Stem cell researchers are often in the news as governments and the general public debate which experiments should be allowed to proceed. They often say — usually with the support of industry groups and patient charities — that cells could one day provide cures for degenerative brain disorders, such as motor neurone disease and Alzheimer's. Of course, this work should be supported, but it is important not to raise false expectations because there is a huge manufacturing challenge involved in bringing cell therapies (especially stem cell therapies) from the lab bench into the clinic.  

Cell therapy is part of a new approach called regenerative medicine, which involves using cells, tissues, and possibly whole organs, to heal and repair the human body. Regenerative medicine may be able to achieve what conventional pharmaceuticals have not — effective, long-term treatments for chronic diseases involving tissue damage. Besides brain disorders, these would include arthritis, diabetes, stroke and heart disease — all of which are increasing as the population ages.  

There are two ways of performing cell therapy, each involving different manufacturing issues. Autologous cell therapy uses the patient's own cells; a biopsy is taken, the cells are manipulated and expanded in the lab, and then returned to the patient. In allogeneic cell therapy, donor cells are used as a source of therapy. Conducting autologous cell therapy on a commercial-scale involves 'scaling out' rather than scaling up; it is similar to procedures that already take place in hospital, such as blood transfusion, and may fit as a 'bedside' procedure into the healthcare system. Allogeneic therapy is more akin to conventional (bio)pharmaceuticals in its manufacture. Scale-up is an issue and one that is made more complex by the end-product being a living cell rather than an inert chemical.  

So far, cell therapies have been applied to more than 250000 patients using products either on the market or in clinical trials. Companies such as Organogenesis, Genzyme, Advanced BioHealing and Intercytex have shown how to manufacture cells for applications as skin substitutes and in-joint cartilage repairs. This, however, represents only a fraction of the potential market, even for well-established indications such as skin and bone. If the market is to achieve its potential, then automation must start to play a much larger role in cell manufacturing. 

"Currently, cell therapy is reliant on the manual 'green fingered' approach," says Professor Chris Mason of the regenerative medicine bioprocessing unit at University College London (UCL). "It has given us a great start, but it won't give us an industry." Automation will give cell therapies better quality and consistency, but the technology needed will be more complex than the robotics used in biopharmaceuticals, and the capital outlay may put investors off. However, some companies are beginning to automate small parts of the manufacturing process, while Advanced Cell Technology and Intercytex have built-in automation from the start.  

In manufacturing a small molecule drug, you can at least be sure of the quality of the starting materials. This is not the case in autologous cell therapy. There is no standardized procedure for surgeons to take the biopsy that starts the whole manufacturing process, and there is always the danger of contamination, which means discarding the product. The growth characteristics of cells also vary with the patient's age and state of health — a factor that no precautions can guard against. Therefore, autologous cell therapy has intrinsic unpredictabilities that manufacturers must deal with.  

Meanwhile, the maturation of the industry depends upon recruiting and retaining people with the right skills, which is proving difficult. There are specialized bioprocessing courses at UCL and some companies offer in-house training. "GMP experience is always transferable," comments Dr Paul Kemp, founder and chief scientific officer at UK company Intercytex, which is pioneering cell therapies for applications as diverse as venous and diabetic ulcers, and hair and skin regeneration. He thinks people from pharma may find adopting a nurturing, 'horticultural' approach to cells difficult; they are more used to regarding them as sources of contamination to be eliminated. Nursing cells can be both tedious and stressful. 

However, Mason argues that increased automation may go some way to addressing this problem. He adds that more needs to be done, for both kinds of cell therapies, on cryopreservation and distribution, if the industry is to reach its potential. At present, products are used 'fresh' and have a limited shelf-life, therefore, creating much wastage.  

There is also the issue of regulation. Currently, the EU is working out its new Advanced Medicinal Product Directive and it should come into force sometime over the next year. "The directive will be good for the industry," says Kemp. "We can jump the regulatory hurdles once we know what they are. With more clarity, we will be able to plan our clinical strategy." In the US (presidential vetos of federal funding of human embryonic stem cells [hESCs] aside) the regulation of the cell therapy industry is ahead of the EU position. One of the market leaders, Geron, has been working with FDA on regulation and is poised to begin the world's first ever clinical trial of hESCs later this year. 

There are also significant scientific barriers to overcome before cell therapy becomes a clinical reality. Much more needs to be known about how to expand and differentiate stem cells. It is also unclear how far allogeneic cell therapies will be immunogenic; that is, whether patients will need to take immunosuppressant medication for life (as with transplant recipients). Some of these issues are being addressed by initiatives such as bioProcessUK, which is creating a research interface between academia and industry to solve these problems.  

 "In cell therapy, the product is the process — the health and quality of the cells are mission critical," says Mason. Ultimately, the challenges of cell therapy manufacture can only be resolved by realising the unique nature of the product.  

Articles

Biopharmaceuticals

... there is a huge manufacturing challenge involved in bringing cell therapies (especially stem cell therapies) from the lab bench into the clinic.

Facing up to the cell therapy challenge

Much has been written about the so-called 'brain drain' that has been plaguing Europe for the past decade. Brain drain is a term used to describe the loss of talented, young European scientists and engineers who are lured to greater opportunities in the US. The exodus of Europe's finest and most talented has been blamed on a variety of factors including a failure of the EU to make adequate R&D investments; outdated immigration policies; antiquated patent and intellectual property (IP) laws; and a lack of financial incentives and innovation that exist at many of Europe's top academic institutions. 

The brain drain dilemma has not gone unnoticed by policy makers at the European Commission. To that end, in 2001 the Commission mandated that all EU member countries should strive to spend no less than 3% of their GDP on research by 2010. Previous reports indicated that the EU spends less of its GDP on R&D (1.9%) than the US (2.8%) or Japan (3% [

]). This policy was designed to create approximately 400000 new jobs for European scientists each year (

). In 2006, the Commission adopted the use of tax incentives to favour R&D and create a new community framework for state aid to R&D and innovation. Unfortunately, only Finland and Sweden have reached the 3% GDP goal for research spending and it is unlikely that other member countries will be able to hit that target by 2010 (

). Not surprisingly, the gap between EU and US research spending continues to widen at an alarming rate. 

