d. Irradiation Technology

Almost a hundred years elapsed since Becquerel, Roentgen, Pierre and Marie Curie and other pioneers discovered the fascinating kingdom of radiant energy, but it is in the 30's and the following decade that the main advances were made, inter alia, with the introduction of Van de Graaff accelerators, cyclotrons and the like. As usual, these developments did benefit from the War and in the 40's and 50's an enormous amount of basic and applied research was done in this field and this within almost every compartment of Science: radioisotopes for medicine, fundamental approach to the structure of atoms and to particle physics, design and operation of nuclear reactors, and last but not least strategic atomic weaponry.

At first glance, all these developments had little to do with the very basic, down to earth needs of the Food Industry. However, this business was still struggling with the adequacy of a geographically large, year round consumer market with seasonal localized natural resources and the profession needed ways and means to collect and preserve these unstable products from agricultural or marine origin. Heat treatment, sterilization or pasteurization, cold storage and distribution were, of course, of current practice but sometimes they did not give all the expected results. This is the reason why, in the early 60's, some pioneers like Henri Vidal, who came from the Refrigeration Industry, thought that, maybe, it could be of interest to try and resort to radiant energy. Numerous tests were done, then, with the Gamma Rays of Cobalt 60 and it was shown, among many other things, that it was possible to prevent germination in stored potatoes, to radio-pasteurize and radio-sterilize natural and processed perishable products. Some years later, the same type of development was achieved with electron beams. Of course, this process was not universal and often it altered substantially the organoleptic properties of the treated goods. There was also, in medical circles, some deep concern about the remnant free radicals generated in the irradiated products: they were supposed to lead to cancerogenesis.

The second input that radiation technology could give to Lyophilization has to do with the products themselves. In a large freeze-drying cabinet holding 10,000 to 100,000 vials, we are not completely sure that all vials are strictly identical at the end of the run and what is valid for their physico-chemical properties, residual moisture and potency is equally valid for their sterility. Can we really rely on a handful of tests on isolated vials to claim that the whole batch is 100 percent sterile? We know, quite well, that the key answer to that question lies beforehand and that the only way to secure final sterility is to make sure that the whole process has been duly carried from the beginning under strictly sterile conditions. This compulsory requirement, unfortunately, is difficult to fulfill and, to the evidence, it adds tremendously to the overall cost.

In a continous process ,complete sterility can be ensured by entering the finished products through a E-beam or window of X-ray tube and can be sterilized in depth,thereby ensuring 100% guarantee of sterility and at the same time substantially decreasing the manufacturing cost. The following parameters are to be considered before we resort to irradiation technology :

1. The dose to be delivered depends upon the initial level of contamination of the product.25 kGy is the regulatory figure but if the processing is done in a clean & hygienic way , even 5 kGy might prove to be enough.
2. The dose delivered to assure sterility does not affect the product in its therapeutic and physiological properties.
3. No foreign element should appear by radiolysis which might have a deleterious effect on health as well as within the other ingredients of the formulation.

For instance, together with Aguettant, that in solution, heparin, morphine and apomorphine were almost totally destroyed by irradiation, but that if they were irradiated in the frozen state, at a low enough temperature, 100 percent of the activity/potency could be preserved. Nevertheless, at these low temperatures, in the frozen state, they became totally sterile under irradiation even after a massive initial contamination with Bacillus Pumillus.

In very recent experiments done in close collaboration with the Center for Biologic Research and Evaluation of the U.S. FDA, Joan May and Louis Rey have been able to demonstrate that the potency and chemical integrity of different polysaccharides of major significance in injectable vaccines can be safeguarded, which had received up to 25 kGy by Gamma Rays (in Celestin, Marcoule and Cigal, Cadarache), E-beam (at Studer's Werk) and X-Rays (at RDI IBA).

The road looks now open. It is rough but a worthwhile exercise, since it can result in a much easier processing of sterile injectables with increased security and a substantial decrease in cost. Moreover, it appears as a smart process easy to couple with continuous freeze-drying.

II. Chemicals and Non-Aqueous Solvents

Water ice is not the unique substance which can go directly from the solid state into vapor and we know that, within different temperature ranges, many organic and some mineral solvents can do it. Thus, actually, there is no reason to limit lyophilization to the single field of aqueous products and, in that move, there is also no reason to restrict freezing and drying to the pharmaceutical, biological and medical fields as well as to cosmetics and food.

