The same phenomena that create lightning and thunderstorms are around us every day, producing incredibly high voltages, which cause sparks and shocks. Static electricity is a mighty force. Each year excessive electrical charge build cause explosions in the grain industry.

  Look around any flammable storage area and you will see both grounding bars on the wall and cables, from the grounding bars connected to the drums of solvents. Take any material safety data sheet (MSDS) for a powder and look in section V; it highlights that any dry powder has the potential to attract and store a charge. 

Yet our understanding of the role of a static electricity build, even in simple blending and handling,

  and its resulting aberrant potency and content uniformity data is under-appreciated on the production floor in both the pharmaceutical and nutritional supplement industries. 

Static electricity is everywhere. Consider the hazards of static electricity in the hospital operating room, the local gas pump. Static electricity can be generated simply when opening a door in our carpeted living rooms or walking around a nonconductive floor. Other industries, such as the electronics industry, use an electrostatic discharge (ESD) shield bag when storing components. Further, employees must wear heel grounders and wrist straps, and the safety checks, procedures and audits that determine how effective they are as a system are continually challenged. The audits may be conducted by an National Association of Radio and Telecommunications Engineers (NARTE)-certified EDS engineer.

Figure 1 Thieves must be verified for proper installation and grounding prior to use.			

On the production floor, consider asking any process operator if he or she has ever been 'zapped' by static electricity when their gloves come in contact with a double-lined plastic bag that lines drums of granulation and tablets and you will discover that operators deal with static electricity every day. The bag manufacturers themselves try to minimize (but cannot totally prevent) static charge build up on the bag rolls.

  Picking up a common plastic bag from a bench generates 1200–20000 volts of static build. Static electric charges can build up on trolleys (tote bins) as you push them around. The charge is usually generated by the combined movement of the trolley wheels and your foot action as you walk — thousands of volts may build up on your body and on the trolley. The charge remains unnoticed until you touch something, which closes the loop, giving the characteristic shock.

  These events are common and drive our point for consideration. 

Electric static charge build and blended powders

Static electricity exists whenever there are unequal amounts of positive and negative charged particles present. It does not matter whether the region of imbalance is flowing or still. Only the imbalance is important, not the 'staticness'.

All solid objects contain vast quantities of positive and negative charges whether the objects are electrified or not. When these quantities are unequal, we say that the object is 'charged' or 'electrified'. Because static electricity is actually an imbalance in the quantities of positive and negative, it is wrong to believe that the phenomena has anything to do with lack of motion, with being 'static'. In fact, static electricity can easily be made to move along conductive surfaces. When this happens, it continues to display all its expected characteristics as it flows, though it does not stop being 'static electricity'. As negative particles are pulled away from the positive particles, equal and opposite areas of imbalance are created (

Triboelectric charges (a transfer of electrical charges) occurs when two or more materials are in contact and then separated.

  One material acquires an excess of negative ions while the second, third or fourth material acquires an excess of positive ions; the force, pressure and separation of these materials are the major causes of industrial static electricity.  

Figure 2 Tote bins must be properly grounded.			

The interparticle forces of these powders are extremely large and cause clumping, sticking and channelling when attempts are made to fluidize them. Although flow problems can be experienced in the transport of free-flowing solids, they pale in comparison to the problems experienced with the flow of cohesive powders. Interparticle forces accounted for 70–80% of cohesive behaviour while mechanical interactions accounted for 20–30%.

  Thus, most nonpharmaceutical process designers have shied away from using cohesive powders. However, these powders are often produced in processes by attrition, and their presence must be acknowledged and addressed for successful processing. The majority of the active pharmaceutical ingredients (APIs) used in pharmaceutical tablets and capsules would be classified as cohesive powders. 

Most APIs are fine powders. Many are, in fact, micronized powders that provide for rapid dissolution profiles. The advantages these powders provide for the end-user can be very problematic when a firm attempts to validate the content uniformity of blender powders and does not consider the potential electrical aspects of fine powder. This becomes even more challenging when the powders are both micronized and cohesive.  

Figure 3 Connect and ground the bin then open the valve and move the powder into the transition piping.			

Cohesive powders are composed of fine particles that tend to attach to one another. Besides particle size, powders may also be classified as 'free-flowing' or 'cohesive'. Colijn defines a cohesive powder as particles generally less than 140 mesh or 100 μm in size.

  One might very well use this definition for many APIs on the market as tablets and capsules. It is, however, important to note that not all cohesive powders will attract and hold an electrical charge; but most cohesive powders do exactly that.  

How does one know that the API or the final blend is cohesive? Confirmation of the particle size distribution may be achieved by applying the Hausner ratio test. The Hausner ratio test is particularly suitable for assessing both the flow and cohesivity of both the active ingredient and the final blend. According to the Hausner criterion, an API or a final blend that has a value greater than 1.40 is deemed cohesive and likely to hold an electrical charge.

 took the position that the pharmaceutical industry as a whole was relatively unattentive to how difficult precision sampling of cohesive powder can be.

The ability of a solid to transmit electric charges is characterized by its volume resistivity.

