solvias prospects - 02/2012

Next Generation Impactor – a novel preparation tech-nique for in vitro analysis of inhaled drugs

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Solvias Ligand Business – managing the ligand patent cliff

Quantitation of amorphous content

2 SoLvIaS ProSPectS — 2/2012

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Preface 4

Solvias Ligand Business – managing the ligand patent cliff 10

Seeing the unseen – 3-D particle characterization 13

Quantitation of amorphous content

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Next Generation Impactor – a novel preparation technique for in vitro analysis of inhaled drugs 23

establishing glass delamination from vials or ampoules by SeM / eDX 26

events

content

3SolviaS ProSPectS — 2/2012

Dr. Hansjörg WaltHer — ceo

Dear reader and valued customer

With autumn approaching, I am pleased to present you with the second issue of our customer magazine in 2012. It covers five really interesting topics and some valuable suggestions.

Many ligand patents are reaching expiration which is excellent news for most of you as ligand users. Matthias Lotz and his colleagues lead us right into the magazine with an article on the Solvias ligand business and how our customers can benefit from the impending situation. the challenging expiration of patent protection means at-tractive price reductions for our customers and you will read about Solvias’s efforts to remain a key player in the upcoming off-patent period in chiral metal complex catalysis.

Imagine being able to see the unseen? that’s exactly what the eyecon 3-D can do! In the segment on Process analytical technology (Pat), we are pleased to announce the collaboration between Solvias’s business unit Process analytical technology and Innopharma Labs to provide expanded Pat solutions. Peter Bauer speaks to Ian Jones, ceo of Innopharma Labs, about their innovative eyecontM 3-D particle characterizer.

Solid-state characterization continues to be a highly relevant field. amorphous fractions can significantly change the physico-chemical properties of active pharma-ceutical ingredients and drug products, and timo rager gives us insights into the ana-lytical methods used to determine the amorphous content of samples.

Pulmonary drug delivery by inhalation is becoming increasingly popular for local and systemic therapies. However, it is equally important to ascertain the efficacy of this form of delivery especially with respect to the drug quantities that actually reach their target. Maria Balaxi introduces Next Generation Impactors, a special sample prepara tion technique for in vitro analysis of inhaled drug products.

Having glass particles enter our system through drugs that are in vials or ampoules is definitely undesirable. It is, unfortunately, a fact that glass corrodes, meaning that parenteral drug products might be affected by the delamination of the inner glass surface and the generation of glass flakes. Bertold Gercken and claudia Müller describe Solvias’s methods for analyzing glass corrosion with scanning electron microscopy and x-ray analysis.

I hope you enjoy and benefit from reading this edition of Prospects, which provides an insight into Solvias’s ongoing efforts to add value to our customers’ business.

I thank you for your loyalty and your confidence in our services, and look forward to meeting you in october at the cPhI exhibition and conference in Madrid!

With my best wishes on behalf of Solvias aG

Dr. Hansjörg Walther


IntrODUCtIOnover the last few decades, chiral metal complex catalysis (cMcc) has become a mature and widely accepted technology within the chemical industry. With this development, the associated technology is now entering the phase of expiring patent protection for numerous proprietary commercial ligand families1. as a conse-quence, the prices for these ligands are expected to decrease significantly in the near future making them a really attractive alternative to ligands still under patent protec-tion. companies such as Solvias therefore need to adapt their business models accordingly. this article highlights the efforts Solvias has made to address this chal-lenge in order to remain a key player in the ever more competitive field of cMcc and ligand sales.

BaCKgrOUnDchiral molecules are of growing importance to the life science industries. today, most new chiral drugs including those under development consist of a single optically active isomer. In addition, other chiral products such as vitamins, fragrances and agrochemicals are produced in ton scale.the established portfolio for the preparation of chiral materials consists of various competing techniques that include: 1. racemic resolution (i.e. formation of diastereoisomeric salts, chiral chromatography), 2. chiral pool synthesis (i.e. using amino acid or sugar starting materials), 3. asymmetric catalysis using chiral chemo-, bio- or organocatalysts. Furthermore, whole cell fermentation (biotechnology) completes the technological picture for the preparation of chiral molecules 2.

cMcc is a mature technology. today, various asymmetric catalytic reactions (i.e. dihydroxylation, epoxidation, epoxide opening and isomerization) have been developed to production scale. However, the most important application of cMcc, both on laboratory and production scale remains enantioselective hydrogenation.

