spectroscopic techniques

Raman Microscopic Applications in the BiopharmaceuticalIndustry: In Situ Identification of Foreign Particulates InsideGlass Containers with Aqueous Formulated Solutions

XIAOLIN CAO,* ZAI-QING WEN, AYLIN VANCE, and GIANPIERO TORRACADepartment of Formulation & Analytical Resources, Amgen Inc., Thousand Oaks, California 91320

Particle identification is an important analytical procedure for quality

control and assurance in the biopharmaceutical industry. Rapid and

reliable identification of micro-particles helps in evaluating the nature of

particle contamination and its consequences on the product quality

regulated by internal and external standards. Raman microscopy is one of

the microspectroscopic techniques that can be used to identify micro-

particles with the advantage of in situ detection. In this paper we

demonstrate that a visible laser Raman microscope was particularly

useful to identify micro-particles that were inside glass containers such as

glass syringes, vials, and test tubes, which are commonly used as

containers for aqueous formulated drugs. The examples include the

identifications of a droplet-like particle inside a pre-filled glass syringe, a

fibrous particle inside a glass test tube, and a white particle inside a glass

vial; all of these examples usually demand challenging or time-consuming

sample manipulation for other techniques. The Raman microscopic

technique was shown to be able to solve these challenging micro-particle

identifications due to its ability to carry out detection in situ. Particularly

in the example of micro-droplet identification, the Raman microscopic

technique was the only choice for a fast and successful particle detection.

For all three identifications, Raman in situ detection has significantly

accelerated particle analysis and avoided potential sample secondary

contamination or losses owing to none or minimal sample manipulation.

Index Headings: Particle identification; Raman microscopy; In situ

detection; Silicone oil; Cellulose; Polypropylene.

INTRODUCTION

Rapid and reliable identification of micro-particles is of greatinterest in the biopharmaceutical industry.1 These micro-particles may generally fall into two categories according totheir origins: intrinsic and extrinsic. Intrinsic visible particlesare formed from drug substance. The sizes and concentrationsof these particles need to be controlled to meet regulationstandards. Extrinsic or foreign particles originate from non-drugsubstances during biopharmaceutical manufacturing process-ing. They could appear together with the product, resulting innon-conformance or deviations from quality standards. Theseforeign visible particles may be present in products at any phaseof the drug lifecycle, from early process development throughcommercial production to final administration to the patient.

Once these foreign visible particles are observed, they must beidentified and the root-cause determined in order to facilitatepost-non-conformance management and prevent future reoc-currence. To identify various particles with different origins, weuse a number of microspectroscopic techniques such as Fouriertransform infrared (FT-IR) microscopy, scanning electronmicroscopy (SEM), and energy dispersive X-ray spectroscopy(EDS) as well as Raman microscopy.

Raman microscopy has been used in many applications,2

including material science, earth and environmental sciences,and medical and biological studies, as well as forensicanalysis.3,4 In biopharmaceutical applications,5–7 it has beenemployed in the determination of the polymorphs of smallmolecule drugs,8 the characterization of the structures of proteintherapeutics9–12 in formulation development, the detection ofcounterfeit drugs,13,14 and the identification of foreign parti-cles.15,16 Complementary to infrared microscopy, Ramanmicroscopy has several unique advantages7 that facilitate thedetection of micro-particles. Firstly, sample preparation isminimized or not required during Raman measurement;secondly, the most common Raman backscattering geometryfacilitates in situ detection of particles. Particularly, Ramanmicroscopy with visible laser excitation is capable of in situdetection of the particles that are inside glass containers (such assyringes, test tubes, or vials) due to the transparency of glass tovisible light. Additionally, in situ particle detection is even moreconvenient when using a Raman microscope with a confocaloptical setup where better axial resolution is achievable and out-of-focus interferences are minimized.