  Powders may be conveniently divided into the following three groups: 					

Low-resistivity powders having volume resistivities in bulk up to 108 ohm-m. Examples include metals, coal dust, and carbon black.

Medium-resistivity powders having volume resistivities of 108–1010 ohm-m. Examples include many organic powders and agricultural products.

High-resistivity powders having volume resistivities above 1010 ohm-m. Examples include organic powders, synthetic polymers, and quartz [NFPA 77 8.4.2.1].

Powders that are free-flowing and are noncohesive have low resistivity. An individual powder, whether present in small or large quantities, but having medium or high resistivity will be prone to effects of static charges and can become cohesive or charged during flow. The charge rapidly dissipates when the powder is conveyed into a storage device or container that is grounded. However, if conveyed into a nonconductive container, the accumulated charge can result in a small spark as the charge in the dust and powder attempts to equalize potential differences during this process (

). The powder blend may also adhere to the container wall causing a slight to high increase in tablet or capsule content as the batch progresses and the active increasingly attaches to the vessel.  

Powder blends may collectively take the profile of an individual active ingredient, regardless of the overall API content and exhibit medium to high resistivity. The charge rapidly dissipates when the powder is conveyed into a storage device or container that is grounded. 

It is important to recognize that mixing cohesive actives with free-flowing excipients will produce a different set of expected difficulties than mixing cohesive actives with cohesive excipients. When a small amount of cohesive active is blended into a much larger amount of a free-flowing excipient, it is imperative to ensure that the agglomerates of the active are effectively comminuted and dispersed into the mixture. When the active and excipients are both cohesive, micromixing (i.e., agglomerate breakup by application of shear force) will be the major performance-limiting phenomenon and must be accounted for throughout the mixing process.

If conveyed into a nonconductive container, such as a blender, drum, tote bin or tablet press, an accumulated charge can result in microsparks as the charge in the dust and powder attempts to equalize potential differences during this process by drawing together. We commonly refer to these pockets as 'hot spots' and they may in part account for the 'erroneous numbers' we sometimes see and cannot understand in our content uniformity results from the laboratory.  

Powder then transiting through a closed feeding system to the die table may attract and retain an electrical charge through contact with container and piping walls. This may take the form of a film inside the tote bin, the transition pipe, the plastic bag in a drum or in the powder hopper on a tablet press. It is easily dissipated when the container becomes grounded. 

Electrostatic charge insulation, grounding and discharge 

Conductors can be insulated (e.g., a jacketed or plastic-coated cable) or uninsulated (i.e., bare conductors). Uninsulated electrical conductors (wires) are recommended, because it is easier to detect defects in them. Charged insulators may attract and retain a charge for multiple hours or even days. Further, opposite charges may exist on an insulator simultaneously. Charges of either polarity will not migrate on an insulator. Grounding an insulator will neither remove nor prevent the accumulation of surface charges. The charge of one of either polarity can remain on a conductor as long as the conductor remains isolated from the ground. But a charged conductor will discharge completely when grounded.

How do we know when a bin, mixer, drum or thief is in fact grounded? Where the bonding/grounding system is all metal, resistance in continuous ground paths will typically be less than 10 ohms (

For all practical purposes, when the terms 'discharge' or 'grounding' are used, they should be thought of as including a connection or path to the earth to put electrically conductive materials at the same potential as the earth. 

Early in product development, a determination of potential cohesivity and static charge attraction must be made. If there is an issue, the firm must begin planning for the day validation and commercial manufacturing commences. Maintaining a high level of attention throughout the scale-up and commercialization process helps prevent procedural mistakes that commonly result in erroneous data. While on the production floor, there are certain procedures that will help the overall effort: 					

A basic fact is to maintain the relative humidity (RH) in the room and in the containers at or above 35% RH and static charges are frequently automatically drained off to a ground. There is usually no need for grounding straps or ionizers.

  However, do not assume that the conditions inside the blender, the tote bin or the drums conform to this guideline or that these conditions were met during both blending and handling.

Most powders meant for tablet compression are, in fact, dry powder with moisture content at or below 5% and they may potentially attract and retain an electrical charge. This is especially true if there is an interaction between a cohesive powder, poorly grounded equipment, bag-lined totes or drums and a sampling thief that is ungrounded.

Our recommended procedure involves the following key steps, usually contained in a standard SOP in the granulating, blending and tablet compression/powder encapsulation departments: 

Use a static meter to determine the polarity and severity of the static charge on a surface. The basic goal here is to create a path of dissipation that will not be subject to the negative effects of pressure piling of electrons. A Static Charge Meter is a noncontact, low cost, static monitoring and locating instrument. It utilizes a centre-zero meter display to indicate the magnitude and polarity of the electrostatic charge.

Treat grounding cables and powder thieves with care. Grounding cable are to be used for drums, totes, transition tubes and tablet presses/encapsulators.

Static electricity is real. Cohesive powders can and do attract and retain small electrical charges. Proper grounding and sampling procedures are necessary to prevent problems. Understanding the nature of static electricity and cohesive powders, and their potential role in solid dosage and hard-shell capsule manufacturing will enable a company to deal with the problem long before commercial manufacturing begins.  

is a guest lecturer at Solid Dosage Training Inc. (USA). 