A number of EU countries recently enacted legislation to make it easier for foreign scientists to enter and remain in countries to conduct research. Also, some EU countries now offer reduced fees, subsidized accommodations and even fast-track language courses for their foreign researchers. Although these inducements are likely to prompt an influx of foreign nationals into some EU countries, they will do little to stop the exodus of native-born scientists who typically leave for opportunities in the US. 

Unlike the US, there is no uniformity among the patent and IP laws that have been crafted by different EU member countries. In general, these antiquated laws tend to financially reward individual scientists rather than the academic institutions that sponsor their work. The lack of financial incentives explains why European universities and academic institutions do not aggressively build patent and IP portfolios. This is in marked contrast with the US where academic institutions encourage patent application preparation, and cover all of the fee and costs associated with patent prosecution. In this case, patents are wholly owned by the sponsor institution and the inventors are awarded a small portion of the royalty payments that are made to the institution when a patent is licensed to a commercial entity. 

The culture at many EU universities and academic institutions rewards older, established scientists. Grants and other forms of funding have often been awarded to well-known scientists with established research track records. Not surprisingly, many junior faculty members are under-funded and frequently exploited by senior researchers because they lack financial independence. Thus, it is not uncommon for junior researchers to toil for many years in suboptimal working conditions and receive little or no recognition for individual scientific accomplishments. This is in stark contrast with the situation in the US where talented, young, academic researchers are nurtured by senior faculty members and are allowed to compete for early funding to 'jump start' their careers.  

So what is the EU to do? It must grant its younger researchers greater scientific autonomy and academic independence. This will encourage younger faculty members to become more competitive, which should drive greater European innovation. EU academic institutions must provide junior scientists with substantially more financial support early in their careers. Further, the salaries of European scientists must become more competitive! EU patent and IP laws must be harmonized and changed so that both academic institutions and individual scientists stand to benefit financially. Finally, all EU member countries must strive to allocate more of their GDPs to R&D to stimulate economic growth and create new jobs. The over-abundance of science jobs in the US, coupled with a shortage of qualified US scientists to fill them, is the single most powerful inducement for immigration of European scientists to the US. Until the EU addresses many or all of these issues, it is likely that the brain drain phenomenon will continue well into the 21st century. 

Manufacturing & Processing

So what is the EU to do? It must grant its younger researchers greater scientific autonomy and academic independence.

Plugging the European brain drain to the US

Dry powder inhalers (DPIs) have advanced significantly over the past 10–15 years.

  This has been driven by the need to reformulate pressurized metered dose systems to chlorofluorocarbon (CFC)-free systems using hydrofluoroalkanes and the potential to deliver a wider range of therapeutics to the respiratory tract (through improved stability and greater dose choice). 

Apart from medication that specifically targets asthma, other therapies under investigation that use DPI systems include both local delivery (for example, mucolytics, antibiotics, antituberculosis and anticancer drugs) and systemic delivery (for example, insulin, analgesics for cancer pain and drugs for erectile dysfunction).

  In simple terms, the formulation of a DPI generally involves: 					

The production and characterization of a powder suitable for respiratory deposition.

The formulation with or without excipients in a device.

The system is characterized by aerosol performance and stability, and optimized through empirical observation. Although highly simplified here, these key stages of development will be discussed in this article.  

Arguably the nature of the active pharmaceutical ingredient (API) particles is the key to effective DPI delivery. The conventional way to produce a respirable size range API (<5 μm aerodynamic diameter) involves the crystallization from a solution followed by high-energy milling. Although this method is still the most common way to produce micronized APIs, it is not necessarily the most attractive as it requires a multistep process, results in particulates with ill-defined geometries and leads to questions concerning polymorphism and the degree of amorphous content. 

The deliberate control of the API's physical form, geometry and surface composition can be dated back to the 1980s when Gonda and co-workers successfully used elongated particles for their advantageous aerodynamic behaviour,

  and particles coated with hydrophobic additives for the reduction of hygroscopic growth in the airways after inhalation.

  Recently, there has been renewed interest in using engineered particles for DPI formulation to improve the powder dispersibility. The objective is to make 'nonsticky' particles that have a small aerodynamic diameter. The approach includes manipulation of the particle size (including nanoparticles), shape, density, surface morphology and other surface properties.

Single-step production methods have been developed with spray drying being the prominent one for inhaled insulin (approved by FDA) as well as inhaled mannitol, which was approved by the Australian Therapeutic Good Administration (TGA). Furthermore, the addition of binary/ternary components into the initial spray dryer solution may allow increased physical stability in primarily amorphous systems and/or the production of specific morphologies with, for example, improved aerosolization efficiency and reduced density. Other methods of API production include supercritical fluid (SCF) precipitation, high gravity precipitation and production through ultrasonic crystallization.

The formulation of many APIs requires the addition of binary or ternary agents after production to improve powder flow, improve dose reproducibility and act as a diluent for highly active compounds to fulfil the regulatory requirements and produce the anticipated clinical outcome. The traditional approach of powder blends containing the API mixed with lactose as a carrier is still most widely used for DPI products. Furthermore, in recent years, this approach has not only been used for dilution and improving the flowability, but also for enhancing the dispersibility of the micronized API. 

There have been significant advances, especially in understanding the role of carrier properties on the aerosol performance of the API. For example, early work suggested the roughness and the specific size of lactose carriers may be related to the aerosolization performance of an API through variation in contact geometry.

Such observations were substantiated and built upon in recent work, which suggests a complex interplay between performance and the degree of fine lactose,

  and aerosol API performance. The exact mechanism of adhesion and drug liberation during aerosolization is still not clearly understood and is a major focus among researchers within the field. 