In the mid 60's Louis Rey et al^11,12 developed freeze-drying from non-aqueous media. The purpose of this article is not to indulge into some considerations on the past, neither to dwell on a topic which lies somewhat outside of the main current of this article, but the ideas are still in the pipe and they might trigger some interesting new ventures even in the most conventional areas of freeze-drying.

Since it is a rather diversified field, we shall try to scan it quickly and spot what appear as promising issues.

1. The Use of Non-Aqueous Co-Solvent Systems

The very basic idea is to freeze-dry aqueous solutions which contain substantial amounts of organic solvents because their presence brings a marked improvement into the results, for instance: increasing the rate of sublimation and chemical stability both of the pre-dried bulk solution and of the dried product; improving wettability, solubility and reconstitution of the dry product; enhancing sterility assurance. Of course, there are also many potential draw-backs: toxicity concerns, operational hazards, presence of undesirable contaminants and, as can be easily understood, an increased cost and adverse regulatory issues. However, in some cases the balance proved to be positive and, in the Caverject‚ Sterile Powder Project, Pharmacia was able to prepare stabilized Prostaglandin E1 by freeze-drying a 20 percent (v/v) tert butanol/water co-solvent system.

Similar cases have been reported for Annamycin (Tert-Butanol, Dimethylsulfoxide, water), Cephalothin sodium (50 percent w / w Isopropyl alcohol / water), Dioleoylphosphatidylcholine (Cyclohexane) and many others.

We can even use a pure non-aqueous solvent alone. In 1964,some successful experiments were done with phospholipids freeze-dried out of carbon-tetrachloride solutions using glycol disterate as the bulking agent.

No doubt that there are a great number of products which could be studied along those lines, still bearing in mind, however, that under present regulations, most of these processes can tumble on pure toxicity and tolerance issues.

These, however, are less stringent for products which are not penetrating the body by the oral or parenteral routes and there are still very large openings for solvent freeze-drying in dermatology or in the cosmetic field. Though, of course, great care has still to be taken of their systemic penetration throughout the skin, if any.

2. Complex Freeze-Drying

In that specific area, freeze-drying can offer even more if we consider the processing of different solvents mixed together. We have discussed the behavior of those systems in 1966 introducing then the concept of "complex freeze-drying" which is closely linked to the physical characteristics of the different solvents.

Depending upon their miscibility, freezing point, and saturated vapor pressure in the solid state, there are two different freeze-drying strategies:

2.a. Joint Complex Freeze-Drying

This refers to solutions made of two or more solvents, either easily miscible, or emulsified together, containing two or more active solutions and their accompanying ingredients (including the ad hoc bulking agents).

After freezing, they can be dried by sublimation under vacuum according to two different modes.

• Simultaneous Lyophilization

Both or more solvents sublime at the same velocity and, thus the freeze-drying boundary moves inside the frozen mass at an even speed. This is typical, for instance, for a mixture of dioxane and water.

• Step Wise or "Scaled" Lyophilization

One of the solvents is more volatile than the other because of its higher vapor pressure in the frozen state and it disappears first, followed at different speeds, by the others. The frozen mass then is "carved" from the inside along the crystallized pathway of the first solvent, and, in this open network, the "islands" of the second solvent are progressively extracted. This is the case for a mixed solution of cyclohexane and water when 100 percent of the cyclohexane has already been sublimated away when more than 85 percent of the water-ice is still there.

2.b. Successive Freeze-Drying

In that process the system under investigation is freeze-dried several times in succession resulting into a final dry product which has an intermingled structure of interlocked membranes. To that end, we should make sure that each "new" solvent does not dissolve the material coming from the preceding ones. For example:

1. A 1 percent solution of polystyrene in carbon tetrachloride is freeze-dried.
2. The resulting plug is impregnated by a 2 percent solution of dextran in water and freeze-dried.

The final cake when imbedded into an acrylic resin and sliced for microscopic examination shows a very nice structure with a regular lattice of polystyrene membranes in which a sub-lattice of dextran membranes is enclosed.

The whole process was observed in real time using a specially built freeze-drying microscope allowing direct observation of the freezing process and of the movement of the drying boundaries in a thin film.