1. A. Castellanos, "Open Problems in Powder Mechanics," Thesis abstract, University of Sevilla, Avenida Reina Mercedes, Sevilla, Spain. 

2. R.E. Freeman, "The Flowability of Powders — an Empirical Approach," International Conference on Powder and Bulk Solids Handling (London, UK, 2000). 

3. National Association of Radio and Telecommunications Engineers standardized tests ANSI/ESD 20.20. 

4. "Static Electricity Stays on the Bags and Rolls Down to the End User," Ion Systems Industrial. 

Articles

Dosage Forms

The same phenomena that create lightning and thunderstorms are around us every day, producing incredibly high voltages, which cause sparks and shocks. Static electricity is a mighty force. Each year excessive electrical charge build cause explosions in the grain industry.1 Look around any flammable storage area and you will see both grounding bars on the wall and cables, from the grounding bars connected to the drums of solvents. Take any material safety data sheet (MSDS) for a powder and look in section V; it highlights that any dry powder has the potential to attract and store a charge.

Static electricity in solid dosage manufacturing

Over the past decade, disposable technologies have become a reality in biotech processes. The use of disposables in research and manufacturing allows high flexibility. 

The aim of cultivation in a bioreactor is to produce biomolecules using animal or plant cells, micrograms, yeast and insect cells. 

Biomolecules are divided into four main classes: carbohydrates, lipids, nucleic acids and proteins.

  The latter is the most common in biotech processes: manufacturing of vaccines, diagnostics or therapeutic preparations.  

Mammalian cells constitute a demanding system for the production of heterologous proteins. Two main formats have been employed for the production of recombinant proteins in mammalians cells: cultures of adherent cells and suspension cultures. The latter is by far the most common.

				 In 2004, mammalian cell-based therapeutic proteins reached a market share of 59% followed by 27% for 

Before production can start, the right cell line is screened out and the cultivation process must be optimized, for example, scaled-up, starting cultivation with a few millilitres and ending up with several cubic metres' cultivation volume. 

For more than 40 years, typical bioreactors for cell cultivation have been made of glass or stainless steel (Figure 1) and have been used in research and commercial production processes. This traditional bioreactor was characterized by substantial investment cost resulting from the necessity for aseptic bioprocess technology, sterilization in place (SIP), cleaning in place (CIP) and validation, all requiring sophisticated instrumentation.  

Figure 1 Classical fermentation system approach.			

Disposable bioreactors with mechanical energy input represent modern alternatives to such traditional cultivation systems for some applications in lab- and pilot-scale. 

The main characteristics of the single-use bioreactor are low cost, ease of operation, time saving and high process security. These parameters shorten the time-to-market. 

Disposable bioreactors can be divided into two groups: 					

Bag reactors with mechanical energy input where the cells are directly in contact with growth media. The most common bag reactor is based on rocking motion, where the wave-induced motion guarantees the energy input.

Membrane cultivation systems with two chambers separated by a semipermeable membrane. The membrane enables the passage of nutrients to the cells, yet the metabolites can leave the cell chamber. High cell densities can be reached, but up-scale capability is limited to the low lab-scale level.

This article aims to outline the potential and limits of the disposable bioreactor based on wave-induced agitation for biotechnology use, while considering the working principle. 

This disposable bioreactor with wave agitation can be used for batch, fed-batch and a perfusion operation mode. It is shown that even fragile cells such as animal cells (CHO, NS0, fibroblasts and hybridoma) human cells (T-Lymphocytes, HEK and Perc.6), insect cells (Sf9, High5 with bacculo virus) and plant cells (hairy root culture, suspension cultures and embryo culture) can be grown in sealed bags made of polyethylene.

Disposable bioreactors based on the rocking motion principle are mechanically-driven reactor systems, which are available in lab- (up to 25 L) and pilot-scale (up to 300 L) versions. They consist of three components: 					

A rocker base unit containing a bag holder with heating capabilities.

The biocompatible disposable chamber with integrated, single use, sensors for pH and DO.

The measuring and control unit (Figure 2). 

Figure 2 BIOSTAT CultiBag RM optical.										

Energy input is affected by rocking the chamber back and forth, generating a fluid movement in the cell culture and medium (Figure 3). In this way, the surface of the medium is continuously renewed so bubble-free aeration can take place. Therefore, a maximum of half of the total bag volume can be filled with the cultivation media. The other half is needed for the overlay aeration. Depending on the sensitivity of the cultivated cell line, the angle and amplitude of the rocking motion, as well as velocity can be varied. Specially designed sterile plastic cultivation bags contain tubing for filling, harvesting and sampling. This allows simple handling and optimal cell growth for different cell types and cell lines.  

Figure 3 Principal of rocking mixing technology.			

The measuring and control unit can facilitate regulation of pH, DO, temperature, rocking rate and gas flow using the gas mixing system. An example for gas supply strategy is shown in Figure 4. The pO

 additionally to base and acid is provided.  

Figure 4 Aeration strategy with BIOSTAT RM Tower optical (Sartorius BBI Systems).			