Recently we have employed Raman microscopy for proteinstructural studies11,12 and for some challenging foreign particleidentifications. In this paper, we report several uniqueapplications involving in situ identification of micro particlesinside glass containers with aqueous formulation by Ramanmicroscopy. These applications included the identification of adroplet-like particle inside a pre-filled glass syringe, a fibrousparticle inside a glass test tube, and a white particle inside aglass vial. All these glass containers were filled withformulated solutions and the micro-particles resided insidetheir respective containers. To the best of our knowledge,similar Raman in situ identifications of the micro-particlesinside glass containers filled with aqueous formulation have notbeen reported in the biopharmaceutical industry.

Received 5 February 2009; accepted 7 April 2009.* Author to whom correspondence should be sent. E-mail: xiaolinc@amgen.com.

830 Volume 63, Number 7, 2009 APPLIED SPECTROSCOPY0003-7028/09/6307-0830$2.00/0

� 2009 Society for Applied Spectroscopy

EXPERIMENTAL

For all three case studies reported here, the particles insidetheir respective glass containers were firstly examined with anoptical steromicroscope (Zeiss Stemi 2000) and their micro-graphs were taken using an AxioCam MRC digital cameraattached to the stereomicroscope. The droplet-like particle

inside the pre-filled syringe was 0.73 mm in diameter, thefibrous particle inside the test tube was approximately 0.1 mmlong, and the white particle inside the glass vial has a length ofapproximately 0.3 mm. The digital photos of these glassdevices with the particles inside were taken with a Nikon D200digital SLR camera. Once the particles were located andimaged, the entire device was placed on the sample stage of theRaman microscope for particle spectral measurements. To dothis the glass device was carefully adjusted so that the particlewas accurately positioned under the selected objective,monitored using the incorporated live imaging system.

All the Raman measurements were carried out with aSenterrat Raman microscope from Bruker Optics. It is thecombination of a dual laser Raman spectrometer and a confocalmicroscope module with three objectives attached to thenosepiece. The 532 nm green laser with a maximum power of20 mW was used for all measurements. The particle wasmonitored and precisely positioned under the laser beam withthe aid of a live-video accessory that utilizes visible illuminationfor viewing by binoculars or digital display. Then the particleRaman signal was checked, optimized, and collected with athermoelectrically cooled charge-coupled device (CCD) detec-tor and recorded for typically 20 scans. For the droplet particle,its spectra were measured at a resolution of approximately 4cm�1 from 70 to 3700 cm�1, and for the other two the resolutionwas 12 cm�1 from 70 to over 4000 cm�1.

The confocal detection for this Raman microscope wasachieved by using a hybrid aperture containing an array ofpinholes and slits serving as the entrance aperture of thespectrograph. The combination of a narrow slit and 203objective (for the droplet-like particle and white particle) and503 objective (for the fibrous particle) were used for thespectral collection. This combination provided sufficient depth(,0.02 mm) and lateral resolutions (,5 lm) for particledifferentiation as well as the sensitivity for detection. Thefrequency accuracy and the stability of the spectral measure-ments were automatically maintained with the internal spectralstandards and calibration program installed in the spectrometer.

FIG. 1. The droplet-like particle (0.73 mm diameter) inside a pre-filled glasssyringe (8 mm diameter); images taken by (a) the Nikon digital camera, and (b)the Zeiss Stemi 2000 stereomicroscope.

FIG. 2. The Raman spectra of (top) the droplet-like particle inside the pre-filled syringe and (bottom) the silicone oil reference.

APPLIED SPECTROSCOPY 831

The spectra were recorded and processed (baseline correctionand cosmic ray removal if needed) with Bruker OPUS 5.5t

prior to the final display.

RESULTS AND DISCUSSION

Identification of Droplet-Like Particle Inside a Pre-filledGlass Syringe. The droplet-like particle in the aqueousformulation was small (less than one mm) and was locatedon the top of the plunger head and near the internal wall of theglass syringe, as shown in Figs. 1a and 1b. The Raman signalof the droplet-like particle inside the pre-filled syringe wasextremely weak and could only be detected when the laserbeam was precisely focused on the particle. With the optimalexperimental setup described above, the Raman spectrum ofthe particle was successfully obtained, shown in Fig. 2 as the

upper trace. The spectrum was first tentatively assigned tosilicone oil (polydimethylsiloxane, PDMS) according to itscharacteristic peaks and comparison with the referencespectrum found in the KnowItAllt Raman databases (Bio-Rad, Enterprise Edition).