This sequenced freeze-drying operation was repeated once more, say with a 2 % maltose solution in diethylamine, resulting in a triple intermingled network of three compounds: polystyrene, dextran and maltose and so on …

These complex porous bodies have interesting properties and could, for instance, be used as "intelligent" filters on gases or fluids, each component of the freeze-dried mass having its own selectivity and retaining those substances with which it can interact. When the filtration is over, it is possible then to extract one by one the three "collections" using the original solvents. That process can be used for the selective retention of aromas and has been patented by the Nestle Company .

3. Mineral Solvents

Organic solvents are not the single ones to be used in freeze-drying, and, at the same period, we devoted a large amount of efforts to evaluate the opportunities offered by some selected fluids from mineral origin. The results were promising and we believe that, today, they can prime new developments in what remains an open and challenging field.

• Ammonia
  
  Liquid ammonia NH₃ is a most interesting solvent where reactions NH₂ – H do occur, similar in a way to the OH – H which are found in water. Alkaline metals can react with ammonia giving solubles amidures which most often do not resist a transfer to room temperature. Many organic substances, too, like amino acids, phospho lipids, certain polymers … are soluble in liquid ammonia at atmospheric pressure at temperatures between –40°C and –70°C. Their solutions can then be distributed in vials, frozen with liquid nitrogen, set into a freeze-dryer and placed under vacuum. Under those conditions, solid ammonia will easily sublime at temperatures as low as –130°C and pressures around 20 mbars leaving a dry porous cake containing the original substance (incorporated if required in an appropriate bulking agent) which being now stable can be brought back to room temperature.
  
  On that basis trials were taken to stabilize transient free radicals generated by irradiation at liquid nitrogen temperature. A 10 percent solution of l-lysine was prepared in liquid ammonia at –70°C and frozen with liquid nitrogen down to –196°C. At that temperature, a dose of 10 kGy of Gamma Rays from a Cobalt 60 source (in CEA, Saclay)was given to the system.
  
  The same experiment was done in parallel with a 10 percent solution of l-lysine in water. In both cases, the activated frozen material gave, at –196°C, a strong paramagnetic signal after irradiation. However, if the temperature rose over –100°C, this signal almost completely disappeared, whilst at the same time, evidence of the recombination of free radicals in the solid state could be witnessed by thermoluminescence, essentially below –100°C.
  
  Thus, instead of rewarming the irradiated solutions, they were placed in two different freeze-drying cabinets and sublimed the solvents under reduced pressure:
  
  - at –30°C and 100 m bar for the frozen water solution;
  
  - at –120°C and 25 m bar for the frozen ammonia solution.
  
  When the products were dried, they were brought back to room temperature, placed under nitrogen gas and tested with an ESR equipment. A very faint signal was detected for the product issued from the water solution (relative intensity: 2,000) whilst the lysine dried from the ammonia solution gave a very strong response, 300 times bigger (relative intensity: 6,000,000).
  
  This result was confirmed with other substances and compared to the evidence that unstable free radicals by irradiation generated at liquid nitrogen temperature could be preserved and stabilized at room temperature by freeze-drying of their solution in ammonia at very low temperatures. This, of course, would be strictly impossible in a water solution.
  
  The physico-chemists can capatalize on the possibilities offered by this technology.
  
  
  
  
  
  
  
  
  
• Carbon Dioxide
  
  Amongst the many solvents which can be used to extract and dissolve natural and artificial chemicals, liquid carbon dioxide is one of the most interesting because, not only of its physical properties, but also because of its lack of toxicity. Sparkling mineral waters and soft drinks are good examples. The only serious draw-back in handling liquid CO₂ is that it is compulsory to operate under pressure .There are two examples which involved Freeze-Drying.
  
  In the first one, a pressure reactor in stainless steel, containing a certain amount of dry black tea, through which liquid CO₂ could percolate at –35°C and 13 bars. Under those circumstances and with continuous stirring, and a loop re-circulation, the solvent extracts a large amount of the main aromatic compounds of tea, essentially the most fragile and volatile ones.
  
  The aromatic CO₂ is, then, separated away and expanded through a nozzle giving a fluffy carbonic snow at –78.8°C. This aromatic snow is, then, mixed on-line, with an instant tea powder coming from a conventional spray dryer and, as it sublimes away, it releases directly into the dry product the delicate aromas extracted from the unprocessed tea. What, indeed, has just been achieved is direct freeze-drying of a frozen carbonic acid at atmospheric pressure and low temperature.
  