The used cultivation bags can be sterilized by autoclaving and then destroyed by incineration. The PE foil from which the cultivation bag is made, is environmentally compatible when incinerated. This is less critical for the environment than the cleaning of conventional bioreactors with alcohol, acids and basic solutions. An additional safety factor is the fact that no cell or protein residues are released into the wastewater cycle. 

The advantages of disposable bioreactors are mainly ease of use, prevention of cross-contaminations, and cost and capital investment savings. The costs compared with conventional stainless steel bioreactors are lower because the capital investment requirement is lower, and the necessity for qualification and validation processes, as well as maintenance, is reduced. A cost reduction of up to 41% compared with conventional stirred bioreactor technology is possible.

  Additionally, the disposable bioreactors enhance process safety because the contamination risk falls under 1%.

  The disadvantage of using disposable bioreactors is the limitation in scale-up (300 L) and aeration capabilities for cultivation of some microorganisms, which require high oxygen uptake rates. 

Oxygenation or aeration can be achieved by three methods, individually or in combination; that is, membrane aeration, surface aeration or direct sparge aeration.  

Microbial fermentations exhibit high metabolic oxygen demand together with high biomass concentration; this combination requires an energy-intensive means of maintaining adequate culture aeration. This aeration is achieved by simultaneously sparging air at high rates and vigorously agitating the culture to increase the bubble interfacial area and affect the mass transfer of oxygen. 

The biomass concentrations and cellular oxygen uptake rates observed in mammalian cell cultures are low compared with microbial systems (Table 1). Even if the cellular oxygen uptake rate is low, it does not mean that oxygenation does not present a problem. The reason is the shear sensitivity of mammalian cells, which prevents the use of vigorous aeration or agitation. As a result, the volumetric oxygen mass-transfer coefficient is low and becomes a limiting factor in scale-up.  

Table 1 KLa and OTR values of different cultivation systems (source: Enfors 2003 and Wave Biotech AG, Switzerland).			

Bioreactors for cultivating mammalian cells are typically aerobic bioreactors and oxygen is usually a limiting nutrient because of its low solubility in culture media. When bioreactors are scaled up from laboratory to production size, their design must meet both oxygen distribution and oxygen mass transfer requirements. 

One engineering parameter that could be used to compare the newly developed disposable bioreactors with established stainless steel cell-culture bioreactors could be the oxygen transfer rate (OTR). Because of its low solubility, only 0.3 mM O2, equivalent to 9 mg/L, dissolves in one litre of water at 20 °C in an air/water mixture. This amount of oxygen will be depleted in a few seconds by an active and concentrated growing culture unless oxygen is supplied continuously. In contrast, during the same period the amount of other nutrients used is negligible compared with the bulk of concentrations. Therefore, most aerobic processes are oxygen-limited. This is the reason why the concept of gas–liquid mass transfer in bioprocesses is centred on oxygen transfer even if other gasses such as carbon dioxide, hydrogen, methane and ammonia can also be involved.  

The mass transfer of oxygen into liquid can be characterized by the OTR or by the volumetric oxygen transfer coefficient (KLa). The larger the KLa, the higher the aeration capacity of the system. These values have been thoroughly examined as a critical parameter for bioreactor function.  

Disposable bioreactors with wave agitation are used for commercial and R&D applications for the production of cell-derived products using animal, insect, plant and virus cultures in good manufacturing practice (GMP) and nonGMP applications. Because of their flexibility, single-use cultivation bags are especially suitable for screening new cell lines, media optimization and vaccine production. Basically, two strategies can be implemented when considering the use of disposable bioreactors:  					

A partially disposable strategy; for example, preparation of inoculum in disposable cultivation bags for process-scale cultivation in conventional stirred tank reactors (Figure 5).

A completely disposable strategy, where inoculum as well as plant bioreactors are disposables based on the rocking-motion principle (Figure 6). 

Figure 6 Total disposable system approach.										

In addition, the use of multiple large-scale disposable bioreactors instead of one conventional stirred tank reactor can be considered, particularly in view of their ease-of-use and process safety against contaminations of disposable bioreactors. Finally, the parameter that will document the merits of using conventional bioreactors or disposable bioreactor systems for biotechnology processes are yield of production, expenditure of time and process costs.

  The cultivation of yeast and aerobic microorganisms is, however, a challenge in disposable bioreactors. As shown in Table 2, because of the published Kla or OTR values, it is unlikely that a wave agitated system can be used for the most common microbial expression systems because of the Kla or OTR values inherent in this method.  

Table 2 Oxygen uptake rate values for mammalian cells and E. coli.			

Even if the market for disposable bioreactors has grown rapidly in recent years, full acceptance will only be reached when the entire production process from media preparation, inoculum preparation and downstream processes can be done using disposable equipment. Some suppliers are focusing on this issue to become a total solution provider for disposable biotech factories.

, Fourth Edition (W.H. Freeman and Company, New York, NY, USA, 1995). 

, "Mammalian cells," in Ed. G. Gelissen, 

., "Catching the Wave: The BEVS and the Biowave", Novartis Pharma Research, Switzerland. 

