The number of biotechnology-based human therapeutic products in the late-stage pipeline along with the average cost to commercialize a biotech product has been steadily increasing with time. In addition, the biotech industry is facing unprecedented challenges of a sagging global economy and rising regulatory expectations. Companies have to continue to evolve their approaches to be more efficient with respect to time, resources and cost. This article describes some of the technologies that can help optimize time and cost of biopharmaecutical manufacturing.

Creating processes that are not only optimal and robust, but also economical in a time and resource efficient manner is the Holy Grail for the biotech industry today. Regulatory initiatives such as Quality by Design and PAT require an improved process understanding earlier in the product life cycle.

Recent advances in fermentation and cell culture processes have increased cell densities and protein titers in process fluids, requiring reliable upstream technologies to handle growing production demands.

 Moreover, within the bioprocessing industry there is a strong trend toward the application of single-use technologies, eliminating the expense of cleaning and cleaning validation, a major cost factor in operating reusable systems. One of the commonly used technologies for primary separation after fermentation is tangential flow filtration (TFF).

 TFF processes have traditionally used cassettes or hollow fiber formats. Typically, TFF cassettes are installed in stainless steel housings that require post-use cleaning and validation of cleaning. The installation and preconditioning steps before process use are time consuming and require a reasonable level of skill to avoid potential malfunction. Hollow fiber filter systems provide an alternative primary separation technology. However, this open channel format tends to require relatively high pumping rates for optimal performance. Achieving these high rates with fully disposable equipment can be difficult.

Membrane chromatography has already proven to be a powerful alternative to traditional packed‑bed chromatography in flow‑through operations, such as polishing for the removal of viruses and contaminants in biologics manufacturing.

 Case studies have been described for the purification of monoclonal antibodies (mAbs) with a high isoelectric point and have demonstrated the popularity of their implementation. As flow‑through utilization has expanded, membrane chromatography applications have also included the capturing of large molecules.

 Such bind‑and‑elute applications imply the demand for higher capacity and larger surface membrane area compared with flow‑through applications.

Manufacturers of biopharmaceuticals are required to characterize the ability of key process steps to clear viruses. Typically, the entire manufacturing process is qualified to attain a cumulative virus reduction factor, which significantly contributes to the documentation of virus safety. In many of these manufacturing processes, a virus retentive filter is used to achieve a robust and effective virus clearance step. The key to implementing virus filtration is the assurance of virus retention and device integrity while maintaining high productivity and ease of use.

While developments in chromatography media (resins) have brought the technique much closer to practical and theoretical limits of binding capacity, future advances are likely to focus on new surface chemistries and a more efficient use of chromatography as a unit operation. One way to gain productivity improvements is to use smaller equipment or the similar size equipment in a more time‑efficient manner.

 series and will present some of the most promising technologies that are being introduced in biotech manufacturing by some of the major biopharmaceutical vendors.

TFF microfiltration capsule for cell clarification and harvest


The Kleenpak TFF microfiltration (MF) capsule from Pall Life Sciences (NY, USA) uses an Ultipleat pleated filter cartridge and Kleenpak Nova capsule with their operation based on TFF principles. The capsule is configured similarly to hollow fibers or cassette formats with feed, retentate and permeate ports. As Figure 1 shows, the pleated construction is used inside the capsule to create multiple parallel flow channels while increasing the effective membrane area in a reduced footprint. It is simple to install and requires limited preconditioning before exposure to process fluid. 

This technology has been tested with representative industrial strains such as Chinese hamster ovary (CHO) cells, and bacteria such as 

 and yeast. Feed volumes of 50100 L have been processed per single full‑scale capsule (0.5 m

), depending on cell type and characteristics. The capsules can be manifolded together in series or in parallel to accommodate larger process requirements; for example, a six capsule assembly can process up to 1000 L. Stable performance has been observed for mAb clarification from CHO cell feed with starting cell counts up to 1x107 cells/mL and step yields of greater than 98% have been obtained. For clarification of hybridoma cells expressing mAbs, filtrate flux rates of 70 L/m

/h (LMH) to a volumetric throughput (VT) of 180 L/m

 have been obtained with a protein transmission of 100%. Protein expression for these cells was approximately 0.1 mg/mL.

A comparison of the Kleenpak TFF technology and two different types of cassettes for concentrating 

 with all three devices incorporating membranes rated at 0.2 μm (nominal) is presented in Figure 2. 

All three devices were operated at a crossflow flux rate (CFF) of 1.5 L/min/ft

. A five-fold volumetric concentration followed by a three‑fold diafiltration buffer exchange was performed. The data show that all three devices can be operated at a flux rate of 30 LMH throughout both concentration and diafiltration stages. Although the start and finish transmembrane pressure (TMP) values are different for the Kleenpak TFF filter compared with the cassette formats, the TMP gradient is similar for all three technologies and suggests stable operation. The flux and throughput performance of all three technologies was similar during these trials. Figure 3 presents a comparison of a Kleenpak TFF MF capsule with a Pall Microza hollow fiber module for clarification of IgG from a CHO cell culture with a cell density of approximately 2x106/mL.

 Each format was operated at a pumping capacity or CFF of 20 L/min/m

. It is seen that the Kleenpak TFF outperforms the hollow fiber module by processing more than three times the filtrate volume for a similar terminal TMP. 

Experiments were conducted with a recombinant 

solution at a cell density of approximately 4% w/w for production of a plasmid used for gene therapy treatment. The primary objective of the application was to concentrate the 

feed by a minimum concentration factor of 10‑fold using a single‑use technology. Initial trials were conducted with the small‑scale device and the filtrate flux rate for concentration was selected to be 22.5 LMH. It was estimated that six full-scale modules (membrane area of 3 m

) will be required to process 500 L of feed at a feed flow rate of approximately 21 L/min (CFF of 7 L/min/m

). To demonstrate scalability to a series and parallel configuration, a trial was conducted to compare the performance of a single full-scale module against the performance of three full‑scale modules operated in series. The filtrate flux rate was maintained at 22.5 LMH for each module throughout the trial. Pressure gauges were installed throughout the system to enable accurate TMP determination across each module. The results shown in Figure 4 indicate that each of the three capsules manifolded together in series provided a VT 

 TMP curve very similar to a single module operated in isolation. 