Solvias Ligand Business – managing the ligand patent cliffAuthors: Matthias Lotz*, Benoit Pugin, Dirk Spielvogel

Dr. MattHIas lOtz — Business Development Manager Matthias Lotz received his PhD with a thesis in Ligand Development in 2002. The same year, he joined Solvias AG as a Project Leader for Catalysis Process Development. After leading the Kilo Laboratory of Solvias AG from 2008 to 2011 he moved to his current position as Business Development Manager for Synthesis & Catalysis.


the classical catalysts for this reaction are composed of rh, ru and Ir complexes with chiral diphosphine and to an increasing extent also monodentate phosphor-based ligands. With the focus on asymmetric hydrogenation, maturity of the technology is evidenced by its routine inclusion during synthesis planning and process design aided by detailed information about scope and limitations (activity, selectivity, productivity, functional group tolerance) available in the public domain. Lastly and equally importantly, many catalysts and chiral ligands are commercially available and several companies offer high throughput screening of chiral catalysts / ligands and /or the development of scalable processes as a service.

the choice of an appropriate ligand, however, is narrowed from a practical point of view. Some common attributes of preferred ligands are (a) generally good catalytic performance, (b) simple preparation in a few synthetic steps, with high yields from inexpensive starting materials, (c) oxidative as well as hydrolytic stability, (d) an established broad substrate scope (e.g. modular ligands whose structure can be optimized with little synthetic effort for a given reaction). From an approximate number of 3,000 literature known chiral diphosphines fewer than 10 % are actually available at multigram scale. For multikilogram requirements the number of readily available systems rapidly drops even further.

a considerable number of relevant chiral ligands were patented in the 1980s and 1990s. With a general patent lifetime of 21 years (including priority year and if upheld by the proprietor until the end) protection on composition of matter of ligands originating from 1990 and earlier is no longer given. Ligands from 1990 and thereafter are now coming off patent on a regular basis. It is important to be aware that the patent priority year refers either to product or to application claims. also, there may be additional later patents with process claims for specific applications 1.

ligandshop.solvias.com – The new platform allows customers to order initial quantities of our chiral and CX-coupling ligands directly online.


eXPeCteD COnseQUenCes WHen lIganDs gO OFF Patentthe impending “freedom-to-use” on an increasing number of off-patent chiral ligands is expected to make the technology more attractive as ultimately classic market laws of supply and demand will determine the price. In addition, one may expect price pressure on systems remaining under patent protection to increase as well as off-patent systems may arguably be allowed to be less selective and efficient but in a bottom-line calculation preferred over their more selective and active proprietary counter-parts. Ultimately, the cMcc user will endeavor to maintain a balance of cost, quality and reliability on procured ligands, depending upon the exact use and the given free-dom to operate. For off-patent systems the preferred source may in some cases continue to be the originator for reasons of reliability etc. In other cases with the ligand price being of preemptive importance and application of the chiral transfor-mation significantly prior to the final step a generic supplier may be preferred.

The fact that numerous proprietary commercial ligand families will lose patent protection in the near future requires

appropriate actions from ligand originators such as Solvias.


Figure 1: Solvias Chiral Ligand Portfolio.

Josiphos

Mandyphos

POX

Trifer Chenphos

UBAPHOX JoSPOphos

Walphos

MeOBIPHEP

Taniaphos

Butiphane


sOlVIas’s ansWer tO tHe lIganD Patent ClIFFthis new situation demands appropriate actions from ligand originators such as Solvias in order to bring the product prices back down to a competitive level. three points of action were identified and successively optimized by Solvias to lower the ligand production costs and therefore allow for more competitive product pricing.

1. Improvement of production processeseven though initial gram demands of a certain ligand for screening and early-phase development purposes can be fulfilled without optimized production protocols the situation changes rapidly when kilogram quantities of material have to be produced. It requires a significant amount of development time and expertise to turn a gram-scale synthesis into a cost-effective and technically feasible production process.considering the importance of elaborated manufacturing processes, Solvias is constantly working on improving production procedures for its marketed ligands. this ongoing effort guarantees optimized manufacturing costs and allows us to offer all portfolio ligands at attractive prices. this holds true not just for soon to be off-patent ligands but enables also a competitive pricing model for ligands that are still under patent protection.

2. Supply chain optimizationthe manufacturing costs of a ligand are composed of labor and infrastructure and of the incorporated raw materials. therefore, a second key issue is ensuring a compe-tent sourcing of raw materials and building blocks for ligand manufacturing.Solvias has successfully established a reliable supply chain based upon a number of key suppliers. Next to pricing there are additional essential selection criteria for sup-pliers such as excellent quality, timing and reliability. a great advantage for Solvias is the fact that most of the marketed ligand families are assembled in a modular fashion which reduces the number of required raw materials considerably and to a large extent facilitates supply chain management.

3. Scale-up to technical scale (kg to >multihundred kg scale)as with any produced material costs are significantly dependant on the production scale. Small quantities (<1g) of ligands such as are used for screening purposes are available from catalogue suppliers for typically 400–1,000 USD/g. these high prices do not reflect the actual ligand cost but rather the cost for the maintenance of a broad ligand platform and for the handling and delivery of ligands. For larger ligand quantities as they are needed in production, the prices are considerably lower. We estimate that 1 kg of a patent-free ligand will be cheaper by a factor of typically 5–30 compared to the given numbers above, depending on the type of ligand and on whether this ligand has already been scaled. In addition, loss of patent protection for successful ligands that are applied in large quantities in industrial processes will lead to competition between ligand providers and manufacturers and we expect that this will eventually result in ligand prices of a few 1,000 USD/kg.