Biopharmaceuticals

Over the past decade, disposable technologies have become a reality in biotech processes. The use of disposables in research and manufacturing allows high flexibility.

Disposable bioreactors based on wave agitation technology

I used to work next door to a microbiology lab where bacteria was grown on brain and heart broth. The smell alone was enough to persuade me to stay with synthetic organic chemistry and keep well away from the black art of cell culture. Brain and heart broth is still used in path labs, but, when it comes to biopharmaceutical manufacturing, cells are having to learn to do without animal components whenever possible. This has undoubtedly been a challenge for the industry, but is leading to a better understanding of cell metabolism and physiology, which, in turn, allows cleaner and more efficient production of recombinant proteins and other biologics. 

It was Harry Eagle of the US National Institutes of Health who pioneered cell culture from the mid-1950s. The medium he devised contains all the different nutrients cells need to grow, such as amino acids, salts, vitamins and glucose. To this he added a liberal dose of horse serum, which contains various growth factors without which the cells die. Eagle's medium was an important breakthrough because a variety of cells were able to grow on it, allowing research in molecular biology to progress and eventually, in the late 1970s, to open the door to industrial cell culture and the biopharmaceutical industry. 

Foetal bovine serum (FBS) became the research and industry standard for supplementing cell culture media, but to this day, no one is quite sure what it contains. This lack of precision and certainty sits uneasily with the demands of good manufacturing practice (GMP) manufacture. So, although cells love to grow on serum, they have to be weaned off it. The BSE epidemic naturally led to concerns about the safety of medicines, such as recombinant proteins, made using bovine serum. In the UK, over 1000 people with haemophilia were infected with HIV through blood-derived Factor VIII (FVIII) treatment. Recombinant FVIII, produced by mammalian cells, is a much safer option; it would be a terrible irony if patients were put at risk again through contaminated FBS. Prions — the infectious agents causing BSE and variant CJD — are not the only potential contaminant in serum; manufacturers must also demonstrate removal of viruses, fungi, bacteria and endotoxins. 

But in fact, the trend towards serum-free cell culture dates from before BSE, and was driven primarily by concern over the cost of serum and the difficulties of obtaining a reliable supply. Moreover, unlike a chemical solvent, serum is not well-defined. Each batch is different, and must be checked and validated. Serum also causes problems in downstream processing because of its high protein content. This can overwhelm the protein product being manufactured during the purification process. That is why foetal, rather than adult, bovine serum has often been preferred for monoclonal antibody production, because foetal serum contains fewer interfering antibodies than adult bovine serum. But foetal serum is harvested from bovine foetuses taken from pregnant cows during slaughter — a procedure that raises animal welfare concerns. 

The three vital growth factors in serum are albumin, transferrin and insulin. The first steps towards serum-free media involved getting rid of the serum and providing these protein ingredients from others sources, mainly blood. Then, with the advent of the BSE crisis, it was thought the proteins ought to be replaced too, to get rid of the animal components altogether. "This proved harder than people thought it would be," says Dr Malcolm Rhodes, technical director of bioProcessUK. "Albumin provides a number of micronutrients, and helps control pH and transferrin binds iron, without which you get oxidation in the cell culture." 

Media supply companies such as Invitrogen and SAFC Biosciences have put years of development work, which is still ongoing, into this area, and there is a range of serum-free, protein-free and chemically-defined media now available for various applications. Serum-free cell culture is often used for research and may still contain proteins. If the protein comes from a recombinant source, then the medium may also be animal-free. The protein-free media could contain yeast or plant — usually wheat or soya — hydrolysates as a replacement for albumin, transferrin and insulin. These are often, but not always, free of animal components. A chemically-defined medium is totally animal-free and is, increasingly, the choice for industrial cell culture. 

Cells do not like change. If they have been growing and producing in a serum-supplemented medium, a sudden transfer to a serum-free medium will be stressful. They need to go through a gradual adaptation process. "It is not well understood what happens to the cells during this weaning process," says Dr Rhodes. "There may be genetic or epigenetic changes taking place." (An epigenetic change affects gene activity, but not gene sequence.) Cells are also more sensitive to changes in pH, osmolality, temperature, mechanical forces and enzyme action in a serum-free medium. It would appear that the protein content of a conventional medium provides the cells with some protection from these changes. Moreover, attachment-dependent cell lines require some kind of extracellular matrix, which serum seems to provide. Without it, surfaces must be coated with something the cells can attach themselves to, such as laminin or fibronectin. 

In brief, moving a cell culture from conventional to serum-, protein-, or animal-free media takes time and money, which is why some older biotech products are still manufactured with serum. The regulatory authorities will accept this — so long as the animal-derived components used in the process come from a source deemed to be safe. But new products must be as free of animal components as possible. Some manufacturers have invested in switching an established product. For instance, Baxter now makes FVIII in a albumin-free medium. 

For conventional biopharmaceuticals, the adoption of more defined media is now well underway. The challenge now is for cell therapies — where the cell itself is the product — to go animal-free. Cells destined as therapies are often grown on a layer of mouse 'feeder' cells and are, therefore, prone to viral contamination. However, Geron (a US company that plans to begin the world's first clinical trial of human embryonic stem cells later this year) has recently published methods of growing cells without the feeder layer. Many other stem cell therapy companies are now starting to develop animal-free culture technologies.  