In summary, the Kleenpak TFF MF capsules’ single‑use TFF technology offers major benefits for cell harvesting and clarification. The self‑contained format eliminates the need to purchase and install holders or housings, and also reduces exposure to biologically active solutions. This is a critical consideration for manufacturers of vaccines and other products that present potential workplace hazards. The optimized feed channel screen provides good performance at low flows and the technology is scalable and reproducible.

Hydrophobic interaction membrane chromatography


Sartobind Phenyl membrane adsorber from Sartorius Stedim Biotech (Germany) uses hydrophobic interaction chromatography (HIC) principles and assembled into a 30‑layer radial flow process capsule (Sartobind). It is based on hydrophilic regenerated stabilized cellulose with the hydrophobic phenyl groups covalently attached to the cellulose matrix. The novel macroporous membrane structure with 13 µm pores has been designed for high flow rates and high binding capacities. As seen in Table 1, the binding capacity for a mAb on HIC membrane is found to be at par with conventional HIC resins even at significantly higher flow rates. 

Table 1: Protein binding capacity on Sartobind Phenyl Membrane and conventional Phenyl Resin. Binding buffer for mAb: 08 M (NH

Articles

Biopharmaceuticals

Upcoming technologies to facilitate more efficient biologics manufacturing

In the business and financial environment we are currently enduring, you wouldn't expect investors to be anxious to buy companies with somewhat uncertain prospects, but this is what is happening in the pharmaceutical services industry. Private equity investors are lining up to own all or a piece of a CRO or CMO business, and the depressed stock market is creating opportunities for them to get in at very attractive valuations. 

The hot interest in pharmaceutical services has some features that make it different from what we saw 2 years ago; for one, the acquisition prices are now a lot lower at the top of the leveraged buyout frenzy. Back then, Blackstone Group (NY, USA) acquired what is now Catalent (NJ, USA) for $3 billion (€2.4 billion) and financed it with $2 billion (€1.6 billion) in debt. The price was 1.6 times sales and 10 times pretax cash flow. Premier Research Group (UK) was taken private by ECI Partners (UK) last year for a similar multiple. By contrast, today's prices are likely to be less than one times revenue with less than half of the purchase price borrowed. 

The lower valuations reflect the restricted availability of debt (when debt was cheap, companies could afford to bid the price up) and the overall uncertain outlook for the bio/pharmaceutical industry. As I've discussed in previous columns, the industry is experiencing a sharp cutback in pipeline development activity as earlystage companies struggle for financing and major companies restructure to improve earnings and cash flow. 

Still, private investors appear confident that the pharmaceutical services business is a longterm winner. Major companies appear committed to outsourcing an ever larger share of their drug development and manufacturing activity. Furthermore, they will increasingly depend on early-stage companies to do the discovery and early development work, and then in-license or acquire the most promising candidates. This means that the earlystage companies will continue to get funding to feed their dependence on contract services. 

The biggest deal to hit the news recently is the acquisition of PharmaNet Development Group (NJ, USA) by JLL Partners (NY, USA), a private equity firm that is also a major investor in CMO Patheon (NC, USA). The total value of the deal will be nearly $250 million (€196 million), comprising $100 million (€78.4 million) for the outstanding PharmaNet stock (valued at $5.00/€3.9per share in the transaction) and $144 million (€112.9 million) to retire PharmaNet's outstanding convertible debt.  

PharmaNet provides Phase I–IV clinical research services, as well as bioanalytical laboratory services. The original PharmaNet Development Group was founded in 1996 by a team that included the founders of what is now Covance, and was acquired by publicly traded SFBC International in late 2004 for $245 million (€192 million). SFBC International changed its name in 2005 to PharmaNet following a scandal surrounding operations at its Phase I operations in Florida (USA). It had revenues of approximately $360 million (€282.1 million) (net of pass throughs) in 2008, putting it among the top 10 clinical CROs by revenue. 

PharmaNet's stock price dropped from more than $40(€31.3) per share in March 2008 to less than $1.00 (0.78) by December 2008. That cataclysmic price drop reflected several factors, including the overall decline in CRO stock prices (Covance dropped from more than $90 (€70.5) a share to less than $35 (€27.4) per share during the period), and a sharp drop in revenues and backlog during the second half of 2008. A complicating factor was the company's inability to get holders of the senior convertible debt to agree to redeem those notes for debt with new terms; under the terms of the notes, the debt holders could force redemption in August 2009 at face value and were in a position to take control of the company if it could not redeem them. 

In a sense, PharmaNet is a victim of the altered financial environment, which starved many of its customers of capital while cutting off its own ability to refinance its debt. At the same time, however, it is a victim of its own problems: the convertible debt was originally issued in 2004 to allow SFBC to acquire PharmaNet, and the subsequent legal problems surrounding the Florida Phase I sites probably distracted management attention from creating a more sustainable business model the way competitors such as Covance and PPD have done.  

The sale to JLL will allow PharmaNet's management team, which is well respected in the industry, to focus on improving the company's operations and, possibly, expand its capabilities through selected acquisitions. In the time it has been an investor at Patheon, JLL has shown itself to be a patient and long-term investor, helping Patheon management complete a necessary, but painful restructuring. JLL has also offered to buy more of the outstanding shares of Patheon, but at a price that may not be attractive to existing shareholders. 