References

1. M. Lotz, D. Spielvogel, B. Pugin, “Chiral Metal Complex Catalysis: From an Innovator to a Generic Business”, Chimica Oggi, in press.

2. For more details please visit www.solvias.com and https://ligandshop.solvias.com

over the past 13 years, Solvias has successfully taken the synthesis of all marketed chiral ligand families (see Figure 1 and Reference 2) beyond kg scale and is currently moving the ligand production on to the next level.

Paying due consideration to commercial demand, Solvias is scaling the production of its most successful ligands (“selected ligands”) up to multihundred kg scale in collaboration with qualified contract manu facturing organizations (cMos). the improved economy of scale thanks to large production volumes allows Solvias to realize a pricing model for the supply of these ligands on technical scale which is also competitive after the mentioned patent expirations. In fact, the vast and ever growing experience at Solvias in the field of ligand synthesis and scale-up makes Solvias an interesting provider also for larger quantities of any patent-free chiral ligand.

the fact that Solvias is not just a pure ligand manufacturer but also an expert in catalysis is another very important business advantage. the ability to perform sophis-ticated in-house use tests for ligands provided on technical scale is a feature which is highly valued by the vast majority of our customers.

COnClUsIOnsIt is to be expected that expiration of patent protection will lead to significant price reductions for catalysts and ligands and as a consequence to improved acceptance of cMcc technology by the chemical industry. this new situation requires appropriate actions from ligand originators in order to stay key players in this field.Solvias is well prepared for this upcoming challenge since production processes are streamlined and all of its commercial ligands have already been scaled to kg quantities and some of the most successful ligands even to >100 kg.as a general conclusion, now is an excellent time for companies that produce chiral products to envisage making use of chiral metal complex catalysis since they can expect the cost for applications of this powerful technology to significantly decrease in the near future. •


Solvias spoke with Ian Jones, ceo of Innopharma Labs, the company that developed the eyecon 3-D, about the benefits of this innovative technology and about their partnership with Solvias.

Ian, in June 2012 Solvias AG and Innopharma Labs announced a collaboration in the field of Process Analytical Technology (PAT). What was the driver for this partnership with Solvias?With the rising importance of QbD (Quality by Design) and PAT in the pharmaceutical industry, Innopharma Labs was looking into a way to develop the market together with a partner with longtime PAT experience.

With the development of continuous manufacturing processes a profound knowl-edge of the raw materials and process intermediates is essential for the quality of the final product. Online particle characterization, together with other well-established PAT techniques like LIF (Light Induced Fluorescence) Sensors or NIR (Near Infrared) and Raman Spectroscopy, helps companies gain the right information to optimize their processes leading to higher yields, less scrap, better product quality and lower production costs.

Can you explain the technology of the Eyecon particle characterizer?The Eyecon TM 3-D particle characterizer technology is based on high-speed 3-D machine vision. It enables the capture of both size and shape characteristics for pharma ceutical particles between 50 and 3,000 microns. A continuous image sequence of the particles is captured using illumination pulses with a length of one microsecond for freezing the movement of particles which are moving with a speed up to several meters per second. The illumination is arranged according to the principle of photometric stereo for capturing the 3-D features of the particles in addition to a regular 2-D image. The particle size is estimated from the images using the 2-D and 3-D information, applying novel image analysis methods and direct geometrical measurement.

Seeing the unseen – 3-D particle characterization the real-time particle characterization capabilities of the eyecon tM 3-D particle characterizer were demonstrated to an interested audience at Solvias’s booth during this year’s acHeMa show in Frankfurt, Germany.With the integration of the 3-D particle characterizer into its process analytical offer, Solvias’s clients benefit from savings in formulation development and scale-up time plus increased process understanding and control during commercial manufacturing.


So what is the difference compared to other existing techniques?As the approach is based on direct measurement instead of indirect, such as laser diffraction, there is no need for material-based calibration. In addition, the method is noncontact and can be applied e.g. behind a view glass on a granulator, mill or spheronizer without significant physical modification of the process equipment.

So the Eyecon TM 3-D particle characterizer can be mounted on process equipment for online particle characterizations. Can you name a few examples of processes within pharma manufacturing where the customer gains process or product knowledge through online particle characterization?All the processes where you change the granule properties, like granulation, milling or spheronization. Let’s take fluid bed granulation, a very popular production process within tablet production as an example. During fluid bed granulation, powders are fluidized and a binder solution or suspension is sprayed onto the fluidized particles, creating liquid bridges between the particles which form agglomerates from the powder. When the desired size of the agglomerates is achieved, spraying is stopped by which time the majority of liquid is evaporated.