According to SAFC Biosciences, the future of media development is towards more and more chemical definition, and will include the use of experimental tools such as RNA profiling to improve our understanding of the metabolic profile of cells during each stage of the cell cycle. "It is a hard road, but there is more potential for optimizing production if you can control individual components in a cell culture medium," argues Dr Rhodes.  

Brain and heart broth is still used in path labs, but, when it comes to biopharmaceutical manufacturing, cells are having to learn to do without animal components whenever possible.

Cell culture must go vegetarian

Different spectroscopic techniques are widely used to help understand the physical solid-state characteristics of drug substances and formulations — and Raman spectroscopy has become a commonly used technique that possesses many advantages over other analytical techniques.  

The principles and applications of Raman spectroscopy have been recently well-addressed in this journal.

  Benefits of the technique are, for instance, that sample preparation is unnecessary or minimal; data acquisition is rapid and nondestructive; and measurements in aqueous environments are possible because water is a weak Raman scatterer (Figure 1). These benefits also allow bulk and final products to be tested directly in their packaging, such as glass bottles.

Figure 1 Examples of Raman spectra of Indomethacin (a) and lactose monohydrate (b) measured from a powder and suspension. For comparison, a spectra of only water is also shown.			

Numerous papers in literature emphasize that Raman spectroscopy is a very attractive characterization tool, specifically because it enables measurements in presence of water, but, surprisingly, few studies have applied Raman spectroscopy in an aqueous environment. This paper focuses on recent uses of Raman spectroscopy to characterize pharmaceutical materials and processes in water. 

Probing critical solid-state processing steps and tablet dissolution 

One of the earliest studies in a pharmaceutical setting demonstrating the use of Raman spectroscopy in crystallization was performed by Findlay and Bugay.

  They studied crystallization dynamics by measuring supersaturated menthol solutions with variable temperature (VT) Fourier Transform (FT)-Raman spectroscopy. Elkordy 

				 reported the measurement of crystallized and spray-dried lysozyme, a model protein, by FT-Raman spectroscopy in 8% (w/v) solutions. They used a relatively high concentration to detect well-resolved Raman bands. After a water background had been subtracted, aqueous state FT-Raman spectra revealed protein conformation changes.  

Raman spectroscopy has been particularly valuable for controlling wet granulation processes. Jørgensen 

				 successfully employed charged coupled device (CCD) Raman and near infrared (NIR) spectroscopy to monitor polymorphic transitions of caffeine and theophylline during wet granulation. They concluded that the symmetric vibrations in the drug molecules could be readily detected and analysed with Raman spectroscopy. As Raman bands were much narrower than those in the NIR spectra (which has dominating OH bands) it was easier to observe the changes in the Raman spectra.  

				 successfully interfaced in-line Raman spectroscopy to a high-shear granulation process and also monitored pseudopolymorphic conversions of anhydrous theophylline. In a pelletization study, Raman spectroscopy, NIR spectroscopy and X-ray powder diffraction (XRPD) were used to characterize process-induced polymorphic changes.

  Samples were collected at the end of each processing stage (blending, granulation, extrusion, spheronization and drying). Raman and XRPD measurements confirmed the expected pseudopolymorphic changes of the active pharmaceutical ingredients (APIs) in the wet process stages. However, a relatively low Raman signal of theophylline (5% [w/w] in the formulation) complicated the interpretation.  

				 Raman spectroscopy was elegantly used to measure the solid state properties 

 during intrinsic dissolution measurements. They showed that the solid-state transformation from anhydrate to hydrate can be quantitatively measured for both theophylline and nitrofurantoin tablets using a Raman probe (Figure 2).  

Figure 2 A schematic of the experimental setup used in a solid-state dissolution study. (Figure modified from reference 9.)			

They concluded that by combining dissolved drug concentration with solid-state measurements, a deeper understanding of the solvent-mediated phase transformation phenomena during dissolution is achieved. 

Controlling the properties of emulsions and suspensions

				 demonstrated the use of Raman to monitor a bench-scale formulation process of topical formulations. They heated the aqueous gel and mixed it with an oil phase to produce an emulsion, showing that a Raman shift of a peak at 925 cm

  was linked to an ionic interaction between Carbopol and triethanol amine. The height of the 925 cm

  increased with greater neutralization, as measured by the pH.  

The authors, therefore, concluded that these peak height ratios can be used to control the amount of neutralizer used during manufacture. Raman spectroscopy in combination with stepwise linear regression has also been successfully used to confirm the polymorphic stability of benzimidazole in a complex suspension emulsion formulation.

  The formulation consisted of active drug suspended in oil droplets, which are emulsified in an aqueous matrix. No polymorphic transition was noted during the duration of the study.  

However, a small sampling area within the heterogenous matrix caused some measurement variability. Thus, when measuring heterogenous samples multiple spectra should be recorded with a sufficiently large sample area to minimize variable and unrepresentative sample analysis. 