Private equity investors may get another bite of the Top 10 CRO apple in coming months if the directors of MDS Inc. (ON, Canada), parent of MDS Pharmaceutical Services (MDSPS), decide to test the market. MDS announced that it has formed a committee of independent directors to review alternative ways to improve shareholder value. The committee will work with outside advisors to review a broad range of options, from doing nothing to sale of business units or the entire company. The company says there is no timetable for the review to be completed. 

As with PharmaNet, MDSPS, which had revenues of $482 million (€377.7 million) in 2008, has faced some particular challenges. Its early development business has been hurt by the reputational and operational implications of quality problems at its Montréal (Canada) bioanalytical lab, which was cited by FDA in 2004 for inadequate investigation of contaminated samples. Management reports that the problems are largely behind it and that it is attracting clients back to the bioanalytical business. In the Phase II–IV segment, MDSPS continues to lose market share to larger competitors, including Quintiles, PPD, Icon and Parexel (NC, USA). It suffers from smaller scale and a more limited global footprint, even though its revenues place it among the Top 10 CROs. 

MDS has ample cash and little longterm debt, so it is not compelled to sell. However, the company's board and management have been under investor pressure for some time to consider strategic alternatives. With three distinct businesses in its portfolio (MDS also has business units that make analytical instruments and radiopharmaceuticals), the company would seem to have some interesting options, including selling one or more of the business units to either strategic or private equity buyers. However, as one analyst told me, the board could find that what buyers are willing to pay for the businesses in the current financial environment is well below what they believe they are worth.  

While publicly held CROs represent the highest profile targets, we are aware of several wellknown privately held CMOs whose owners have put them on the block and which are attracting considerable interest from investors. Private investors aren't the only buyers either: publicly held CROs such as PPD (NC, USA), which recently acquired the Eastern Europe CRO AbCRO (Bulgaria), are also taking the opportunity to make deals that help flesh out their capabilities. 

This doesn't mean that investors aren't being picky, however. Investors are focusing on CROs with strong market positions and viable business models, mindful that the services industry will experience a shake out in coming months. We know of several highprofile companies that have been unsuccessful at finding private investors in the last year. 

The lesson is that despite the doom and gloom in the financial media, there are still good businesses to invest in and institutions with money to invest in them. The smart money seems to be saying that pharmaceutical services will be a great business well into the future. 

Outsourcing review

Despite the market downturn and financial meltdown, private equity investors are lining up for pharmaceutical services businesses.

Pharma's silver lining

Many companies are adopting or extending PAT solutions to achieve product innovation and process improvements through engineering development and manufacturing performance. Typically, quality units Quality Assurance or Quality Control operate within budgets representing 20–30% of manufacturing costs, but industry statistics indicate that adopting PAT could reduce this figure to <5%. Simultaneously, PAT could enhance product yields, enable faster product timetomarket, and improve operational performance by increasing plant and process line key performance indicators (KPIs). 

(GEORGE DOYLE & CIARAN GRIFFIN/GETTY IMAGES) 			

Employing the PAT principle of having a scientific understanding of how critical process parameters impact product quality attributes is fundamental to drive operational excellence programmes and lower the risk, as well as cost, of manufacturing. Despite these benefits, many companies are concerned about embracing PAT solutions for all aspects of the drug life cycle because of the potential risks at the different levels:  					

Supply chain and business level — risks include excessive raw material inventory acquisition, storage or usage, throughput and product delivery time, and loss of product quality excellence. 

Programme or project activities at midmanagement level — risks revolve around organizations because new cross-functional teams must be created, which could diminish other activities and cause personal relocation or dislocation. 

Project or plant level — risks include developing new complex process development techniques, operator training and capability enhancement.

Operational excellence enables an organization to manufacture products to exact quality specifications in the shortest period of time, consume the least amount of resources possible, address regulatory requirements and satisfy customer demand. Goals will vary for each company depending on operating constraints, but referencing 'world class' benchmarks can provide insight as to what is possible. 

Business level risks may cause companies to struggle in finding the best way to utilize PAT. A company may not have a clear manufacturing technology strategy or may possess a 'wait and see' attitude regarding PAT, or adopting PAT may involve undefined expenses. Management may also consider PAT initiatives as labour reduction exercises, which are counterproductive at all levels in the organization as labour reduction in terms of rationalization or downsizing may be perceived as a large risk by personnel. Additionally, PAT can cause a paradigm shift in an unprepared business. New personal skills and capabilities may be required to support Quality by Design (QbD) for new products or building quality into the manufacture of existing important drugs.  

Organizations must be willing to make appropriate changes to benefit from utilizing PAT in any of the three areas presented by FDA:

Designing quality into the processes for existing products.

Maintaining cGMP operations throughout the manufacturing floor.

What are the risk factors at each of the operational management levels? 

Global competition, rising costs, timetomarket constraints, governmental regulations, political pressures and supply chain management are driving companies to develop new products to meet market needs, and ensure product quality and patient safety, while also increasing shareholder value. Today's businesses require: 					

Comprehensive understanding of process and product quality relationships.

Costeffective process solutions investments.

Implementing PAT programmes into process facilities can be challenging and may pose a risk to the business. Because new processes change the way operations are performed, there is an associated risk to product quality and regulatory compliance. Implementing the proposed changes may, therefore, be met with some reluctance. Additionally, each business must keep existing operations running, continue to manufacture quality products and allocate resources for future projects. By considering the risks and benefits associated with their vision for a 'desired state', a comprehensive plan can manage risk during PAT project implementation, and utilize company resources effectively and efficiently. 

With operating efficiencies increasingly dependent on 'right first time', there is a need to change the 

 in the way processes are controlled. These changes impact product life cycle management, resulting in a risk to operations and overall organization. 