The majority of challenges during fluid bed granulation result in either undergranulation or overgranulation. Undergranulation means that the particles have not bound sufficiently together and their morphology and mean particle size has not reached the optimal range for further processing. This phenomenon will result in reduced yields and ultimately ineffective compression activities.

Overgranulation may result from inaccurate identification of the granulation end point leading to excessive binding of particles. Overgranulation means that the particles have converted into oversized granules and may have returned to smaller particles again through the generation of fines following attrition between over-sized particles. In addition, these oversized granulate masses may not effec-tively blend with the lubricant within the formulation as insufficient surface areas are presented for interaction with the lubricant. This may cause challenges during compression.

Adds another dimension to Solvias’s already existing wide range of QbD/PAT applications: EyeconTM's 3-D particle characterizer.


If we look at continuous granulation techniques, like dry granulation and twin-screw wet granulation, they are always followed by a milling step to reduce and standardize the particle size and shape. Why is it so important to control the particle size and shape?As the granule properties play a pivotal role in the downstream production steps and affect the final product properties a real-time monitoring of the milling process is im-portant. In most applications, the compacted product must be subsequently milled to a uniform particle size distribution. The mill operates by feeding material uniformly into a chamber in which a rotating blade assembly reduces the particles of the material by cutting or impacting them. The material discharges through a screen which regulates final particle size at the outlet of the milling chamber. The blade and screen act simultaneously to determine final product sizing. This assures a consistent presentation of material for subsequent manufacturing steps like blending with further excipients and ultimately compression.

The majority of challenges associated with milling result in variability of particle size and the generation of fine particles. This variability in particle size may lead to downstream segregation of materials or ineffective secondary blending and com-pression processes.

So how can the Eyecon TM 3-D particle characterizer help customers understand these processes?In using the Eyecon TM 3-D particle characterizer, customers will understand the physical properties of granulated, milled or spheronized materials which significantly help them during formulation development, scale-up and routine commercial manu-facture. Our customers are realizing significant benefits through using our characterizer – one customer estimates that they have saved just under €1M over 12 months through yield improvements and a reduced number of process interventions and investigations.

I am really delighted that we can extend our existing range of PAT techniques with the Eyecon TM 3-D particle characterizer. We are confident that the Eyecon TM 3-D particle characterizer will provide our customers with an additional tool to increase product quality and optimize their processes to stay competitive in the market.

Thank you for your time and for sharing your thoughts on the Eyecon technology.


crystalline substances are often less than 100 % crystalline. the crystallization process itself may already produce a fraction of amorphous solid, e.g. because insufficient time is given to the molecules to arrange themselves in perfect order. Furthermore, subsequent processing steps such as desolvation of a solvate or milling can intro-duce disorder by destroying the original crystal structure.

amorphous impurities are usually undesired because they have very different physicochemical properties than when in a crystalline state. Possible differences lie in faster chemical degradation, increased hygroscopicity, a different dissolution profile and an inherent tendency to recrystallize over time because of its metastable, high-energy character. as a consequence, the solid-state form of the drug substance would be no longer well defined, its storage stability will be reduced, and the aggre-gation behavior of a powder may be changed.

a control of the amorphous content of a drug product requires that appropriate analytical techniques are available for quantitation. Indeed, a number of techniques are sensitive to the particular properties of amorphous materials and can be used for this purpose:

Quantitation of amorphous contentamorphous fractions can significantly change the physicochemical properties of active pharmaceutical ingredients and drug products. Solvias offers a variety of analytical methods to determine the amorphous content of your samples.

Dr. tIMO rager — Project Leader Timo Rager received his PhD with a topic in polymer chemistry in 1997. Following several postdoctoral stays, he joined Solvias in 2005 as a project leader for solid-state development.

The selection of the preferred method will mainly depend on the physicochemical properties of the substance under investigation

and the required accuracy.


one established method for the determination of amorphous content is based on x-ray diffraction. the periodic variation of the electron density of a crystalline solid leads to a selective diffraction of the x-ray radiation at specific angles. contrary to this, the random distribution of electron density in an amorphous material does not bring about such angle selectivity and a diffuse halo of diffracted radiation is ob-served instead (Figure 1). after appropriate calibration, the ratio of the sharp reflections to the diffuse background signal can be used to quantify the amorphous content. the application of this method is very general and nondestructive. on the other hand, the sensitivity of the method is rather low because of the low intensity of the diffuse background signal from the amorphous fraction. Its accuracy may suffer from variations of the signal from the crystalline part, which serves as an internal reference. Such variations may be caused by differences in the size or orientation of crystals for example.