Clearly, by controlling such parameters, the aerosolization performance of a particular API may be 'fine tuned' to acquire the chosen respiratory deposition, while maintaining product stability. Subsequently, many relevant carrier engineering technologies have been reported to improve specific performance parameters. 

Some of the key developments include the improvement of API aerosolization performance by controlled crystallization of the carrier: 

 Lactose crystallized from Carbopol gel achieves more regularly shaped lactose crystals with a smoother surface compared with the control lactose, resulting in higher efficiency and reproducibility of drug delivery by DPIs.

 The engineering of lactose carrier surfaces using a proprietary particle smoothing process results in significant differences in surface morphology when compared with the 'as supplied' starting material, and leads to improved aerosolization efficiency from commercially available DPIs.

Controlled etching of the carrier particles to reduce variation in API adhesion.

 Treatment of the lactose carrier by solution-phase variable temperature dissolution with a suitable liquid that can control the particle surface dissolution, combined with the removal of fines produce materials that increase aerosolization of the active from a single dose inhaler.

 Addition of ternary agents, such as magnesium stearate and/or L-Leucine, reduce API carrier adhesion; that is, the use of spray-drying produces a specific physical form of L-Leucine that is suitable as a flow aid during aerosolization.

 Addition of carrier fines can reduce the variation of API-carrier adhesion by filling active sites and/or by forming complex binary/ternary mixtures; that is, investigating the order of mixing of ternary components, examining particle interactions between drug and fine excipient particles (intrinsic or externally added ternary particles); and investigating adhesional properties of the lactose carrier surface using specific techniques such as the atomic force microscopy (AFM) colloid probe technique (CPT).

The inhaler device is as important as the powder formulation for the successful development of DPI products. Powder dispersion in DPI devices is accomplished either passively by the patient's own inspiration, or actively by a suitable component, such as an electronic vibrator or impeller, in the DPI device. Because micronized particles are difficult to deaggregate, the efficacy of passive DPI devices depends critically on a patient's inspiratory flow rate. 

As a result of the recent focus on tightening regulatory requirements, it is desirable for the performance of (breath-actuated) passive inhalers to have minimal dependence on the patient's inspiratory air flow. Furthermore, these DPI devices are now much more sophisticated than they used to be when only single-dose, capsule-loading systems existed. 

From the development perspective, 'passive' multidose DPIs fall into two main categories. They either measure the dose from a powder reservoir or they disperse individual doses that are premetered into blisters by the manufacturer. In general, the premetered drug-in-blister/capsule approach is easier to develop because the reproducibility of the metered dose can be ensured during the drug formulation and packaging, but for the purpose of this article the authors will mainly focus on novel multidose devices. 

One of the most recently marketed multidose DPI products in the US, Asmanex Twisthaler (Schering-Plough, Kenilworth, NJ, USA), for the delivery of mometasone furoate has shown highly reproducible results on emitted dose and fine particle fraction (FPF) using a patented agglomerate formulation.

  In the Twisthaler, the DPI nozzle comprises two parts: a lower swirl chamber and upper chimney in the mouthpiece. A patented fluted chimney design was used to produce a stronger vortex with an increased number of particle collisions with the chimney for deagglomeration.

The Novolizer (Meda Pharma BV, Amstelveen, The Netherlands) is a multidose DPI device already marketed in Europe.

  The Novolizer works on the cyclone principle and air classifier technology. These are robust concepts involving both centrifugal force and drug force acting on the particles swirling in a cylindrical chamber (cyclone), which coupled with collision force achieves drug particle detachment from carrier surfaces.

Another multidose device using a cyclone (in the mouthpiece) for drug deagglomeration is the Next DPI.

  The Otsuka DPI (ODPI [Otsuka Pharmaceutical Co., Ltd, Tokyo, Japan]) disperses a freeze-dry cake containing the active drug by inspiratory air flow of the patient during use.

  This is particularly suitable for delivering biological products, as each dose is individually contained in a vial which eliminates problems caused by contamination and microbial growth. 

Other recent devices include the Acu-Breathe multidose DPI by Respirics Inc. (Raleigh, NC, USA),

  a prototype DPI by Oriel Therapeutics Inc. (Research Triangle Park, NC, USA) using a piezoelectric polymer, which vibrates to deliver the powder,

  and a patented powder dispensing device based on the rotating fluidized bed principle.

  While most DPIs deliver relatively low doses of drug, the Actispire by Britannia Pharmaceuticals  (Redhill, UK) is suitable for high dose delivery (e.g., 20–250 mg of a synthetic lung surfactant).

  This device uses a pressurized gas to aerosolize micronized powder contained in the individual vial. 

There are numerous studies comparing the 

 performance of DPIs, but few provide insights into the mechanisms of powder dispersion in the inhaler devices. Such information is beneficial and would allow rational design of a more efficient inhaler with enhanced performance. The designs of the Twisthaler and the Next DPI were aided by computational fluid dynamics (CFD), but unfortunately, very little details were provided.

Recently, the Aerolizer (Figure 1) was investigated in a series of fundamental studies linking experimental powder dispersion data with CFD simulation of air flow in the inhaler.

  These results provided a deeper understanding of how the inhaler performance is influenced by DPI design features, including the design of a grid, the presence of a capsule and the dimension of the air inlet — all these are common features present in DPIs. 

 The grid was found to have three functions: 					

Helps straighten the air flow in the mouthpiece.

Generates small eddies immediately above the grid in the mouthpiece. 

Allows high-velocity collision between the particles and the grid to occur. A high FPF can be achieved using a grid that will generate more energetic turbulence and higher frequency of particle–grid collision.

 A number of passive DPIs use a capsule to contain a unit dose, followed by emptying the powder into the air stream inside the inhaler after piercing the capsules. It was found that the presence of a spinning capsule in the Aerolizer significantly reduces the turbulence (i.e., the potential for powder deagglomeration). However, the dimension of the pierced hole in the capsule is critical because deagglomeration occurs when the powder is sheared through the holes when it is emptied from the spinning capsule. 