PAT programmes can help companies achieve their desired state, address today's business requirements and mitigate business risks. As described in FDA guidance,

  PAT must be based on a scientific understanding of how development formulations and manufacturing process factors impact product performance and quality attributes. Understanding the relationship between these concepts is essential in applying PAT during the entire product life cycle (Figure 1). 

PAT methods should be identified that analyse parameters or variables that control critical process parameters through defined process models and produce specified product quality characteristics with continuous verification. PAT is both strategic and tactical: strategic in that it establishes plans to achieve company goals, and tactical in that it defines specific projects that are aligned with strategic plans. 

Implementation and automation solutions may be defined through an applied systems development life cycle (SDLC) that: 					

Assures organizational buyin at senior management level.

Involves multidiscipline teams, such as R&D, process engineering, quality, operations and maintenance.

Engages stateoftheart applications, including multivariate analysis, design of experiments (DoE), predictor–corrector algorithms and advanced process controls. 

Creates offline research systems for process development.

Is scalable from pilot projects to manufacturing and the enterprise. 

Integrates into online systems for process/production line control.

Is compatible with company IT infrastructure, network and data storage.

Communicates with applicable governmental agencies such as FDA and EMEA.

Continues to consider all relevant governmental agency regulations and guidance, particularly for making and reporting manufacturing changes.

Provides for continuous quality improvement in the product life cycle. 

Through multivariate analysis in R&D, a correlation between critical process parameters and product quality attributes can be established. This type of analytical approach is a basis for identifying parameters that should be controlled through scientific understanding of what factors contribute to product quality. In turn, the performance of DoE in R&D can be implemented to optimize equipment setup and operation before transferring processes to manufacturing. These activities foster an environment for producing product QbD that reduces startup time and inprocess development to drive operational excellence directly through R&D endeavours. 

This is a means for applying knowledgeable change to increase right-first-time performance indicators. Incremental process improvements to existing process automation and control systems, such as closed loop predictor–corrector algorithms, can be implemented as they are identified. This approach provides an opportunity to analyse processes and implement applicable PAT methods. 

Managing the risks associated with PAT initiatives provides a framework to drive operation excellence through a scientific understanding of critical process parameters as they relate to, and influence, product quality attributes. In addition, properly applied PAT will mitigate the risk associated with regulatory compliance and meet business objectives (Table 1). 

Table 1: Potential benefits of PAT initiatives.			

 Once a company is committed to using PAT, a programme or project must be initiated. Unfortunately, there are PAT projects that have been started without reaching their full potential as there was no real cohesive business strategy for implementing them. When executing the planning phase, the risks in the project preparation phase, as well as in future development phases, must be recognized. 

It is important to build a crossfunctional team with executive sponsorship. The executive sponsor should also be the project champion. Without the commitment and active participation of the sponsor, the project has a high risk of failing.  

The project team will need to identify the control system that will be the foundation of the process line. IT systems that can integrate the company's process systems, databases of historical data and other information generated by PAT technology should also be identified. The resulting planning, implementation and management will extend during the life of the project, and different groups will take the lead role throughout the various phases of SDLC for the product, process or both. Losing project sponsorship could lead to a lack of focus, resulting in inefficient resource or higher funding requirements. 

Operational excellence awaits, but only if you can implement PAT successfully.

Managing the risks of PAT

Do I need particle size specifications for my pharmaceutical product?

Specifications define the critical to quality factors to which a pharmaceutical product must conform to be acceptable for its intended application. Quality by Design highlights the importance of identifying and controlling 

 the parameters that can influence final product performance: ICH Topic Q6A identifies particle size as a potentially important variable.

  The specific need for a particle size specification is determined by assessing if and how this parameter affects performance. 

For solid or suspension delivery systems, bioavailability is often directly related to particle size because it controls dissolution/solubility characteristics. Dissolution rate is directly proportional to particle surface area (Noyes-Whitney equation), so a finer particle size promotes faster drug dissolution. Particle size distribution is also relevant as a narrow distribution produces more uniform dissolution. Formulations with even a small number of relatively large particles may take some time to dissolve completely, but this may be the design intent. 

For suspensions, stability is an important issue because if the active ingredient settles there is a greater chance of non-uniform delivery. Stokes' law relates settling velocity to the physical characteristics of the fluid and the size of particles in the suspension. The relationship here is a strong one: velocity correlates with the square of particle diameter. For suspension stability, a very low settling velocity is preferable and is more easily achieved with finer particles. 

Perhaps less obviously, particle size may also affect a formulation's behaviour during processing and, ultimately, its content uniformity, which is critical. Take, for example, the widespread operation of direct compression tableting; particle size can influence segregation behaviour, the ease with which powder flows through the press and the compressibility of a formulation. In turn, these factors affect the consistency of tablet weight and composition, how the press operates and the mechanical properties of the finished product.  

Figure 1 shows a near-infrared chemical image of a recalled tablet. The active ingredientrich domains (the red areas) are much larger in the right side of the tablet than in the left, demonstrating that composition is not uniform. The product was recalled in this case as the larger particle size may have caused some unit doses to fail potency specifications. Poor control of particle size clearly resulted in a substandard product.  

As analytical techniques become more sophisticated, researchers are increasingly able to show that particle shape, as well as size, can be influential. The tableting example illustrates this point as segregation, flow properties and compressibility are a function of both size 

 shape, not size alone. It is not uncommon to find particle size specifications supplemented with complementary shape criteria. 

In summary, if any critical performance attributes depend on particle size or shape, a suitable specification is needed. If other variables can fully define performance, then none is necessary. In practice, particle size specifications are common within the industry for tablets, dry powder inhalers, suspensions and creams. 

What issues should be considered when selecting a particle sizing technique/instrument?