The polymorphic composition of L-glutamic acid crystals has been monitored during batch cooling of saturated solutions using Raman spectroscopy 

  By using peak height ratios, the α and β forms were quantified and at 25 °C in aqueous solution, the α form dominated. However, a solvent mediated transformation to the β polymorph followed, with the conversion accelerated at higher temperatures. This 

 quantification allows the optimization of both crystallization kinetics and polymorphic purity.  

Raman spectroscopy offers an attractive possibility for direct and nondestructive quantitative measurements in an aqueous environment. Raman spectroscopy has been successfully used to determine the amount of medroxyprogesterone acetate in an aqueous suspension.

  Besides the fact that water does not interfere, an advantage of Raman spectroscopy is that it allows direct measurement of suspended drug concentrations, without the need to know the exact volume or density of the sample suspension, as would be the case with HPLC. Thus compared with HPLC, Raman spectroscopy provided a more reliable quantitation method for the amount of medroxyprogesterone acetate in a suspension.  

Raman spectroscopy can also be used to detect changes in dissolved compounds. Li 

				 used FT-Raman spectroscopy to examine the acid-base characteristics of various citrate buffer systems. The results indicated that the ratio of ionized and un-ionised species remained the same in the solution, the frozen solution and the amorphous state. Quantitation was based on the FT-Raman peak intensity ratio of the -COO to -COOH carbonyl stretching vibrations at 1400 cm

, respectively. The signal to noise ratio was largest in the frozen samples, which was probably because of the shorter acquisition times that were necessary to prevent the samples melting. 

Studying water sorption and hydration phenomena 

Raman spectroscopy has been widely used to detect changes in the solid-state of a drug because of water sorption into the material. Airaksinen 

				 used Raman spectroscopy to determine the effect of excipients on hydrate formation of theophylline during wet granulation. Despite the excipients in the formulation, Raman spectroscopy could still detect the theophylline anhydrate to monohydrate conversion without excipient interference.  

A blue shift of the O=C-N bending band at 550 cm

  and changes in the C=O stretch region at about 1700–1650 cm

  were used to detect the hydrate formation. Raman spectroscopy has also been used in-line for quantitation of changes in the amorphous content of spray-dried amorphous lactose and trehalose that were stored in high relative humidity (RH [Figure 3]).

  The quantitation was based on multivariate analysis. Both of the amorphous sugars recrystallized during storage at 85% RH. Amorphous materials are very hygroscopic and absorb moisture when stored in high RH. Unlike with NIR, the absorbed water did not interfere with the Raman spectra and thus Raman spectroscopy was more suitable than NIR spectroscopy for the crystallinity quantitation.  

Figure 3 Following the conversion of amorphoustrehalose to trehalose dihydrate at high relative humidity (85% RH) using in-line Raman spectroscopy; (a) illustrates the Raman spectra and (b) shows the increase in crystallinity determined by a PLS model. (Data modified from reference 16.)			

Raman spectroscopy has been used to study the gelatinization and liquefaction (hydrolysis) process of starch.

  An aqueous starch suspension was heated above the gelatinization temperature and spectra recorded in-line. During gelatinization, the uptake of water into the crystalline starch caused inter- and intramolecular hydrogen bond breakage and new hydrogen bonds were formed with water molecules.  

This could be observed in the Raman spectra because the bands at 1633 cm

  increased while the others decreased. The band at 3213 cm

  was attributed to hydrogen bonding with water and the band at 1633 cm

  to decreased starch crystallinity. After gelatinization, the liquefaction was initiated by addition of α-amylase. Significant changes in the spectra were used to monitor the liquefaction. The formation of free hydroxyl groups because of the hydrolysis reaction was noted by the increase of the bands in the region of 1057 cm

In addition to solid-state transitions in aqueous environments, Raman spectroscopy can be used to study the molecular interaction between water and different substances. Raman spectroscopy has been used to determine the changes in the strength of hydrogen bonding between water and different pharmaceutical polymers.

  Raman spectroscopy could be used to study the hydrogen bonding as the studied polymers, PVP, PVAc and PVP/VA were unable to form intra- or intermolecular hydrogen bonds because of the lack of acidic protons. Therefore, any changes in the spectra of these polymers, when in solution, were as a result of hydrogen bonding with water molecules. However, the spectral changes were because of not only the changes in the hydrogen bonding, but also the plasticizing effect of water, which increased the molecular mobility. The increased molecular mobility affected the C-H and C-C vibrations. These groups form the backbone of the polymers. 

In studies on nanostructured lipid carriers it has been shown that Raman spectra of lipids are sensitive to conformational, packing and dynamical changes involving hydrocarbon chains.

  Therefore, this technique can be applied to provide more detailed information on the microstructure of colloidal lipid dispersions such as nanoemulsions (NE), solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC). Investigations on SLN based on glycerol behenate and NLC (mixtures of glycerol behenate and medium chain triglcyceride [MCT]) resulted in the conclusion that MCT loading did not lead to changes in order or packing behaviour of the lipid chains, meaning that oil incorporation could not be confirmed.