 All DPIs contain some form of air inlet to allow entrainment of air into the device for powder emptying and dispersion as the patient inhales. If the air inlet dimension for the Aerolizer is reduced, it takes longer for the air flow to be fully developed in the inhaler, and the capsule spins faster (as a result of the higher inlet air linear speed) to empty the powder out into the air stream sooner. 

When the powder is emptied into the air stream before the air flow is fully developed, powder deagglomeration will be less effective, making the inhaler performance actually poorer with a smaller air inlet. This was observed for the Aerolizer operating at 60 and 90 L/min with a one-third or two-third inlet size. The results, therefore, highlighted the importance of synchronizing powder release and turbulence build-up in the inhaler. 

The market is rapidly expanding and many novel inhalation devices are in development. Several companies are active in the area, including Aradigm, Batelle Pharma, Chrysalis Technologies, Boehringer-Ingelheim and MAP Pharmaceuticals.

  All are developing 'active' inhalers that offer the advantage of dosing precision and an energy source to enable patient-independent, reproducible aerosol production. 

Recent research into multidose DPIs has been presented in this article. The aerosolization performance of a DPI is a complex system that clearly requires more fundamental studies. It is anticipated that future development of DPI products will rely more on rational design of both the inhaler device and powder formulation properties to attain the optimal products. 

Is a lecturer all at the Faculty of Pharmacy, University of Sydney (Australia). 

Is a former PhD student at the Faculty of Pharmacy, University of Sydney (Australia) and currently senior scientist at Pfizer Ltd (UK). 

Drug Delivery

There have been significant advances, especially in understanding the role of carrier properties on the aerosol performance of the API.

Dry powder inhalers: challenges and goals for next generation therapies

Unlike the traditional outsourcing of 'back-room' functions, many purchases of outsourcing services relate to 'representative' activities such as sales and merchandising, and other activities requiring contact with patients and the public. They tend to involve a supplier providing a visible workforce. 

The reputation of a purchaser is, therefore, associated with the actions of the service provider's staff. The potential purchaser of these services, therefore, must be confident in its provider. The focus of this article is on contractual issues that typically arise in these more outward looking services.  

This is a key area and an agreement should deal comprehensively with personnel issues, including:  					

Experience, qualifications and  general 'quality' issues.

A right to require the removal of individual  members of staff.

Controls on conduct, for example, by reference to a purchaser's employment handbooks or other contractual guidelines on behaviour, dress and so forth.

How bonuses will be filtered down to staff.

Rights to engage the provider's staff (considering relevant employment agency law).

Protection against the effects of Transfer of Undertakings Protection of Employment (TUPE) on  termination.

This last point can be particularly problematic if the parties do not clearly agree what is to happen.  

When a service is being provided internally by employees, although staff may have job descriptions, it is unusual for there to be much detail about what their daily tasks  consist of. One of the problems with outsourcing of all types is that the  service description setting out the tasks of the service provider is often too vague.  

Some services lend themselves to greater precision than others, for example, research and clinical trials. The position tends to be less clear with 'softer' services such as sales and merchandising. Sometimes the service can be described merely by reference to the resources to be  provided, such as numbers of account managers and sales executives. Ideally, the agreement would specify what the purchaser actually requires.  

Consideration is, therefore, needed of the activities that can be measured and targeted. For example, contact with a 'decision maker' may be required, rather than a simple site visit.  

Where teams are engaged to provide professional services such as assisting with clinical trials or are interfacing with patients, the agreement must be completely clear on how these  activities must be conducted, such as obtaining any required consents, ensuring appropriate patient selection, accurate data collection and audit trails. 

Generally, the provider must ensure that its staff complies with all relevant codes and guidelines, such as the ABPI code, and guidelines of the General Medical Council (GMC). 

The clinical trials disaster of the TGN1412 trial demonstrates what can happen when things go wrong. Appropriate legal protections should be included in the Agreement for loss or damage suffered by the  purchaser as a result of breaches and other  misconduct by the supplier's staff.  

From the supplier's point of view, it is unlikely that it will be prepared to accept all liability arising from its default and will normally seek to include limitations on its  liability, reflecting the degree of risk it is prepared to accept in return for the fees it is to receive.  

Liability issues are often  controversial, but much depends on the nature of the agreement. Some transactions are 'riskier' than others and proper debate is required to see what each party can sensibly accept rather than simply 'locking horns'. 

The insurance position on the  service supplier should also be checked and the supplier must undertake to carry the agreed levels of insurance.  

The agreement must clearly specify how the service charges are calculated. Charging structures vary, but a common one is an 'actual cost' basis whereby all costs in providing, for example, a salesforce and its  accompanying equipment, are simply passed through to the  purchaser, plus a management fee. 

Alternatively, a fixed fee per member of workforce may be agreed, depending upon seniority and skills, and is inclusive of certain types of expenses.  

The key is to try to ensure there are no surprises and that flexibility is realistically achieved ; for example,  discounts are available and if  reductions are required, charges are not penal.  

Actual costs contracts can work well provided it is clear what is a properly recoverable cost and whether any agreed budget is in fact a maximum. This can be a particular issue regarding employee costs; for example, who bears the costs associated with redundancies or legal claims by the team?  

If the purchaser does have to accept this type of cost liability, then it cuts deeply into one of the reasons for not wishing to take on a workforce itself directly. 

To achieve flexibility, if the purchaser is required to commit to take a level of resource for a given period, then that period needs to be suitable and not overly tying. Watch in particular for automatic rollovers of agreements for set periods. You may also want to consider an express right to cancel and negotiate a cancellation fee if this is important to you. 

Use strong confidentiality provisions to protect all information provided to supplier's staff, whether on  products, plans, working practices and so on.  

If the service provider's staff have access to the purchaser's systems and databases (e.g., to upload data), appropriate IT security arrangements are required to limit any potential 'snooping' or damage. 