Once the need for a particle size specification is established, it must be defined in the most relevant way. Here, an understanding of different particle size measurement techniques (operating principles and the reported variable) is essential. There are many ways of defining particle size, but only perfect spheres, which are rare in the manufacturing environment, can be described fully using a single number (a diameter). For particles with irregular shapes, there are multiple, equally valid measures of size including:  			

equivalent volume diameter (diameter of a sphere with the same volume)

circle equivalent diameter (diameter of a circle with the same projected area)

longest dimension (arguably most relevant when particles are highly irregular — long and thin).

The ideal technique will report data that correlate closely with a given performance attribute. Measured parameter is one aspect of this, but the basis for the distribution is also important. With a number-based particle size distribution, the number of particles in each size range is reported; with a volume-based distribution, it is the volume, or for particles of constant density, the mass of material. In the case of pharmaceutical products, a mass or volumeweighted distribution is often the most relevant descriptor of the content of active ingredient as a function of particle size.

  Volumebased methods may be more sensitive to the presence of larger particles and aid the detection of the end point for unit processes, such as milling. However, number-based methods may improve detection of small amounts of fine particles, which may impact properties such as powder flowability and dissolution rate. Therefore, the choice of technique depends on the application.  

More pragmatic issues include the speed of measurement and how easy it is to transfer the technique from the laboratory to the manufacturing floor. During the early stages of a development, longer analytical times may be acceptable, particularly if the results are information rich. For particle characterization, image analysis can be an excellent choice because it provides statistically relevant size and shape data as well as visual images, which in combination give detailed insight into the product. Measurement times are relatively long (approximately 15–20 min), but still much shorter than using manual microscopy. 

In contrast, PAT must offer much more rapid analysis because the aim is measurement of a dynamic process. Where extremely fast analysis is required, other techniques become more valuable. Laser diffraction, for instance, provides a complete volumebased distribution in milliseconds. This makes it extremely productive for particle characterization in the laboratory and equally valuable for the realtime monitoring of processes, including spray events. 

Whatever the analytical method, many of the issues regarding instrument choice are shared — ease of use, flexibility and reproducibility are all key. For laser diffraction, decades of development have streamlined analytical methods; the best instruments now offer fully automated operation driven by standard operating procedures (SOPs). The repeatability of such systems is exceptionally high. Advances in the core technology have also delivered improvements in fundamental performance, broadening measurement ranges. A single instrument can be used for many different samples, building greater value into the investment. 

How do I develop and validate a methodology for particle size analysis?

If a particle size specification is required, then so too is an analytical method that will accurately reflect whether or not a material meets the specification. Measurement should be repeatable, reproducible and robust.  

If a method is highly repeatable it means that running the 

 instrument produces very similar results. Repeatability is determined by instrument performance and the state of dispersion of the sample. Typically, the aim is primary particle size measurement, in which case it is necessary to disperse any agglomerates/aggregates prior to analysis. Research has shown that inadequate dispersion is the greatest source of measurement error for particles with a diameter of 20 μm or less (Figure 2).

Laser diffraction can be used for either wet or dry analysis. Wet sample dispersion involves the use of a suitable dispersant to separate the particles, followed by stabilization with admixtures or surfactants to prevent recombination of the particles during measurement. Vigorous stirring or ultrasound treatment promotes initial dispersion. With a dry system, the air pressure used to draw the sample into the measurement zone can be manipulated to control the degree of dispersion. 

A wellengineered instrument will produce repeatable measurements provided dispersing conditions are appropriate and well defined, and the concentration of particles in the measurement zone is suitable. However, reproducible measurement is a more exacting requirement. A highly reproducible procedure gives accurate and precise results for different samples, so sampling methodology is a contributory factor. The US and European pharmacopoeias specify a target reproducibility of 10% for the volume median diameter (Dv(50)) for particles >10 μm in diameter and 20% for those that are smaller.

  This figure takes into account all the errors associated with the entire analytical process. 

Many analysts underestimate errors that arise from sampling. Laboratory analysis involves measuring a small amount of material to determine the properties of a much larger bulk. Ensuring the sample is truly representative is essential. It is estimated that for systems containing particles >75 μm in diameter, sampling is the greatest source of error.

  As with dispersion, it is sensible during method development to investigate the impact of sampling technique on size measurement and to closely define a procedure.  

Comparison of different measurement techniques can provide valuable insight for method development; for example, image analysis provides size and shape data that verify effective dispersion. With dispersion, the aim is to apply sufficient energy to disperse the particles without breaking them up. Image analysis is a relatively straightforward way of determining whether or not this aim has been achieved. Comparing image analysis and laser diffraction data is also a good way of confirming that the optical properties being used for diffraction analysis are correct. 

Validation is the final step in the development of a particle size specification and an associated measurement method. FDA guidance states that, for particle sizing, "methods validation usually involves an evaluation of intermediate precision and robustness".

  Intermediate precision is related to both repeatability and reproducibility, and is tested by measuring the same sample, on a different day, using a different instrument. Robustness is assessed by evaluating the impact of small changes to the methodology on the results. 

FDA guidance also states that "assurance should be provided that the data generated are reproducible and control the product's quality".

  Effective method development, particularly when implemented through SOPs, will give reproducible analysis. For the particle specification to control product quality, it essential to have effective definition that considers the variability associated with measurement. Understanding the particle size range that gives acceptable performance boundaries for a product enables the specification to be set. The specification will be narrower than the true window of acceptability by an amount that depends on the variability of the analytical method (Figure 3). If the product meets this specification then it will deliver the required performance.  

is Product Manager Diffraction Systems at Malvern Instruments Ltd (UK).

1. ICH Topic Q6A — Specifications: Test Procedures and Acceptance Criteria for New Drug Substances and New Drug Products: Chemical Substances, December 2000. 

3. H. Merkus "Quality Assurance in Particle Size Measurement" from Improving Standards in Particle Size Distribution Measurement, Engineering Research Centre for Particle Science and Technology (Gainsville, FL, USA, February 1997). 

