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Formulation Matters

Jorge Sassone of Improvement-Proquimo explains the formulation stage in lyophilisation, its constituent parts and its importance to the process as a whole

Product formulation is by far the most important part of lyophilisation as a whole since it is where the chosen production method, the utilised equipment and the necessary instrumentation are decided upon. Furthermore, each formulation is a unique system and as such should be treated as an individual case. Nevertheless, we intend here to put forward a general guide for the procedures used in preparing formulations of healthcare products before they are lyophilised. It is important to highlight that the general guide set forth in this article is made from a scientific, riskbased assessment of the formulation. Lyophilised healthcare products may, for convenience, be divided in the following way:

  • Pharmaceutical products
  • Diagnostic products (in vivo or in vitro)
  • Biotechnologic and biologic products
  • Veterinary products

All of these products share the same characteristic. When formulated in an aqueous medium, their stability is limited to a few days or perhaps weeks, even when stored at 4°C. Some of them, when stored at -70°C for instance, exhibit major stability or are actually entirely stable.

FORMULATION

Each formulation is composed of at least one key constituent called the active ingredient; in addition, it might be necessary to include other constituents provided that each one has some proven function in the formulation. In fact, using any additional component in the that does not have a verified function is not recommended as it may alter the thermal properties of the formulation.

These additional constituents often include, but are not limited to:

  • Bulk agents
  • Active ingredient stabilisers
  • Buffers
  • Solvent and co-solvents
  • Cryoprotectant
  • Lyoprotectant
  • Isotonic agents
  • Antioxidants

Characterisation of the formulation is necessary to assure reproducibility from batch to batch, so it is not only important to control active ingredient potency, but the colligative, optic and electric properties of the formulation should also be within certain specified values. The more information there is regarding these properties, the better the confidence in batch-to-batch reproducibility. Among the physical properties for characterising formulations are colour, conductivity, refraction index, turbidity, freezing point depression and osmolarity. During development of a formulation, the principal goal has always been to achieve a stable environment for the active ingredient; it is a pity that key factors such as the thermal property of the formulation, which serves as a starting point for the lyophilisation process, are invariably forgotten. Lyophilisation scheduling is the longest single operation involved in the manufacture of lyophilised products; therefore optimisation of its duration will greatly benefit the costs and yields of a company. That is why care should be exercised in developing formulations with such thermal properties, so that a short lyophilisation cycle can be achieved.

SCIENTIFIC FOUNDATION FOR FORMULATION OF LYOPHILISED PRODUCTS

Active Pharmaceutical Ingredient (API)

In view of the impact they may cause to the thermal properties of the formulation, we can classify APIs into two main categories:

Synthetic APIs
Synthesised through organic or inorganic chemical reactions, they have two great advantages; one is that they can be prepared to a high degree of purity, and may be obtained in crystalline form (crystalline states tends to be more stable than amorphous ones). Oxaliplatin, Letrozole and Leuprolide acetate are examples of this kind of API.

Natural APIs
Obtained by natural processes such as fermentation, or by growing in a culture medium, the API sometimes contains a constituent from the medium it comes from, which might vary in concentration and composition in batch-to-batch production. In other cases however, the API is obtained in a highly purified state. Vaccines are an example of a biological API since they are produced by growing viruses that have infected a chicken embryo. Proteins are examples of biotechnological APIs, since they are normally produced in production stages that are highly complex and may involve sequential steps, such as fermentation or extraction, followed by solvent recovery, HPLC purification, precipitation, filtration, second purification from IIC or gel permeation and so on. In addition, biotechnological APIs include those derived from genetic engineering. In reality, there is still no agreement between scientists on what defines biological and biotechnological APIs, what their differences are, and where the boundary would be between them – if indeed there is one.

In lyophilisation, the formulation tends to form glassy states in the interstitial region of the matrix and the API will be present in an amorphous rather than a crystalline state; there are certain methods for transforming the lyophilised API from an amorphous to a crystalline state. We can highlight the use of organic solvents as a water co-solvent, as they decrease the dielectric constant of the system, promoting crystallisation of the API during the freezing stage. Humidification post-lyophilisation for obtaining crystalline hydrates from previously amorphous lyophilised APIs has been used successfully in several instances such as cyclophosphamide. However, annealing is the most popular method, whereby the API is recrystallised during the freezing step by successive warming and cooling close to the glass transition temperature (Tg) or collapse temperature (Tc). Annealing has also shown a certain level of efficiency in removing some metastable states.