The collection of personal information, whether from patients or other  individuals, will have data protection implications. The purchaser must control the way in which data is  collected, set out any requirements for obtaining specific consents and ensure that the service provider is appointed as a 'data processor' for the agreement; that is, so that it is only acting on behalf of the purchaser and has no right to itself manipulate or use the data collected.  

Finally, in any large-scale service  procurement, the issue of ongoing management can be overlooked. Sometimes outsourcing can be seen as the panacea to end all problems. In fact, managing outsourced service providers can be a major activity in its own right. One of the main reasons for project failure is a lack of commitment by the purchaser to ongoing management. This can either leave a service provider  disenchanted, in a desert, or over-powerful with no controls on it.  

The worst examples of this can arise where payments against  milestones are made — in poorly managed projects, the payments may be made as a matter of  administrative course, rather than there being any assessment as to whether the milestone has actually been achieved.  

There are many challenges  surrounding the outsourcing of  client-facing services, and while this should not put businesses off from making a commercially sound decision,  thorough precautions should be taken to avoid potentially costly  mistakes.  

One of the key stages in the  decision-making process should involve careful evaluation of possible areas of concern. These can then be addressed in the outsourcing contract, and typically include staff 'quality' issues training, TUPE, compliance and liability, confidentiality and many others. Planning is key to the success of any outsourcing project and should not be undertaken  half-heartedly.  

is a partner at the corporate and commercial team of Stevens and Bolton LLP (UK). 

Outsourcing

Liability issues are often controversial, but much depends on the nature of the agreement.

Contractually speaking

When developing active pharmaceutical ingredients (APIs) it is essential, first, to screen the most suitable polymorph for the formulation and, second, to be able to preserve its properties when the entire multistep manufacturing process is completed. However, it is well known that during processing, APIs and excipients can be subject to mechanical and/or thermal stress, and exposed to aqueous environments, which can cause transformations in the solid state.

  These process-induced transformations (PITs) cannot be neglected as they can lead to changes in processability, bioavailability and stability of the drug.

  The properties mentioned can change as a result of polymorphic interconversions, desolvation, alteration of the crystallinity or formation of hydrates. 

Solid state characterization with various complementary techniques is required to generate meaningful supporting data throughout the manufacturing process to assess concerns regarding PITs. Among a wide range of analytical techniques, Raman spectroscopy (RS) and near infrared (NIR) spectroscopy have attracted growing attention as they have great potential in process analysis and control. The recent developments in optics and probe design have made timely measurements easier in dynamic process conditions, such as moving powder beds and agitated suspensions, and the number of papers dealing with these issues is constantly growing. This paper lists some examples of PITs and how they can be monitored, with a focus on recent applications of Raman and NIR spectroscopy in different processing steps of pharmaceutical solids (Figure 1). 

The first unit operation during drug manufacture is often crystallization. To ensure the right solid form and avoid setbacks at later stages caused by undesirable solid forms, crystallization has to be performed in a highly controlled manner. As crystallization is a very complex process, several parameters have to be monitored to achieve a well-controlled process. 

Different kinds of PITs may occur during crystallization; for example, if the API being crystallized is enantiotropic (a system in which the stable polymorphic form is not the same above and below a specific transition temperature), temperature changes during the crystallization process can induce solid phase transformations. Transformations during crystallization may also occur because the first solid form to crystallize is often a metastable form that converts to a more stable form as the crystallization process proceeds. 

 used RS to simultaneously measure the desupersaturation profile (drug concentration in the solution) and polymorphic form of an enantiotropic API, flufenamic acid, during the crystallization process.

  Raman spectra were recorded in the study with a fibre-optic immersion probe in the crystallization vessel. Peaks characteristic of different phases of flufenamic acid (polymorphic forms I and III, and the solute) were identified in the spectra and quantitative information of the liquid and the solid phase was derived from the same spectra. Quantification of the solid phases was based on ratios of characteristic peak intensities. The Raman spectral analysis of drug concentration in the solution was in agreement with ultraviolet (UV) spectroscopic verification, and a temperature-induced interconversion between the polymorphic forms during the crystallization process was quantitatively monitored. 

 studied solid state transformations during crystallization and filtration processes with NIR spectroscopy.

  The study was performed with an API (SaC) appearing in two crystalline forms (SaC I and SaC II) and one amorphous form during processing. Crystallizations were performed in acetone solutions, and NIR spectra were recorded with a transreflectance probe immersed in the solution during crystallization. The downstream filtration process was monitored with a diffuse reflectance probe above the filter cake. Quantitative analysis was based on a multivariate method, principal component regression, using only a small number of calibration samples. Transformations from SaC I to SaC II (the stable form) were observed at different rates depending on various parameters (seeding, temperature and solvent composition). The transformations were successfully measured with NIR spectroscopy, and the coarse calibration with only few data points was found satisfactory for the measurement of polymorphic composition during processing. 

Milling or grinding is an important manufacturing process for solid dosage forms. A possible unwanted milling-induced phase transformation is the production of small levels of disorder or amorphous material in the milled crystalline material. This is usually found predominantly at the surfaces of crystals or powders, and these can lead to significant chemical and physical instability and change the processability of the formulation. However, a change from crystalline to amorphous can also be deliberately made by grinding when the aim is to produce an amorphous drug. 

 investigated the effect of morphology on milling induced changes.

  They showed that succinic acid with a plate-like morphology has more tendency to disorder than a needle-like morphology of the drug. It was also found that the type of mill influences the amount of change in crystallinity. Of the methods used, solution calorimetry, proved to be useful in quantifying small levels of amorphous material, which X-ray powder diffraction (XRPD) and disc-spinning calorimetry (DSC) could not detect. Mackin 

 used isothermal heat conduction microcalorimetry and dynamic vapour sorption to quantify low levels of amorphous material within a crystalline API after micronization.