another common method relies on differential scanning calorimetry (DSc). It is characteristic for amorphous substances to exhibit a glass transition step between the glassy and the rubbery (and finally liquid) state. this transition is characterized by an increase in heat capacity of the material as a result of the mobilization of rotational and translational degrees of freedom. above the glass transition temperature, the amorphous material will possibly recrystallize and then melt at a still higher tempera-ture. this recrystallization is kinetically driven and may not take place at all. an evaluation of the amorphous content in mixtures can be based either on the height of the glass transition step or the size of the recrystallization and melting peaks, whereas the contribution of the crystalline fraction has to be taken into account as well. the method is obviously limited to substances that are chemically stable in the

Figure 1: Typical x-ray diffractograms of an amorphous sample (orange) and a crystalline sample (blue). A structureless halo is observed for the amorphous sample as opposed to a well-resolved diffraction pattern for the crystalline one.

Intensity

angle 2.0


relevant temperature range. the sensitivity depends on the ability to determine the exact heat capacity change or the transition enthalpies. In particular, the glass transition step is a small signal (typically on the order of 0.5 J/(g K)) and may be spread out over a broad temperature range, which causes a significant uncertainty for quantitation.

a method based on dynamic vapor sorption (DvS) takes advantage of the increased hygroscopicity that is frequently observed for amorphous samples as compared to crystalline ones (Figure 2). the amorphous content can be quantified when the difference in water uptake between crystalline and amorphous material is known for a given relative humidity and scanning rate. the sensitivity of the method will change from substance to substance depending on its hygroscopicity. Furthermore, the result may be affected by particle size and by the rate of recrystallization of the humidified amorphous material, which is typically connected to a release of ab-sorbed water.

the same property that underlies the application of DvS, i.e. the absorption of water or solvent vapor, is also utilized for the determination of amorphous content by microcalorimetry(μCal).However, it isnottheweightchangethat isdetected,butrather the release of latent heat from solvent-induced recrystallization that is observed in this case. the success of the method depends on the identification of suitable crystallization conditions, which can be very challenging. the recrystalliza-tion process should be neither too fast (which would cause an overlap with the initial transient event) nor too slow (which would impede the detection of the re-crystallization end point). Finding a suitable solvent to meet these criteria can be tedious or even impossible. on the other hand, the method is very sensitive when successfully implemented.

Figure 2: Comparison of the water vapor sorption isotherms of an amorphous sample (orange) and a crystalline sample (blue) of the same substance. With increasing relative humidity, the weight of the amorphous sample increases due to water uptake before it suddenly drops as a result of recrystallization.

Water uptake recrystallization with release of water

relative weight

relative humidity


Last but not least, the enthalpy difference between amorphous and crystalline material is also detectable by solution calorimetry. Instead of converting crystalline material into amorphous (DSc) or amorphous material into crystalline (microcalorimetry), both solid-state forms are transformed into a solution as a common reference state (Figure 3). the dissolution will release different amounts of latent heat depending on the energetic state of the starting material. this heat can be detected as a temperature change of the solution in the 10–100 mK range. the sensitivity of the method is high but depends to some extent on the selected solvent. Finding a suitable solvent for fast dissolution can be a problem. In addition, similar to DvS and microcalorimetry, the method is not specific for amorphous impurities. almost any chemical or physical impurity can provoke thermal effects, which may be misinterpreted as higher or lower amorphous contents. It is therefore advisable to combine the method with complimentary techniques (e.g. HPLc and XrPD).

as briefly outlined above, each of the methods for the quantitation of amorphous content has its strengths and drawbacks. It is therefore helpful to have several methods to choose from. the selection of the preferred method will mainly depend on the physicochemical properties of the substance under investigation and the required accuracy. •

Figure 3: Quantitation of the amorphous content with thermal methods.

enthalpy

amorphous crystalline solution

DsC µ

Cal

sol C

al


MetHOD aDVantages lIMItatIOns tyPICal lIMIt OF DeteCtIOn (lOD)

x-ray powder diffraction (XrPD)

• nondestructive• fast• specific• universally applicable

• requires internal referencing; no absolute values

• low sensitivity• adverse effect on

accuracy by crystal size and orientation possible

10 %

Differential scanning calorimetry (DSc)

• fast• specific• only small amount of

substance required• uncritical kinetics

• limited to temperature-stable substances

• low sensitivity

10 %

Dynamic vapor sorption (DvS)

• sensitivity can be high• simple sample

preparation• relatively simple

method setup

• depends on hygro-scopicity of the amorphous material and its tendency to recrystallize

<1%

Microcalorimetry (μCal)

• high sensitivity• simple sample

preparation

• requires suitable crystallization kinetics

<1%

Solution calorimetry (Solcal)

• high sensitivity • requires rapid dissolution

<1%

coMParISoN oF cHaracterIStIc ProPertIeS oF SoMe MetHoDS For aMorPHoUS coNteNt DeterMINatIoN