4. General Chapter <429> (2008), "Light Diffraction Measurements of Particle Size," United States Pharmacopeia. 

5. European Pharmacopoeia, Chapter 2.9.31, Laser Diffraction Measurement of Particle Size, Supplement 5.6 (2006), p 4429. 

6. FDA Draft Guidance — Analytical Procedures and Methods Validation, August 2000. 

How to Establish Manufacturing Specifications

Analytical

An expert on diffraction systems offers insight on particle size specifications and analysis.

Particle size analysis

The formation of a partnership, whether personal or professional, is risky business. Usually, a partnership is entered into by two parties seeking to benefit from the collaboration in some way, and it is only with time, dedication, common interests and hard work that a partnership is likely to succeed. When you're talking about a partnership between multibillion dollar firms, the stakes couldn't really get much higher. 

We have only just reached the end of Q1 for 2009 and already this year will probably go down in corporate and pharmaceutical history as the year of the mega mergers. Pfizer started in January by announcing its intentions to acquire Wyeth for $68.1 billion (€52.8 billion). This was followed some weeks later by Merck's surprise announcement that it was to acquire ScheringPlough for just over $40 billion (€31 billion. Then, Roche agreed to buy the remaining 44% of shares in US biotech giant Genentech. 

Imminent patent expirations, dry late-stage pipelines, market withdrawals, highprofile lawsuits and the economic downturn have all affected the majority of Big Pharma, but is this wave of acquisitions a sign of strategic brilliance or sheer desperation? And should all Big Pharmas be considering similar acquisitions simply to survive? 

On the face of it, each of these acquisitions makes good financial and strategic sense. Pfizer's purchase of Wyeth will create one of the most diversified companies in the global healthcare industry, with no drug expected to account for more than 10% of the combined entity's revenues by 2012. In ScheringPlough, Merck will not only double its pipeline of late-stage compounds, but could also save the company approximately $6 billion (€4.7 billion) annually after 2011.  

Genentech, conversely, has been spurning the advances of Roche since last summer, but it seems that every company has its price and Genentech settled on $95 (€73.6) per share, allowing Roche to purchase the remaining 44% of the firm. This widely anticipated deal will see Roche gain maximum exposure to the fastest growing section of the global pharmaceuticals market — biotechnology and cancer medicine. The world's largest biotechnology company, Genentech, has been majorityowned by Roche since 1990 and, during this time, Genentech has developed some blockbuster brands that have been the envy of the industry. It will be interesting to see how this outright acquisition will alter Genentech's culture of innovation. Time will tell. 

One thing that is certain, I don't think 2009 has seen the last of the pharma powerhouse mega mergers, which begs the question: who's next? 

The formation of a partnership, whether personal or professional, is risky business.

A question of strategy

Amorphous materials in pharmaceutical formulations yield complex and challenging problems concerning the performance, processing and storage of these products.

  The presence of amorphous materials can be wanted or unwanted, depending on the desired or undesired unique properties of the amorphous state. Additionally, during the processing of pharmaceutical solids (e.g., milling, spray drying, tablet compaction, wet granulation and lyophilization), various degrees of disorder in the form of crystal defects and/or amorphous regions may be generated. Even relatively low levels of amorphous material (< 10%) may have a detrimental impact on the stability, manufacturability and dissolution characteristics of the formulated drug product. Amorphous materials are inherently metastable and tend to revert to a more thermodynamically stable, crystalline form. As this instability has a potentially negative impact on storage and drug potency, it is important to quantify and control the amorphous content of pharmaceutically relevant materials. For these reasons, investigating the amount and stability of amorphous material is critical. 

Gravimetric vapour sorption methods have been widely used to study amorphous materials; for example, several different gravimetric vapour sorption techniques have been developed to quantify amorphous contents below 1%.

  Also, gravimetric vapour sorption has been used to investigate moisture-induced phase changes,

  as well as vapour-induced crystallization kinetics.

  This paper outlines several recent applications of gravimetric vapour sorption techniques to characterize amorphous materials.  

Gravimetric vapour sorption instruments are well-established methods for determining vapour sorption isotherms. These instruments measure the sample mass change as the vapour environment surrounding the sample is altered. An increase in mass is typically associated with vapour sorption, while a decrease is caused by vapour desorption. The vapour concentration around the sample is controlled by mixing saturated and dry carrier gas streams using electronic mass flow controllers.  

In this study, all experiments were performed using a DVS-Advantage (Surface Measurement Systems, UK). The system utilizes an ultramicrobalance with 0.1 μg sensitivity, allowing the measurement of small vapour uptakes. The instrument can actively measure and control the concentration of water and a wide range of organic vapours by using a proprietary optical sensor that is specifically tuned for a range of solvents. This technology allows the instrument to measure and control organic vapour concentrations in real time. 

There are numerous techniques available to quantify moderate-to-high levels of amorphous material in powders. Some of these techniques include differential scanning calorimetry (DSC), density, powder x-ray diffraction, thermal stimulated current spectroscopy, solution calorimetry, FT-Raman, near infrared and modulated DSC. However, there are few techniques that can quantify amorphous contents below 1%. These techniques include solid state nuclear magnetic resonance, microflow calorimetry and parallel beam powder x-ray diffraction. One of the most sensitive techniques is gravimetric vapour sorption.  

There are several methods in the literature using gravimetric vapour sorption techniques to quantify amorphous contents.