  They found that amorphous material can be generated with extremely small changes in the operating conditions of the micronizer and were able to detect 0.5% levels of amorphous material with the above mentioned techniques.  

The aim is to monitor changes from crystalline to amorphous during processing, but so far very few examples of real-time measurements of phase transformations during milling exist in the cited literature. Only very recently Savolainen 

used on-line NIR and RS together with multivariate modelling tools to monitor process-induced changes from the crystalline to amorphous form for a crystalline starting material, lactose monohydrate.

  They concluded that spectroscopic techniques do not only provide a tool to monitor the changes in the amorphous content during milling, but they are also applicable as fast and uncomplicated methods to monitor amorphous content at different locations of the milling bowl. The variation in amorphous content in the bowl could also be used to describe the state of the process and to determine the end-point of milling. 

APIs are exposed to water in many pharmaceutical processes and the subsequent API–water interaction may induce phase transformations. As a typical example of this, wet granulation processes provide an ideal environment for the crystallization of new phases of the API. In fact, a number of PITs can occur during different process steps of wet granulation, which include wetting, mechanical stress and drying.  

Granulation or the manufacture of pellets using the extrusion–spheronization technique includes several process stages (blending of the dry mass, wet granulation of the mass, extrusion of the moist mass, rotation of the extrudate by spheronization and drying). The amount of wetting liquid in the powder mass in pelletization by extrusion–spheronization is relatively high compared with other granulation methods. Consequently, depending on the drug substance and excipients processed, solution-mediated polymorphic transformations are probable. Previously, Sandler 

have investigated phase transitions occurring in nitrofurantoin and theophylline formulations during pelletization by extrusion– spheronization.

  An at-line monitoring approach was used to increase the understanding of the solid-state behaviour of the APIs during the process. RS, NIR spectroscopy and XRPD were used in the characterization of polymorphic changes during the process. Samples were collected at the end of each processing stage (blending, granulation, extrusion, spheronization and drying). Water induced a hydrate formation in both model formulations during processing, and NIR spectroscopy gave valuable real-time data about the state of water in the system, but it was not able to detect the hydrate formation in the theophylline and nitrofurantoin formulations during the granulation, extrusion and spheronization stages because of saturation of the water signal. Raman and XRPD measurements confirmed the expected pseudopolymorphic changes of the APIs in the wet process stages. However, a relatively low Raman signal of theophylline (10% [w/w] in the formulation) complicated the interpretation of spectra. 

RS has been shown to be very helpful in controlling wet granulation processes. Jørgensen 

successfully used (off-line) charged coupled device (CCD) RS and NIR spectroscopy to investigate solid phase transitions of caffeine and theophylline during wet granulation.

  They concluded that the symmetric vibrations in the drug molecules could be effectively detected and analysed with RS. 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. Wikström 

successfully interfaced in-line RS to a high-shear granulation process and also monitored solid phase transformations of anhydrous theophylline.

  They also studied effects of several processing parameters, namely the mixing speed and monohydrate seeding. It was found that the mixing speed had the greatest effect on the phase transformation, showing that an increase in mixing speed shortened the onset time of conversion and increased the rate of transformation to the monohydrate. However, the seeding with monohydrate or a change in the incorporation of the binder to the system did have an effect on the transformation profile. 

After wet granulation or pelletization, the granules/pellets are usually dried to a certain moisture content. The drying can be performed, for example, in an oven on a tray or in a fluidized bed dryer. Either way, heat is brought to the system to evaporate the excess moisture from the particles, but in fluidized bed drying moisture removal is made more effective by blowing an air stream through the fluidized particles. The drying processes can cause phase transformations, in particular dehydration of hydrate forms. 

used in-line NIR and RS to quantitatively monitor the solid phase of an API during fluidized bed drying.

  Theophylline monohydrate granules were fluidized in a microscale fluid bed dryer and NIR and Raman spectra were recorded through a quartz sight window in the dryer wall (Figure 2). The quantification of the solid phase was based on a multivariate method, partial least squares regression. During fluidization, theophylline monohydrate lost its water from crystallization. The loss of water was clearly visible in the NIR spectra and quantification of monohydrate content was accurate. However, the real-time Raman spectra of the fluidized granules exhibited apparent features not present in Raman spectra of pure monohydrate or anhydrate form. These features were linked to the intermediate metastable anhydrate form upon dehydration of theophylline monohydrate to theophylline anhydrate. It was concluded that as a method sensitive to water, NIR spectroscopy detects the dehydration, and as a complementary method sensitive to alterations in the crystal structure, RS detects the anhydrate formation. 

Even though dissolution is not a manufacturing process, similar transformations can occur during dissolution of solid material. When a dosage form or an API is introduced to the gastrointestinal tract, or dissolution media mimicking it, a transformation from a metastable to a more stable form with lower solubility and bioavailability is possible. Such transformations have traditionally been detected as dissolution rate changes during intrinsic dissolution rate tests. However, the solid phase transformations during dissolution involve several overlapping factors affecting the dissolution rate that can hinder the detection of the transformation. For example, changes in the crystal habit and specific surface area as well as the concentration of the dissolution medium have an effect on the transformation kinetics and the overall dissolution rate. 

combined intrinsic dissolution rate testing with simultaneous 

 solid phase analysis of the dissolving sample to directly measure solid phase transformations during dissolution testing.

  The solid phase was quantitatively analysed by RS through a quartz window and the quantification was performed using ratios of characteristic peak intensities of each solid form in the Raman spectra. Dissolution behaviour of two hydrate-forming APIs (theophylline anhydrate and nitrofurantoin anhydrate) was investigated. Both APIs transformed to monohydrate forms during dissolution, but their dissolution behaviours were very different. Figure 3 shows results of an intrinsic dissolution rate test of theophylline anhydrate and, for comparison, theophylline monohydrate. The dissolution rate of theophylline anhydrate decreased clearly as the solid phase transformation to monohydrate form proceeded. The transformation was also very fast (~7 min). Dissolution rate changes of nitrofurantoin were more complicated and the transformation took much longer (~130 min). The incorporation of real time solid phase analysis to dissolution testing enables extensive knowledge of dissolution behaviour of drugs and is an improvement to prevailing practice, as it provides accurate information of the whole dissolution process, and not only the drug concentration in the dissolution medium. 