Delivery of drugs to the human lung by inhalation is becoming increasingly popular for local and systemic therapies. Locally, inhaled drugs have been extensively used for a number of years throughout the world for the maintenance treatment of asthma and chronic obstructive pulmonary disease (coPD) mostly using bronchodilators and corticosteroids. Pulmonary drug delivery is used also as an option for the local delivery of antibiotics or antiviral agents to the lung. as lung epithelium can serve as an injection-free portal of drug entry to the blood stream, inhaled products can be used for systemic treatment as well1, offering a range of advantages and attracting increasing interest from pharmaceutical companies. the strength of the pulmonary route lies in its efficacy in targeting drugs directly to the airway surfaces resulting in a rapid action onset, leading to lower dosage requirements when compared to the oral route with relatively lower incidence of side effects. Not least, inhaled drugs do not undergo hepatic first pass metabolism which can partially inactivate them. For drug delivery to the lung a number of different administration technologies have been developed. Metered Dose Inhalers (MDIs) and Dry Powder Inhalers (DPIs) are the most popular devices for patients because of their convenience in use, their portability, robustness and potentially multidose.

Dr. MarIa BalaXI — Project Leader Maria Balaxi completed her PhD in Pharmaceutical Technology in 2009. After a postdoctoral internship at Novartis Pharma in the field of Pharmaceutical and Analytical Development of Dry Powder Inhalers she joined Solvias in 2012 as a Project Leader for NGI Analytics.

Next Generation Impactor – a novel preparation technique for in vitro analysis of inhaled drugsDrug inhalation is growing in popularity because of its efficacy in targeting drugs directly to the airway surfaces for a quicker action onset and thereby reduced dosage requirements. In vitro testing of medical inhalers is highly demanding both in terms of time and personnel expertise and requires close attention to detail to ensure accurate and reproducible measurements.Author: Dr. Maria Balaxi


In vitro testing of inhaled products seeks in general to ensure that they contain and emit the correct amount of the appropriate drug or drugs upon inhalation. In particular, in vitro tests aim to determine the following:

• ensure that a reproducible mass of drug exits the inhaler’s mouthpiece each time the inhaler is used by the patient.

• assess the fraction of the drug that is in a size range small enough to deeply penetrate the lung.

• establish the presence and possibility of subsequent inhalation of toxic impurities and microbiological contamination during storage or use.

conditions applied during an in vitro test (i.e. temperature and relative humidity, air flow, time of inspiration etc.) are selected to be as close as possible to those occur-ring when the patient uses the device in reality according to the manufacturer’s instructions. Matching these criteria, however, is difficult because several unstable and unpredictable variables can impact the performance of an inhaled product, the most important being the inhalation characteristics of the patient. It has also been proved that variables such as humidity, temperature, prior handling of the device or delay between shaking and actuating can influence performance during in vitro testing. all these variables cannot be evaluated during routine in vitro tests due to the complexity of the test procedure, but the variables are standardized for each in vitro test in order to acquire reproducible results, sacrificing some “realism” along the way. For this reason, in vitro testing of inhaled drug products should be considered as a means of collecting comparative data rather than to simulate and predict in vivo performance2.

among the FDa’s recommended in vitro tests for inhaled drug products the most critical ones are considered to be DD (Delivered or emitted Dose) and aPSD (aerody-namic Particle Size Distribution). Indeed, the number of aerosol particles deposited in the lungs and their exact deposition sites depends on their aerodynamic particle size, the mode of inhalation and the morphology of the respiratory system3. For an aerosol particle, its aerodynamic diameter is the most appropriate measure because it relates to its aerodynamic behavior, in other words how the particle behaves in an air stream, and is the most relevant to the lung delivery and final therapeutic effect4.


the instruments of choice when measuring the aerodynamic particle size of medical inhalers for both US and european Pharmacopoeias are cascade Impactors (cI). cascade impaction functions as a size classification of incoming aerosol particles in an air stream, moving at constant velocity. It can be considered as a particle “sieving” analogue which separates particles while they are in motion based on their aerody-namic diameter – which is a function of properties such as density and physical dimensions – and not simply based on their geometric size.

Next Generation Impactors (NGIs) are the most recently developed cI instruments used widely for evaluation of inhaled products (Figure 1, Figure 2). an NGI consists of a series of stages each comprising one or more nozzles, the sizes of which decrease progressively through the impactor. Under the nozzles at each stage there are collec-tion cups for the recovery of drug particles. once the inhaler is discharged the aerosol cloud is drawn into the impactor by means of a vacuum pump connected to the outlet of the impactor by a vacuum tube. the aerosol is introduced into the main body of the NGI through the induction port, often known as USP throat. Its purpose is to detain from the impactor the fraction of particles that are considered too large to enter the lungs. Between the induction port and the main impactor a preseparator may interfere and its purpose is also to exclude the very large particles, usually origi-nating from the carrier (i.e. lactose, manitol etc.). this is why a preseparator is mainly adapted to the impactor when carrier-based inhaled products such as Dry Powder Inhalers (DPIs) are analyzed whereas it is not necessary when liquid propellant-based inhalers that do not contain any solid carrier particles such as Metered Dose Inhalers (MDIs) are tested.