  In another set of experiments, more connected with the pharmaceutical or chemical industries, an active substance was dissolved - or a more complex formulation including a bulking agent - in liquid CO₂ under agitation. This operation was carried in a special stainless steel tray sitting on a shelf inside a pressure vessel. As in the preceding case, the operation was done at –35°C under 13 bars with a given volume of liquid CO₂. When it was felt that the dissolution is done, liquid nitrogen was fed into the underlying shelf and the solution slowly frozen. During that time and before reaching –78.8°C, the decreasing CO₂ pressure was compensated by a controlled injection of nitrogen gas to keep the vessel pressure around 10 bars. Finally, near –80 °C, the whole material was frozen hard and it was further sub-cooled to –100°C / -110°C. The nitrogen pressure was released and the vessel brought back to 1 bar.
  
  In the stainless steel vessel, we have now a solid disc of deep frozen carbonic acid ice which is ready to sublimate if we provide the appropriate energy .Then solid CO₂ evaporates and the drying boundary recedes progressively inside the frozen slab leaving behind a dry solid porous cake. What is remarkable is that, right to the very last core of sublimating ice, this operation is done at temperatures below or at least equal to –78.8°C and at atmospheric pressure at a relatively good rate. Indeed, depending upon the nature and concentration of the starting solution, the freeze-drying boundary sinks inside at a speed of 3 to 15 mm/hour.
  
  These are only two examples of what can be achieved with CO₂ and it is our firm conviction that many new products and/or ingredients used by the pharmaceutical and especially the cosmetic industry could be processed successfully with this technology.
  
  
  
  
  
  

3. Freeze-Drying and the Major Chemical Industry

Switching from water to co-solvents, then to organic solvents and further to mineral solvents we have progressively shifted from classical pharmaceutical practices to more advanced "exotic" technologies which open the door to major chemical industries.

Indeed, if we can make an indiscriminate use of solvents - except for the usual regulations applied to chemical plants - we can visualize many potential areas in which freeze-drying has already brought and could bring new exciting developments. A few examples are quoted below:

Preparation of porous, expanded, fats, polymers and fibers.

• Preparation of catalysts by freeze-drying their metallic salts which, when expanded as dry porous cakes, can be turned into oxides by heating, then reduced by hydrogen as dispersed metals.
• Preparation of filters in slabs or in powders susceptible to be placed on air channels in fluidized beds or else as selective adsorbents on process lines. In the recent years, interesting approaches were made in this field using freeze-dried silica and alumina for the retention of radionuclides, acid droplets and contaminants in the ventilation ducts of nuclear plants. Besides their great efficiency and low pressure drop, a most interesting feature of these freeze-dried filters is that they can withstand extreme pH conditions and high temperature.
• As a side line, preparation of "absolute" filters with the same freeze-dried silica beads to control air ventilation in highly sensitive laboratories for bacteriology, virology, genetic engineering, P.4 labs. …
• Preparation of pigments and dyes for cosmetics, paints and coatings. Exploratory work has proved very promising. Freeze-dried pigments (most often from their organic solutions) are of upper quality, bright, glittering, with fresh colors
• Decontamination of low to medium activity liquid wastes from nuclear centers. A very advanced work has been done in the past by CEA on that issue and the results were excellent since decontamination factors of 106 or even better could be achieved (Rey, Cerré and Mestre), much better than by single effect or even multiple effect evaporation.

This listing, of course, is in no way limitative and there are many other areas in the chemical industry where Lyophilization could be used. Is this century old technology susceptible to meet this challenge? Shall we not tumble on pure engineering problems and cost as we enter an industrial area where the daily batches might be several orders of magnitude higher than in the food industry? The Lyophilisation industry is definitely positive. Yes, Lyophilization can afford to enter these new areas. The continuous process developed for pharmaceutical and foods can be easily scaled up to handle hundreds of tons per day if needed and the components industries are already able to deliver the corresponding equipment. Large vacuum plants are no threat to the different manufacturers who already build them for steel degassing and casting. As far as the energy requirements are concerned - and since they play a critical role in freeze-drying where we pay four times the change of state (to freeze – to sublimate – to condense the vapors – to defrost the condenser!) - we can find a solution if the freeze-drying plant is positioned in close vicinity to one of these huge storage areas devoted to LNG (Liquefied Natural Gas). Indeed, there we have very low temperatures available at almost no cost, since the liquid methane has to be converted into a simple gas to be fed into the network. Today, this major refrigeration capacity is poorly used. Moreover, the liquefied gas can be boiled under pressure and, thus, provide mechanical energy to drive turbines, alternators, pumps … Finally, the outgoing gas can still be burned to deliver the necessary heat for sublimation. Whilst neither a food industry nor a pharmaceutical plant manufacturing ethical products will ever settle on the premises or even in close vicinity of a refinery or a petro-chemical complex, it is pretty clear that a pure chemical industry would do it right away.