  Most of these methods are based on the amorphous phase sorbing more vapour than the crystalline phase. Amorphous materials typically have a higher surface area and vapour affinity than their crystalline counterparts. For vapour sorption methods, a calibration of known amorphous contents is usually necessary. The equilibrium vapour uptakes at a particular vapour concentration are then plotted against the known amorphous content, and the result is a straight line to which unknown amorphous contents can be compared. Figure 1 shows the octane vapour sorption isotherms (a) and calibration curve (b) for lactose samples with varying amorphous contents. The error margins in Figure 1b were based on the first standard deviation for repeat measurements (n = 3). Amorphous standards were created by making physical mixtures of 100% amorphous and 100% crystalline lactose. The resulting calibration curve and correlation coefficient (R

 0.9994) suggests that amorphous contents below 0.5% were achievable with an accuracy of ± 0.3%. Other vapour sorption approaches have also been able to quantify amorphous contents below 1% with similar accuracies.

As an amorphous material passes through the glass transition, it often transforms from a glassy, hard, brittle material to a less viscous, 'rubber' state.

  Additionally, there is a shift in the molecular mobility of amorphous compounds at the glass transition.

  Above the glass transition, the molecular mobility increases as evidenced by a decrease in viscosity and increasing flow. Temperature often forces the transformation at a characteristic temperature or temperature range (i.e., Tg). Water is a common plasticizer for a range of materials,

  thus the water content in amorphous foods, polymers and pharmaceutical materials can have a significant lowering effect on the glass transition temperature.  

If the humidity surrounding an amorphous material is linearly ramped from 0% relative humidity (RH) to a humidity above the water-vapour-induced glass transition, then a shift in vapour sorption characteristics will be evident. Below the glass transition, water sorption will typically be limited to surface adsorption. As the material passes through the glass transition, molecular mobility increases, allowing bulk water absorption. Therefore, the shift in sorption characteristics can be used as a measure of the glass transition.  

Above the glass transition, some amorphous materials will relax to their more stable, crystalline state. When the material undergoes an amorphous to crystalline transition, the water sorption capacity decreases drastically, resulting in an overall mass loss as excess water is desorbed during crystallization. This transition mass loss can be used to isolate the particular humidity at which crystallization occurs.  

Figure 2 displays a humidity ramping experiment for an amorphous lactose sample. Below the glass transition, water sorption will be dominated by surface adsorption. Above the glass transition, water sorption is dominated by bulk absorption. The transition point between these two regimes is taken as the vapour-induced glass transition. In Figure 2, this occurs at approximately 30% RH. This technique has been used previously on different materials with results similar to those found by DSC.

  As the humidity is increased and water vapour further plasticizes the sample, some low molecular weight materials may relax to the crystalline state. This is observed in Figure 2 by the sharp mass loss at approximately 60% RH. Again, this correlates well to other vapour studies on amorphous lactose.

  The physical changes inferred from the gravimetric data can be further supported by 

 microscopic images collected during the experiment. Figure 3 shows 100x images of amorphous lactose taken at 0% (A), 50% (B), 60% (C) and 90% RH (D). By 50% RH, the sample has changed to the glassy form because the humidity-induced glass transition has been reached. By 60% RH, the sample begins to crystallize and at 90% RH crystallization of amorphous lactose is evident by an increase in the opacity of the image. When combined with the change in mass results in Figure 2, the images shown in Figure 3 identify different humidity-induced phase changes.  

As previously stated, when an amorphous material undergoes a vapour-induced crystallization, there is often a sharp mass loss caused by the lower surface area, surface energy, and/or void spaces in the crystalline phase. This mass loss can be used to monitor the crystallization reaction. Figure 4 shows the moisture-induced crystallization behaviour for amorphous lactose exposed to 55% RH at 25 °C. The distinct mass loss feature observed at approximately 900 min is caused by crystallization of the amorphous lactose. Crystallization is complete by ~ 1200 min in Figure 4.  

If experiments similar to that shown in Figure 5 are performed at different temperature or humidity conditions, then the crystallization kinetic mechanism can be elucidated, and crystallization behaviour can be predicted with a wider range of vapour concentration and temperature conditions. Figure 5 shows the crystallization behaviour for an amorphous lactose sample at 51% RH and different temperatures. The scale has been normalized such that time equal to zero is set to the point at which crystallization begins. Moreover, the y-axis is normalized to fraction crystallized (1 = completely amorphous; 0 = completely crystalline). When processed with a mechanistic modelling package, the data in Figure 4 resulted in a mechanism with two competing, two-step reactions. Complete details on the crystallization mechanism can be found in the literature.

  Once the mechanism is determined, it is possible to predict the crystallization behaviour of the amorphous phase with a wide range of storage, processing and packaging conditions. This approach can also be used to study other solid-state transitions, such as dehydration, hydration, or solvation.

Characterization of amorphous materials can be greatly enhanced by using gravimetric vapour sorption techniques. Quantifying amorphous contents below 1% is possible using a range of gravimetric methods and phase change behaviour can also be characterized. This could be in the form of a vapour-induced glass transition or crystallization. Finally, crystallization kinetic modelling can be investigated. Gravimetric vapour sorption techniques can be used for a range of other pharmaceutical applications including: hygroscopicity, hydrate/solvate formation, solid-state transition kinetic modeling, BET surface areas, pore size distribution and diffusion measurements.  

is Head of Application Science for Surface Measurement Systems Ltd (PA, USA).

is Global Product Manager at Surface Measurement Systems Ltd (UK).

is Founder and CEO of Surface Measurement Systems Ltd (UK).

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3. G. Buckton and P. Darcy, Proc. 1st World Meeting APGI/APV, (Budapest, Hungary, 9–11 May 1995). 

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11. D. Lechuga-Ballesteros, A. Bakri and D.P. Miller, 

12. E.A. Schmitt, D. Law and G.G.Z. Zhang, 

14. L.H. Sperling, Introduction to Physical Polymer Science (Wiley & Sons, New York, NY, USA, 1986). 

15. Y.H. Roos, Phase Transitions in Foods (Academic Press, New York, NY, USA, 1995). 

18. D.J. Burnett, F. Thielmann and T.D. Sokoloski, 

Even small amounts of amorphous materials can have a significant effect on the drug product. Are gravimetric vapour sorption techniques an effective solution to characterize amorphous materials?