This overview shows that process-induced transformations are likely to occur during manufacture and end-product analysis of solid dosage forms. RS and NIR spectroscopy often provide suitable solutions for process monitoring, but the choice of the process analytical technique has to be made carefully to obtain meaningful data from the process. Developments in analytical methods and the immense activity in the field will hopefully result in a large amount of new examples of PIT monitoring during processing of solid dosage forms in the near future.  

is a postdoctoral researcher at the Division of Pharmaceutical Technology, University of Helsinki (Finland). His research area is solid phase analysis, with a special interest on the development of spectroscopic tools for real-time process analysis. 

is a senior scientist at AstraZeneca R&D (UK) in the area of material science supporting product development projects. He has experience in applications of image analysis in solid dosage forms process monitoring and solid state characterization with spectroscopic tools. 

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

Dosage Forms

When a dosage form or an API is introduced to the gastrointestinal tract, or dissolution media mimicking it, a transformation from a metastable to a more stable form with lower solubility and bioavailability is possible.

Monitoring of phase transformations during processing of solid dosage forms

Antibody-based therapeutics are expected to continue to be a major source of new therapies for the next decade. However, monoclonal antibodies (MAbs) are among the world's most expensive drugs. Pressure from healthcare providers is driving the need to lower the costs of manufacturing MAbs by as much as an order of magnitude — from $1000 per gram to $100 per gram.

So far, the most significant improvement is the increasing titres from concentrations of low milligram to multigram per litre (Table 1).

  This increased upstream productivity has subsequently created a demand on downstream processes to provide enhanced capacity and speed. In addition, because only a small proportion of biotherapeutics that enter clinical trials make it to market, companies tend to populate the candidate pipeline quickly, meaning more processes require development in shorter timeframes.

  To solve these multiple demands, several solutions have been developed, including the use of platform technologies and chromatography resin improvements. 

Table 1 Upstream productivity increases 1994â2004 (g/L).			

A downstream platform technology is based on experience with purifying a class of products with similar properties. With more than 500 MAbs currently in preclinical studies, monoclonal antibodies represent the greatest number of biotechnology products to which similar purification processes can be applied. In particular, the unique selectivity of protein A has enabled the development of platform technologies for the purification of monoclonal antibodies. 

Platform technologies facilitate rapid and economical process development and scale-up, potentially allowing evaluation of a larger number of product candidates, speeding market entry and even reducing validation. Familiarity with a process can also result in more robust processes and better technology transfer from development to manufacturing. Platform technologies create predictable activities and durations that result in a generic timeline.

  Further advantages are the use of established vendors for raw materials and standardized waste disposal procedures. Platform technologies are composed of a number of unit operations and methods, and not just purification steps. An example of a platform technology for a monoclonal antibody is shown in Figure 1.  

Monoclonal antibodies usually differ from one another in surface charge and glycosylation. By selecting and adjusting unit operations, selectivity is achieved for each MAb. Typically, the order of anion and cation exchange steps might be reversed, hydrophobic interaction chromatography might replace cation exchange chromatography, or hydroxyapatite columns might be used. Before adding or substituting a chromatography step, it is usually advisable to first try optimizing the process by evaluating column load, wash and elution buffers, and pH. Of particular importance is pH, as some proteins tend to aggregate when exposed to low pH. Downstream platform technologies thereby simplify development across the breadth of MAb products.  

Most host cell impurities will be very similar to process impurities. By extending the platform to the upstream processes of cell culture and cell clarification, even greater success rates can be achieved for the downstream process. Platform analytical approaches are also being applied to the development of monoclonal antibodies, reducing the time to toxicological and first-in-human (FIH) studies. Other important efficiency gains include fewer protocols and lower complexity of multisite operations, more lenient preliminary specifications for investigational new drugs (INDs), and more robust FIH methods and testing.

Platform technologies can also reduce validation costs. The use of platform technologies, combined with the precision and sensitivity of quantitative polymer chain reaction (QPCR) has helped the evaluation of the efficiency of generic and matrix viral clearance. Anion exchange steps in the platform are used as polishing steps; that is, to remove impurities such as DNA, host cell proteins and adventitious viruses.  

Because anion exchange chromatography is commonly used as part of the platform purification process for monoclonal antibodies, a great deal of knowledge has been gained in this area. Most monoclonal antibodies have basic isoelectric points, typically greater than 8, so they do not bind under neutral pH and low conductivity conditions. Experience with fast flow agarose anion exchangers combined with QPCR analysis has shown that bracketing and generic approaches demonstrate efficient removal 

from the process of a nonenveloped virus, SV40.

Another potential improvement in validation is minimizing resin lifetime studies for protein A and anion exchange chromatography. Studies with anion exchangers in flow through mode have demonstrated that surrogate measurements such as DNA clearance, back-pressure, and band spreading can predict when columns will no longer provide sufficient virus clearance.

  It has been shown that when Protein A columns are multiply cycled, antibody step yield and breakthrough are performance quality attributes that decay before a decrease in retrovirus log reduction values (LRV).

Today, some chromatography resins can achieve binding capacities greater than 250 g/L of protein. Fast mass transfer rates enable the use of more than 80% of the capacity after only 1 min, and flow velocities can now exceed 1000 cm/h. For ion exchangers, the most commonly used type of resin, these improvements have been achieved through a greater understanding of resin structure and surface chemistry that enables bead design with appropriate mechanical properties, porosity and distribution of charged functional groups.  