Bulk Agent

It is generally agreed that the bulk agent has the function of increasing both the sustainability and the mass of the lyophilised products, mainly in products with low API doses. We should also consider that bulk agents may function as stabilisers in formulation – for example, acting as a crystallisation bed for APIs and even as a buffer in other cases (amino acids). As a result they should not be overlooked in the lyophilisation process. The necessary characteristics of a bulk agent are as follows:

  • Inert before contact with the API in both liquid and dried forms
  • Physicochemically compatible with every other excipient present in the formulation
  • Non-toxic; no threat to human health
  • Capable of producing the highest possible Tg for the formulation
  • Preferably able to produce a high degree of crystallisation or promote euthectic formation

Manitol, lactose, maltose, dextrose, glycine, CMC, PVP K-17 or -12, sorbitol, sodium chloride and many more have these characteristics. It is important to remark that the total solid concentration of a formulation should range from two per cent weight per weight to 30 per cent weight per weight. Solid concentrations lower than two per cent will promote a poorly self-supporting cake and may also allow solid matter to escape out of the vial during primary drying, resulting in a lack of uniformity of contents in the whole batch produced. On the other hand, a percentage of solid higher than 30 per cent probably yields a dense cake which may cause an impediment for vapour flux during primary drying, resulting in expansion of the process and high residual moisture in the final product.

Buffers

The function of buffers is to stabilise both the liquid and solid phases of the formulation, avoiding incompatibilities between the API and its environment due to incorrect pH levels or pH fluctuations in the formulation. A buffer is a system formed by a weak acid and its conjugated base or by a base and its conjugated acid, so that there is a certain capacity to regulate the pH of the solution between pre-established values when a particular amount of acid or base is added to the system. The capacity of the buffer system will depend on its acid and basic reserves (CA and CB respectively). In lyophilisation, when we have to set the pH level inside the range of 3-10, we always use buffers. In general, in formulation for lyophilisation, the CA and CB range from 0.001M to 0.1M. Examples of such systems are NaH2PO4 /Na2HPO4, citric acid/mono sodium citrate, tartaric acid/sodium tartrate and acetic acid/sodium acetate.

Although in analytical chemistry, strong acids or bases are also considered to be buffers, since they resist the addition of small quantities of bases or acids without pH modification (due to their high degree of dissociation), it would be correct to refer them as pH-modifiers. For instance, NaOH 0.5875M is used in lyophilised Acyclovir, producing the pH value of 11.5 which is necessary to dissolve the API; in formulation of methotrexate NaOH is used as well.

Stabilisers

The stabilising agents may stabilise the API in both the liquid and/or solid phase of the formulation. There are different mechanisms by which they can stabilise the API, so the formulator should know the API physicochemical properties in order to develop the correct mechanisms and establish which stabiliser should be used. In stabilisation by chemical equilibrium, the stabiliser interacts with the decomposition equilibrium of the API, displacing it towards the formation reaction by way of the Lavoisier principle. One example is the use of NaCl in the formulation of Cis Platin, where the chloride ion acts in hydrolysis equilibrium with the API, preventing its hydrolysis. Another mechanism is stabilisation by dissolution co-adjuvant, which involves the use of tensioactives for enhanced dissolution of the API in the solvent liquid phase. Castor oil ethoxilated, sorbitan carboxilates, sodium lauryl sulphate, macrogol 15 hydroxistearate and PEG are examples of such stabilisers. The last mechanism to mention is crystallisation induction, where excipients are used that aid active crystallisation through various possible processes:

  • The excipient forms a complex with the API that crystallises during the freezing step: for example amino acids such as glicine, alanine, seryne and methionine
  • The excipient crystallises during freezing and serves as a seed for posterior API crystallisation. Manitol possesses this property for several APIs
  • The excipient forms an euthectic with the API during freezing

Concerning the crystallisation of the API, one has to take into account that, in the case of proteins, it may result directly in a loss of stability due to protein oligomer formation, with the result that the protein molecules that crystallise over an excipient bed are concentrated in excipient crystals, instead of dispersed in amorphous media. One solution would be to avoid excipient crystallisation by using sugars such as sucrose to form a glassy state, but unfortunately this method is not always successful and some proteins continue to form oligomers. Protein oligomer formation can be limited by using tensioactives, which form a hydrate layer on the protein molecule surface that prevents approximation to each other, in turn preventing polymerisation.