Lyophilization will not remain an exclusive technique for a selected few. It will move as cryogenics did and today when companies deliver liquid oxygen by the hundreds of tons we are pretty far away from the isolated dewar of liquid air prepared a century ago by Georges Claude. There should be not the slightest doubt that Lyophilization can follow this route.

References

1. Bordas F, m .d’Arsonval C.R, Acad.Sci. 1906,142:1058,1079;143:567.
2. Flosdorf E .W, Mudd S: Procedures and apparatus for preservation in ‘lyophile’ form of serum and other biological substances , J.Immunol. 1935,29,389.
3. Carpenter J.F, Pikal M.J, Chang B.S, Randolph T.W: Rational design of stable lyophilized protein formulations: Some Practical Advice, Pharm. Res. 1997,14,969-975.
4. Gombotz W.R, Pankey S.C, Bouchard L.S, Phan D.H, Mackenzie A.P. Stability, characterization, formulation and delivery system development for transforming growth factor beta. In: Pearlman R ,Wang Y.S, eds. Formulation, Characterization and Stability of Protein Drugs, New York : Plenum Press, 1996; 219,245.
5. Mohan ,Yakahin ,Kenkyosho, Japanese Patent No.11114019A
6. Degrazio F, Flynn F: Closures for protein based drugs, J. Parent. Sci. Tech. 1991,46(2),54-61.
7. Lee S.S, Merck and Co. Inc, World Patent No.9925629 A1
8. Chang B.S, Kendrick B.S, Carpenter J.F: Surface induced denaturation of proteins during freezing and its inhibition by surfactants, J. Pharm. Sci. 1996 , 85,1325-1330.
9. House J.A, Mariner J.C: Stabilization of rinderpest vaccine by modification of the lyophilization process, Dev. Biol.Stand.1996,87,235-244.
10. May J.C, Wheeler R.M, Etz N, Grosso A Del: Measurement of residual moisture in freeze dried biological products, Dev. Biol. Stand. 1991,74,153-164.
11. Rey, L.R .Les orentations nouvelles de la lyophilization.In: Rey L.R ,ed. Research and Development in Freeze Drying, Paris : Hermamn, 1964:621-653.
12. Rey L.R, Un de`veloppement nouvean de la lyophilisation : la cryodessication des systemes non aqueux. Experimentia .1965;21:241-246.

Kachhwaha S.J^*, Mandal J.K , Goswami J.M, Sumitra Pillai.

Astron Research Limited, Premier House I, Sarkhej-Gandhinagar Highway, Bodakdev, Ahmedabad-380054, India.

* For Correspondence & Reprints.

Sandeep J. Kachhwaha

M.Pharm.,Ph.D. (Pharmaceutical Sciences) from University of Pune, Pune.

Industrial Experience – 6 years

Assistant Group Leader, Astron Research
Limited, Premier House , S.G. Highway, Bodakdev, Ahmedabad, India.

Publication: 3

Phone : 91 79 6853518,6853492, Fax : 91 79 6840224, Email: skachhwaha@astron-research.com

Left to Right are : Jitendra Goswami , Sandeep Kachhwaha & Sumitra Pillai.

Jitendra M. Goswami

M. Pharm., Pharmaceutics & Pharmaceutical Technology from L.M.College of
Pharmacy, Ahmedabad

Industrial Experience – 4 years

Research Associate, Astron
Research Limited, Premier House ,S.G. Highway, Bodakdev, Ahmedabad, India.


Publication: 1

Ms. Sumitra Pillai

M. Pharm., Pharmaceutics & Pharmaceutical Technology from L.M.College of
Pharmacy, Ahmedabad

Industrial Experience –5 years.
Research Scientist, Astron
Research Limited, Premier House, S.G. Highway, Bodakdev, Ahmedabad, India.

Jayanta Mandal

M.Pharm (Pharmaceutics) from Birla Institute of Technology, Mesra, Ranchi

Industrial Experience –12 years

Assistant Director, Astron
Research Limited, Premier House ,S.G. Highway, Bodakdev, Ahmedabad, India.

Publications: 3

Publication: 1