Figure 1: Next Generation Impactor (NGI) known as USP Apparatus 5 (without preseparator) or USP Apparatus 6

(with adapted preseparator).

as the name implies, the basic principle of size separation in a cascade impactor as well as in the lung is the inertial impaction (Figure 3). Whether or not a particle impacts on a stage depends on its aerodynamic size. as the aerosol-particles-filled air stream is drawn through the NGI stages, those particles with sufficient inertia won’t follow the streamlines to the next stage and they will impact on the relevant stage collec-tion plate while smaller particles with insufficient inertia will remain entrained in the air flow and pass to the next stage where the same process is repeated. as the nozzles

Induction port ( USP Throat)

Preseparator NGI Body

Impactor outlet/Connection to vacuum pump


Figure 2: The Next Generation Impactor (NGI) with its stage nozzles and collection cups.

Figure 3: Particle sepera tion in the impactor’s stages due to inertial impaction.

of each stage get smaller, the air velocity through the nozzles increases and smaller particles are collected. at the end of the analysis the particle mass deposited at each stage is recovered from the collection cups with the appropriate solvent and analyzed usually using high-performance liquid chromatography (HPLc). each stage corresponds to a specific aerodynamic size range and the particle size distribution is yielded in terms of mass collected per size range. although NGI separates particles based on their aerodynamic behavior exactly as they are being separated in the lung and with the same principles, a cascade impactor should not be considered as an in vitro simulator of the lung since it operates at a constant flow rate in contrast to the continuously varying flow rate over a breathing cycle5.

It is generally agreed that NGI tests are labor intensive and require well-trained labo-ratory personnel as well as good experimental design, but at the present time cascade impactors represent the only method of determining aerosol Particle Size Distribution (aPSD) of inhaled products. During an NGI test there are numerous vari-ables to consider making the sample preparation a special process.

Stage nozzles Collection cup

Stream linesJet exit

Trajectory of particles too small to impact

Trajectory of impacted particles

Impaction plate


Whereas for example the choice of the appropriate solvent and technique for the drug recovery is pharmaceutical product dependent, there are technique-depen-dent variables which should be taken into account and be established by the operator in every NGI test. Some of them are briefly discussed below:

collection surface preparation: coating of the collection cups with a special agent is necessary prior to testing, to prevent bounce and reentrainment of impacting particles.

Flow rate control: a very important variable that influences the size-discriminating ability of the NGI by influencing particle velocity through the nozzles. It is important to be constant during the testing time. the NGI is designed to be able to operate at flow rates between 15–100 L/min.

actuation time: once the flow rate is established it is necessary to control the volume of the air drawn through the inhaler in the impactor during testing. according to Pharmacopoeias the total drawn volume when DPIs are tested should be 4 liters and 2 liters according to FDa guidelines. this is in order to simulate as closely as possible the in vivo inspiration volume of the patient and is achieved by setting the duration of the inhaler’s actuation for the specific flow rate. For example, for 60 L/min flow rate the actuation time should be 4 seconds in order to achieve a 4 L inspiration volume. For MDIs the actuating duration is not specified.

temperature and relative humidity: Processing conditions also have to be established especially for DPIs. Powder formulations can be subject to capillary forces due to the presence of moisture or electrostatic forces. Low moisture may help to reduce the capillary forces but can also raise the surface electrostatic forces. Dry fine particles are very sensitive to electrostatic charging which may occur via triboelectrification during the contact between particles as well as between particles and inhaler device surfaces. on the other hand, high relative humidity conditions increase the risk of particle sticking and agglomeration due to moisture presence which can have an impact on a product’s performance. therefore temperature and humidity are critical and should be controlled in order to avoid any impact on the inhaler’s performance.

Because of the complex coexistence of many factors that need to be considered and the nature of the equipment used, i.e. using NGI or another impactor, in vitro testing of medical inhalers is highly demanding both in terms of time needed and personnel expertise and requires close attention to detail to ensure accurate and reproducible measurements. •

References

1. Byron, P.R., and Patton, J.S., “Drug delivery via the respiratory tract”, Journal of Aerosol medicine, 1994, 7, 49–752. Clark, A.R., and Borgström, L., “In vitro testing of pharmaceutical aerosols and predicting lung deposition from in

vitro measurements”, in Drug Delivery to the Lung, Bisgaard, H., O’Callaghan, C., and Smaldone, G.C. (eds), 2002, New York: Marcel Dekker, 105–142