Cryoprotectant

This is a substance that protects the specimen being lyophilised during the freezing stage. Living cells are of this kind of specimen, so cryoprotectants must be present in all living cell formulation – which includes live viruses. In these cases, water inside the cell must be kept in a glassy state so the cell can survive the freezing step. Ice formation inside the cell produces an increase in NaCl concentration that leads to protein denaturalisation, by increasing in ionic strength and causing cellular death. In addition, because ice has a lower density than water, ice formation will cause an increase in cellular volume which results in high pressure on the cellular membrane, promoting its rupture.

Consequently an excipient that prevents water crystallisation should enter the cell; examples of these are glycerin, dimethylsulfoxide, glucose, fructose and urea among others. On reconstitution, they cross over the cellular membrane again to the outside of the cell, avoiding haemolyses.

Cryoprotection will also depend on the freezing rate, which should be high in order to promote glassy formation. It should be noted that by means of this rationale, one could also use some substance that does not enter the cell but has the ability to form a glassy state as a cryoprotectant – for instance PVP at 4-10ºC/min freezing rate. Other examples would be sucrose, starch, raffinose (trisacharide) and dextran. This sort of cryoprotectant does not destroy the cell by thawing or lyophilisation because it never penetrates the cell in the first place. Ultimately, cryoprotection depends on the specimen to be lyophilised.

Lyoprotectant

This is a substance that improves API stability, preventing its degradation during sublimation and the secondary drying steps in the lyophilisation process. Historically, lyoprotectants have been referred to as ‘protein protectors’ during lyophilisation because of the difficulty faced when working with proteins due to their variable physicochemical complexity. The mechanism used by these lyoprotectants in protecting the proteins seems to be the formation of a border around the protein that supplies a source of water molecules. This means that denaturalisation due to loss of the ‘water bridge’, which may cause changes in protein configuration, whether secondary, tertiary or quaternary, is avoided. Disaccharides, trisaccharides and polysaccharides are suitable as lyoprotectants. Those saccharides with 1-1 bonds seem to be more effective because they are not reducing agents, and so cannot also reduce proteins. Trehalose dyhidrate is a well known disaccharide that can act as a lyoprotectant; sucrose and maltose have also been used.

We also should include in this category the surfactants or tensioactives that use the same mechanisms previously mentioned above to form a hydrate layer on protein surfaces, preventing oligomer formation. Examples of these are sorbitan carboxilates, sodium lauryl sulphate, macrogol 15 hydroxistearate and PEG.

Isotonic Agents

Sometimes inorganic salts are used in formulation to control isotonicity in the blood. Examples of such salts are sodium chloride, sodium nitrate, potassium chloride, ammonium chloride, calcium chloride and so on.

Solvents

This constituent is often overlooked because it is merely thought of as a liquid medium that will be eliminated during the primary and secondary step of the lyophilisation process. Water is the main solvent used in lyophilisation, but there are also other solvents that can be used, alone or added to water, to solve several problems in formulation.

Because of its chemical structure, the water molecule possesses a high dipolar momentum. This momentum generates hydrogen bonds, which are the cause of high surface tension, high fusion heat, high boiling point, and high dielectric constant. The last of these enables water to dissolve hydrophilic molecules, as well as molecules with polar groups such as HO-, -CN, -NO2, -COOH, despite being symmetric molecules. Water, generally, can also dissolve the substance by solvation, whether it is formed by ion, polar or sometimes non-polar molecules. Solvation is the process by which ions or molecules are surrounded by water molecules due to their electrostatic field; in such a way, a layer of water is formed around each type that prevents approximation between them. In protein formulation, it is best to use tensioactives that remain hydrated through the hydrogen bonds on the surface of proteins, as this avoids protein-protein interaction during freezing.

Because of this mechanism, water is an excellent solvent for excipients, proteins (giant ions), DNA, viruses and cells. However, as mentioned before, there are at least two reasons for using solvents other than water. The first one would be the need to crystallise an API, so some organic solvents can be incorporated as a co-solvent of water for promotion of crystallisation when the water is separated from a formulation solution as ice during freezing: therefore the organic solvent is concentrated and the API crystallises. Ter-buthyl alcohol, acetone and acetonitrile are examples of such an application. The second reason for use of organic solvents is the need to stabilise the API in the liquid phase by increasing its solubility, hence a vast list of organic solvents can be mentioned as being used in lyophilisation, including ter-buthyl alcohol, n-propanol, acetone, DMSO, acetonitrile and dioxane.