When investigating the same systems, but using cetyl palmitate as solid lipid and MCT as oil component, Raman spectroscopy revealed that the NE was characterized by random coiled chains, while SLN and NLC formulations clearly showed sharp bands at 1128 cm

  indicating the high conformational order of the acyl chains.  

To determine whether the NLC-formulation spectrum is a simple mixture of spectra of SLN-formulation and nanoemulsion, a spectrum was calculated as a linear combination of 0.9 times the pure SLN spectrum plus 0.1 times the pure nanoemulsion spectrum. This calculated spectrum was compared with the NLC spectrum. The main peak positions did not change and subtraction of the normalized spectra revealed no major differences between the experimental and calculated spectra, suggesting that the SLN in the NLC particles is completely unperturbed by the presence of the oil.

This paper has demonstrated the versatile use of Raman spectroscopy in pharmaceutical analysis. The technique offers a major advantage that spectra can be obtained noninvasively in the presence of water. However, the full potential of Raman spectroscopy analysis in aqueous environments is still to be realized, and it is envisaged that the number of application and studies in this area will grow rapidly in the near future.  

is a senior scientist at AstraZeneca R&D (UK).			

 is a research scientist at the Division of Pharmaceutical Technology, Faculty of Pharmacy, University of Helsinki (Finland). 			

 is a research scientist at the School of Pharmacy, University of Otago (New Zealand). 			

is chief research scientist at the Division of Pharmaceutical Technology, Faculty of Pharmacy, University of Helsinki (Finland). 			

is a Professor at the School of Pharmacy, University of Otago (New Zealand).			

5. A.A. Elkordy, R.T. Forbes and B.W. Barry, 

7. H. Wikström, P.J. Marsac and L.S. Taylor, 

Analytical

Raman spectroscopy has become a commonly used technique for physicochemical analysis that possesses many advantages over other analytical techniques. It is a very attractive characterization tool, not least because it enables measurements in water. However, very few examples of its application in an aqueous environment exist in literature. This paper provides some recent applications of Raman spectroscopy in pharmaceutical material and process characterization when water is present.

Applications of Raman spectroscopy in aqueous environments

Skin is a barrier protecting the organism against physical, chemical and biological agents, and mechanical injuries.

				 This organ maintains equilibrium between the organism and the external environment. The skin consists of the epidermis with the stratum corneum (SC) and the dermis (Figure 1).  

				 and is composed of collagen fibres and a small amount of elastin. It contains connective cells, capillaries, nervous ends and appendages, such as hair follicles and sweat ducts. The deeper subcutaneous layer mainly consists of fat lobules.  

Figure 1 A schematic structure of the skin (adapted from reference 24).			

The dermis connects with the epidermis via the stratum basale.

  The epidermis consists of keratinocytes, arranged in layers. As a result of the keratinization process the region of dead cells (SC) is formed. Because keratinocytes of the SC are cornified, they are also termed 'corneocytes', which are filled with the protein α-keratin.

  The SC comprises 15–20 layers of corneocytes. The water content in the SC is 10-80%. The SC thickness depends on the hydration level, but is usually 10–100 μm. 

Intercellular spaces of the SC are filled with lipids arranged into ordered laminar structures, where the fundamental unit is the lipid bilayer.

				 SC lipids are primarily ceramides, cholesterol and its esters, fatty acids and triglycerides. Hydrophilic polar groups (heads) of lipids are directed to each other and a monomolecular aqueous layer appears between them. (Figure 2).  

Figure 2 An arrangement of intercellular lipids (adapted from reference 25).			

Blood vessels, coming across the dermis and reaching up to the border with the stratum basale, deliver oxygen and nutrients to the skin, and remove toxins and metabolites.

				 These vessels also deliver substances that permeated the SC barrier directly to the blood circulation. 

The ability of xenobiotics to permeate through the skin depends on the following two processes:  					

the substance in the dissolved form must be released from the carrier 

Both processes are closely related and depend on the physicochemical properties of the penetrant, the type of carrier and the penetration pathway. 

There are two skin penetration pathways for the exogenous substances: the transepidermal route through the skin cells and the transfollicular route through the skin appendages.

				 The transfollicular route has little significance because the appendages of the skin occupy less than 1% of the total skin surface, and the penetration can be moderated by the secretive activity of the appendages.The principal pathway for the skin penetration of xenobiotics is the transepidermal route.

  In the protein hydrogel of the viable skin layers (epidermis and dermis [ED]) the penetration rate can be considered as a constant and unlimited. These layers do not influence the rate of the penetration process and the permeated amounts.  

The real barrier for the skin penetration is the dead SC layer. The substance permeates this layer mostly via a tortuous intercellular lipid pathway (Figures 1 and 2).  

Terpenes are widely used in topical preparations. This paper focuses on the skin penetration of terpenes depending on their physicochemical properties and influence of the type of dermatological vehicle that was used. The structure of human skin and the skin penetration process is also briefly described.

Pharmaceutical Technology Europe-02-01-2007

Static electricity in solid dosage manufacturing

Disposable bioreactors based on wave agitation technology

Cell culture must go vegetarian

Applications of Raman spectroscopy in aqueous environments

Skin penetration of terpenes