3. Dolovich, M.A., “Influence of inspiratory flow rate, particle size, and airway caliber on aerosolized drug delivery to the lung”, Respiratory Care, 2000, 45, (6), 597–608

4. Telko, M.A., and Hickey, A.J., “Dry Powder inhaler Formulation”, Respiratory Care, 2005, 50, (9), 1209–12275. Mitchell, J.P., and Mark, W.N., “Cascade Impactors for the Size Characterization of Aerosols from Medical Inhalers:

Their Uses and Limitations”, Journal of Aerosol Medicine, 2003, 16, (4), 341–377


Glass delamination in vials and ampoules, also known as glass corrosion, can seriously endanger patients’ safety as parenteral drug products enter the bloodstream directly. Special care must therefore be taken to establish glass delamination if and when it occurs. visible particulates in parenteral formulations are not at all acceptable, and, there is a regulatory expectation that packaging (in this case, vials or ampoules) in no way diminish the quality of the product.

Pitting of the glass surface could be related to, or even be the initial stage of glass delamination. as pits grow larger and/or appear in higher numbers, they usually begin to form a flat surface resulting in the flaky appearance that finally delaminates. Glass vials shed under certain conditions thin, flexible fragments called “glass lamellae” or “glass flakes”. these glass flakes from the inner surface of the container are difficult to detect in pharmaceutical liquids by visual inspection. Depending on the product, the process of glass corrosion can take weeks or months.

Some parameters that affect the durability of glass for pharmaceutical purpose are: • Sterilization by autoclaving• thermal exposure during manufacturing• chemical composition of the glass • treatment of the surface • chemical and physical properties of the pharmaceutical liquid

establishing glass delamination from vials or ampoules by seM / eDXDelamination of glass, or the generation of glass flakes from vials or ampoules containing parenteral drug products, continues to be a persistent problem for the pharmaceutical industry. Scanning electron microscopy (SeM) at Solvias is the method of choice for examining glass vials and ampoules for pharmaceutical purposes.

Dr. ClaUDIa Müller — Scientist Microscopy Claudia Müller received her PhD with a thesis in EXAFS (extended x-ray absorption fine structure) on photosystem II in 2006. In 2007, she joined Solvias AG as a scientist for physical chemistry analysis. In 2009 she moved to her current position as a scientist for light and electron microscopy.

Dr. BertOlD gerCKen — Senior Lab Manager Bertold Gercken received his PhD with a thesis in analytical chemistry in 1989. After a year of postdoctoral research at UMASS, he joined Ciba-Geigy AG in the year 1991 as a lab manager of the ICP-MS lab. After leading the team for ultra trace analysis of Solvias AG from 2001 to 2011 he moved to his current position as head of the team for elemental analysis & microscopy.


Dissolution of the glass surface and the formation of subvisible glass particulates occur well in advance of the appearance of visible glass flakes, and should be consid-ered as leading indicators for the loss of glass chemical durability. the formation of glass flakes on the vial interior can be observed by scanning electron microscopy (SeM; see images in Figure 1 to 4). It takes a significantly longer time until visible glass particles can be detected in the pharmaceutical liquid.

SeM at Solvias is the method of choice for examining glass vials and ampoules for pharmaceutical purposes. SeM is used to characterize the morphology of the inner surface of selected glass vials before and after contact with the pharmaceutical solution. this characterization of the vial interior provides the most direct evidence that the glass and the liquid it contains are interacting. However, SeM is not suitable for detecting glass flakes directly in pharmaceutical liquids.

Figure 1: Glass surface with pitting. Figure 2: Glass surface with bubbles.

Figure 4: Glass surface at an advanced stage of delamination. Figure 3: Onset of delamination.

30 μm20 μm

10 μm 40 μm


early stages of delamination can be observed on SeM images. Figure 1 shows a glass surface with pits with a size of less than 5 µm. Pitting could be the initial stage of glass delamination. Figure 2 shows a glass surface with a bubble-like structure. the size of the bubbles is also less than 5 µm. Such bubbles could be considered as evidence of interaction between the glass surface and the vial content. Formation of bubbles indicates the loss of chemical durability of the glass. In Figure 3, we can see the onset of delamination. the entire structure looks like a circle with a diameter of approximately 30 µm. In the center, a glass flake just prior to delamination can be observed. this structure could have developed from a bubble that has collapsed. Figure 4 shows glass delamination at an advanced stage. In the center of the image glass flakes are already visible. at that stage of glass corrosion glass flakes are also detectable in the solution.

Glass flakes can be separated from the vial content by filtration under clean room conditions. For SeM / eDX (energy dispersive x-ray) analysis the filter is coated with graphite to prevent charging. Flakes are analyzed for their elemental composition by eDX. the eDX spectrum in Figure 5 shows, besides the carbon peak (from coating), signals of such elements as Si, al and Na, all well-known elemental components of glass. •

Figure 5: EDX spectrum of a glass flake.


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