Binding capacity can influence process economics in several ways. High capacity can reduce column size requirements, which in turn can reduce space requirements and water consumption for buffers and cleaning solutions. Binding kinetics also influence raw material costs per gram of product, though this area is rather complex. In the example shown in Figure 2, resin A has a high total capacity, slow kinetics and low maximum flow velocity compared with resin B, which has low total capacity, fast kinetics and a high maximum flow velocity. At very short residence times, resin B gave higher productivity than resin A; but when residence time was optimized for resin A, it provided a more economical solution. The example also points out the importance of process development for each individual biotherapeutic. 

Improvements in resin design can be illustrated by comparing a classic fast flow, agarose-based resin with a recently introduced resin designed to improve productivity. The increased flow velocity that can be achieved with this second-generation anion exchanger is demonstrated in an industrial column with a 20 cm bed height and a 1 m internal diameter (i.d. [Figure 3]). A constraint of the industrial production environment is the need to work with moderate pressures to achieve adequate flow rates. Realistically, very large columns for capture applications should not need to handle more than 1–2 bar to avoid weight and handling issues. Designing an ion exchanger for capture means striking a balance between bead properties that give high capacity and high resolution, and properties that allow high flow rates at low back-pressures. The packed bed of the new resin enables a linear flow rate of more than 700 cm/h at a back-pressure of less than 3 bar. High flow rates can improve turnaround time in downstream processing by decreasing washing, cleaning, and re-equilibration times. In most cases, sample loading and elution times can also be reduced, though this reduction depends on the properties of the target molecule and impurities. These time savings can result in the production of more batches to meet the quantity demands for high-dose biopharmaceuticals. 

High flow velocity alone does not make a productive process. Binding capacity at high speeds, however, is essential for capture steps. The second-generation resin provides significantly higher dynamic binding capacities compared with classical fast flow agarose anion exchangers (Figure 4).  

A high binding capacity of 80–120 g/L immunoglobulin G (IgG [at residence times of 2–6 min and typical pH and conductivities]) could be obtained with a novel, recently introduced cation exchanger belonging to the same high flow agarose generation.

By combining speed and binding capacity, overall productivity is enhanced, as seen in Figure 5, which compares high-scale productivity for the capture of a target protein from an 

 homogenate on three different resins. This study demonstrated that it is feasible to purify more than 100 kg of target protein product in 24 h.  

Selectivity plays a major role in platform technologies because it reduces the number of purification steps, thereby shortening overall production times and increasing recovery. Specific binding of the target molecule to a ligand-substituted resin enhances productivity by rapidly removing the bulk of the impurities from the feedstream and concentrating the product in one step. This point is supported by the successful use of immobilized protein A as the capture step in the production of most approved therapeutic monoclonal antibodies as well as hundreds in development. Protein A binds human IgGs at the Fc region, which facilitates the isolation of monoclonal antibodies from cell culture harvest. Purity levels are 98% or greater after this one unit operation, greatly simplifying the challenges further downstream and allowing robust, economical purification. Successful therapeutic applications of MAbs have put great demands on production quantities, where expression levels of more than 1 g/L are commonly reported. A new generation of high flow agarose-based protein A resins has been designed to handle large volumes of feed with high titres (Table 2).   

Table 2 Challenges of downstream processing to produce large amounts of MAbs, addressed by second-generation, high flow agarose protein A resins.			

The potential for carryover of product and impurities from one run to another must be addressed for packed columns, and the cleaning protocol must be shown to remove residual materials. Many cleaning and sanitization protocols use NaOH. Another factor is the stability of resins, columns and associated equipment in sufficiently harsh conditions for cleaning and sanitization. In addition, column packing at large scale is time-consuming and costly; maintaining a packed column is essential for economical production of consistent product. Repacking columns requires large quantities of water and buffers column qualification exercises, and time. If reserve columns are not available, the failure of a column because of bacterial contamination, for example, could result in production downtime that alters in-process holding times, complicates scheduling in the plant and creates an overall decrease in productivity. Furthermore, effective cleaning and sanitization, with preserved performance, are essential to reduce the raw material costs of resins. Protein A-based resins used for the capture of MAbs are more expensive than ion exchangers, but their use becomes economically favourable if they can be used for many production cycles. An example of how increased resin stability can extend the number of cleaning cycles that can be performed 

 is shown in Figure 6. Although the dynamic binding capacity of a conventional protein A resin was below 90% of the original capacity for a polyclonal antibody after approximately 20 cycles, the novel protein A resin, designed to tolerate NaOH, was observed to maintain a capacity of 90% up to almost 200 cycles when 0.1 M NaOH was used, and up to about 60 cycles when cleaned with 0.5 M NaOH for 15 min. The resin uses a genetically engineered protein A ligand designed for improved stability under alkali conditions. Other properties were also optimized for MAb capture. 

In a case study, a clarified Chinese hamster ovary (CHO) cell culture feedstream containing IgG

 was purified on a column packed with the alkali-stabilized protein A media for 150 cycles, with cleaning-in-place every cycle for 15 min with 0.1 M NaOH. The yield was consistently 95–100% and purity levels were consistent, as measured by removal of host cell proteins. There was no detectable carryover and no signs of discolourations or deposits on the resin. The dynamic binding capacity remained at greater than 85% of the initial value. 

Improvements in feedstream titres translate into the need for improvements in downstream processing productivity. A multiprong approach has enabled downstream process scientists to achieve the necessary productivity. The proven strategies include the use of platform technologies consisting of second-generation resins with increased capacity and velocity, and enhanced stability to harsh cleaning and sanitization conditions. 

The PCR is covered by patents owned by Roche Molecular Systems and F Hoffmann-La Roche. A licence to use the PCR process for certain research and development activities accompanies the purchase of certain reagents from licensed suppliers. 

is product manager, bioprocess, both for the life sciences business unit of GE Healthcare (USA). 

Pharmaceutical Technology Europe-04-01-2007

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Plugging the European brain drain to the US

Dry powder inhalers: challenges and goals for next generation therapies

Contractually speaking

Monitoring of phase transformations during processing of solid dosage forms

Improving productivity in downstream processing