Characterizing amorphous materials with gravimetric vapour sorption techniques

The value of counterfeit drugs sold globally is estimated at more than $35 billion (€27.5 billion) and the US Center for Medicines in the Public Interest recently predicted that this figure could reach $75 billion (€58.9 billion) by 2010.

A report by the International Medicinal Products Anti-Counterfeiting Taskforce (IMPACT) — created by the World Health Organization (WHO) in 2006 to mobilize awareness and action in the fight against counterfeit drugs — stated that 10–30% of medicines for sale in much of the developing world are counterfeit. For medicines purchased via the internet from sites that conceal their physical location, IMPACT estimates that up to 50% may be counterfeit.

Counterfeit medicines can range from 'copycat' medicines that contain the declared active ingredient, to those with inactive preparations or random mixtures of toxic substances. All counterfeits are dangerous because their content and quality are unknown. WHO has documented thousands of cases worldwide of counterfeit medicines causing serious illnesses or death because of substandard, ineffective or toxic ingredients. The threat is particularly acute in developing countries where counterfeit treatments for lifethreatening conditions such as malaria and HIV are commonly available.  

In addition to their impact on global public health, counterfeit drugs pose a serious threat to the pharmaceutical industry — both to the sales of specific products and to the reputation of the industry as a whole. With the tremendous costs and challenges of bringing new therapies to market, pharmaceutical companies cannot afford the additional threat of having a product's efficacy or safety called into question because of ineffective or dangerous counterfeits. There has been much progress within the industry to test sophisticated technologies such as radiofrequency identification (RFID) to combat counterfeiting, but widespread implementation of any technological solution is still some time away. However, there are numerous steps a company can take immediately to help prevent counterfeiting, as well as improve the detection of counterfeit products that reach the market. These efforts are essential for companies to protect patients, maintain the value of their assets, and retain public trust of the industry and its products. 

Any effort to improve anticounterfeiting measures should be incorporated into a pharmaceutical company's overall risk management and pharmacovigilance efforts. While pharmacovigilance programmes have traditionally focused on quality assurance and postmarketing safety surveillance of legitimate products, counterfeit drugs pose a substantial safety threat and financial risk. The risk is especially elevated for higher value products that are in strong demand; for products that are widely distributed in markets where counterfeiting is common; and for lifestylerelated therapies that are not covered by health insurance. As part of a continuing risk assessment of its product portfolio, a pharmaceutical company needs to evaluate and understand the potential counterfeiting risk factors of every product, including its overall value to the company, the markets where it will be sold, price and expected sales volume. With that information, a company can implement an appropriate risk management strategy that focuses on the products most likely to be targeted. 

Many companies are changing the focus of their pharmacovigilance programmes to a more proactive approach that emphasizes risk assessment at the earliest stages of product development and integrates a companywide 'safety culture' into every part of the development process. In the same way, an effective anticounterfeiting programme requires an integrated, crossfunctional, and systematic approach that continuously evaluates the counterfeiting risk and incorporates prevention efforts into the early stages of a drug's development and formulation. Counterfeiting awareness should be established as a corporate priority, with highlevel support and clear goals for which managers are held accountable. This effort must be maintained throughout the product life cycle to ensure counterfeiting issues are kept to a minimum. 

The corporate commitment to the anticounterfeiting aspect of risk management and pharmacovigilance also requires investment in the appropriate people, governance, processes and technology needed to make the programmes succeed. Rather than being an overall cost or a liability, these investments enhance the value of the company by protecting one of its most important assets: its products. 

Efforts to control the threat of pharmaceutical counterfeiting can be divided into two major categories: prevention and detection. Prevention programmes encompass all activities that contribute to making a product harder to counterfeit, as well as efforts to maintain the security of the entire supply chain. Detection programmes incorporate activities leading to the uncovering of counterfeit products that reach the market, such as marketplace sampling and analysis of adverse events or other reports from physicians or patients. 

There are two key elements in the fight to prevent drug counterfeiting: using various technologies to make a product harder to copy and bring-to-market undetected; and implementing tighter security throughout the supply chain to ensure that drug supplies, packaging, manufacturing processes and other production components are only accessible to authorized personnel.  

Anticounterfeiting technologies include: 					

 This approach is already in common use and involves the inclusion of authentication markers on products and/or packaging that allow distributors, healthcare providers and others involved in the pharmaceutical supply chain to ascertain that the products they receive are genuine before they reach patients. These technologies include colour-shifting inks, fluorescent material, holograms, tamperevident packaging and chemical markets. They can be either overt or covert. Authentication technology provides an important layer of protection for drug authenticity, although some counterfeiters have learned to copy markers as well as products. 

 Another technology being used on a limited basis is the data matrix, also referred to as a 2D barcode. This technology enables additional information to be imbedded in a special barcode, such as an individual serial number for each pack or other data that aid in tracking and authentication. However, this technology requires an investment in a new type of barcode reader and standards for the coding system have not been agreed upon across the industry. 

 This technology, which is still in the pilot stage for pharmaceutical products, has initiated widespread discussion. It utilizes low-power radio frequencies that can be 'read' to reveal specific information about both the product's manufacture and its electronic 'pedigree'— a record of the chain of custody of the product from the point of manufacture to the point of dispensation. While there is general agreement that RFID is a promising technology for tracking and tracing drug authenticity, it also has significant drawbacks including cost, its unknown effect on sensitive products such as biologics, data privacy and the lack of industry data standards that would be needed to support such a technology. Even if these issues can be resolved, it will be several years before RFID is ready for widespread use in the pharmaceutical industry. 

Steps companies can take to help safeguard patients and the pharma supply chain.