Once again, we should point out the importance of obtaining adequate thermal properties in formulation – one has to be careful to choose the organic solvent correctly at the time, otherwise it will strongly alter the Tg and Tc. Ethanol is an example of solvent that decreases Tg, even at very low concentrations.

Care must be taken when using organic solvents due to the environmental and product residual problems that they can cause. For instance, these problems may occur when using dichloromethane, acetonitrile, DMSO and DMFA. These reasons highlight the importance of fully understanding the API’s physicochemical properties before considering the use of organic solvents in its formulation.

THERMAL PROPERTIES

Glass transition temperature, collapse temperature, degree of crystallisation and supercooling are by far the most important thermal properties altering both the user required specification (URS) of the freeze dryer needed, and the lyophilisation schedule to be followed.

Thermal properties are dependant on the composition and concentration of the constituents of the formulation. Thermal properties will govern the cosmetic aspect of the final product too. The Tg and/or Tc will determine both the chamber pressure (Pch) and the shelf temperature (Ts) necessary to obtain the correct product temperature (Tp) throughout the lyophilisation schedule.

Consider the following example. Suppose we obtain a certain formulation with poor solubility of the API and a low Tg (less than -45ºC). It will require a high fill volume (say 25ml per container) and a large energy demand for refrigeration power, since Ts must be less than -55ºC to maintain a Tp of less than -50ºC and a condenser temperature of lower than -70ºC. In addition, with such a low Tp, primary drying will be prohibitively long, and with an increasing process time, there will be a higher probability of an adverse event occurring (from energy defaults to refrigeration problems); the latter can compromise the quality and API potency of the formulation. It should also be noted that the freeze dryer needed for such a formulation would be very expensive, and by changing the formulation to achieve a better Tc, one would use cheaper equipment and attain a safer product in a shorter period of time.

CONCLUSION

The formulation stage of lyophilisation has a significant impact on the process as a whole. Important decisions that rely on a number of factors are made during this stage, from the solvent to the buffer used, that are greatly influenced by the properties of the API. Consideration of these factors early on in the formulation process may well be advantageous not only to the study, but also in achieving a safer and more cost-effective product.

References

  1. Sassone J, Bases cientificas e pratica da Liofilização, EPUB, RJ, 2009
  2. Smith AU, Aspects Theoriques et Industriels da la Lyophilisation (Rey L, eds), Herman, Paris, 1964
  3. Greaves RIN, Biological Applications of Freezing and Drying (Harris RJC, ed), Academic Press, NY, 1954 
  4. Sassone J, Citostaticos, Hormonios e Antivirais, Tecnologia de fabricação e controle, EPUB, RJ, 2006 
  5. Jennings TA, Lyophilization – Introduction and basic Principles, Interpharm/CRC, 2002
  6. MacKenzie AP, in Freeze Drying and Advanced Food Technology (Goldblith SA, Rey L and Rothmayr WW, eds), Academic Press, NY, 1975
  7. Pikal J, Dellerman K and Roy ML, Formulation and stability of freeze dried proteins; effects of moisture and oxygen on the stability of freeze dried formulations of human growth hormone, Developments in Biological Standardization, 1992
  8. Rey LR, International Symposium of Freeze Drying of Biological products, 1977
  9. Wang DQ, Freeze-drying cycle development for Kogenate-FS, International Conference on Lyophilization and Freeze Drying, Dublin, Ireland, 2007
  10. Bakaltcheva I, Freeze-Dried Plasma for Field Utilization, International Conference on Lyophilization and Freeze Drying, Dublin, Ireland, 2007
  11. Ward K, Advanced Uses of Freeze- Drying Microscopy for Product and Lyo-Cycle Development, International Conference on Lyophilization and Freeze Drying, Dublin, Ireland, 2007

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Jorge Sassone received his PhD in Chemistry from Buenos Aires University in Argentina. Between 1989 and 1994, he was Professor of Advanced Qualitative and Quantitative Chemistry, Radiochemistry and Nuclear Chemistry at FCEN-UBA, Argentina. His current position is Chairman at Improvement-Proquimo in São Paulo, Brazil. He has over 20 years’ experience in R&D in pharmaceutical and veterinary manufacturing. He is a member of the ISL PAT executive committee, and has published two books.
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