To further confirm the identification, we also measured theRaman spectrum of silicone oil with the same spectrometer byplacing a small droplet of it on a Raman slide (SpectRIMTM

plate from Tienta Sciences, Indianapolis, IN). The Ramanspectrum of silicone oil, measured under identical conditions,is shown in Fig. 2 as the lower trace. The comparison of thetwo Raman spectra in Fig. 2 shows that they are virtuallyidentical within the spectral noise level: the four characteristicstrong peaks located at 488, 709, 2906, and 2965 cm�1 fromthe silicone oil reference were present in the Raman spectrumof the particle at the same positions. Moreover, the relativeintensities and patterns of the peaks at 488 and 709 cm�1 and2906 and 2965 cm�1 also resembled each other between thetwo spectra. Finally, the remaining weaker bands in the particlespectrum also matched those present in the reference siliconeoil spectrum. Based on these detailed spectral comparisons, thedroplet-like particle inside the pre-filled syringe was confi-dently identified as silicone oil.

Silicone oil17 is an optically clear liquid and is generallyconsidered to be inert, non-toxic, and non-flammable. It is usedcommonly as a lubricant inside glass syringes for the purposeof smooth liquid injection. The silicone oil droplets mightcome from the non-uniformly distributed silicone oil on theinternal wall of the syringe barrel. Here we demonstrated thatthe silicone oil droplet inside the pre-filled syringes can bedirectly confirmed by Raman microscopy, the only applicabletechnique for in situ detection without the need for particleisolation and further manipulation.

Determination of Fibrous Particle Inside a Test Tubewith Aqueous Formulation. In the second case, a tiny fibrousparticle was observed inside a test tube with the aqueousformulation. The digital photo of this test tube (15 mm

FIG. 3. The fibrous particle inside a glass test tube (15 mm diameter); imagestaken by (a) the Nikon digital camera; and (b) and (c) the Senterra Ramanmicroscope.

FIG. 4. The Raman spectra of (top) the fibrous particle inside the test tube and (bottom) the cellulose reference.

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diameter) with the solution is shown in Fig. 3a. The fibrousparticle was examined under the Raman microscope and itsmicrographs are shown in Fig. 3b and in Fig. 3c with anexpanded view. Evidently the isolation of this tiny particle witha size of 0.1 mm from solution was challenging as well as timeconsuming. Therefore, Raman microscopy was utilized againfor the in situ identification of the tiny particle. To do this, thetest tube was placed directly on the sample stage of the Ramanmicroscope, under the objective (503 magnification for thissmall particle), and the laser beam was focused on this particle.

It was seen under the microscope that the fibrous particlewas in the vicinity of the internal glass wall. This wasconsidered to be important for the Raman microscope to ‘‘see’’the particle, as the working distance for an appropriateobjective is limited. The objective with longer workingdistances might not have enough light collecting power todetect the weak Raman signal from a micro-particle. With theright objective selected and the laser beam precisely focused onthe particle, the ‘‘check signal’’ mode was activated to optimize

the particle Raman signal by fine tuning its positions with themicrometers attached to the stage. Under these optimizedconditions, the Raman spectrum of the fibrous particle wascollected from 70 cm�1 to over 4000 cm�1, shown as the uppertrace in Fig. 4. The lower trace in Fig. 4 is the cellulosereference spectrum from the KnowItAllt Raman database. Thecellulose peaks in the fingerprint region were characterized bythree separated groups (I, II, and III) centered around 1380,1096, and 380 cm�1, respectively. The spectral agreementbetween the fibrous particle and the cellulose reference isevident: essentially all the peaks in the fingerprint regionmatched well between the two spectra. This included severalpronounced bands at 1480, 1380, and 1340 cm�1 from group I;1152, 1124, and 1096 cm�1 from group II; and 460, 436, and380 cm�1 from group III, in addition to a singly positionedband at 900 cm�1. Therefore, the fibrous particle inside the testtube was identified as cellulose based on their spectralagreement. This case study shows that the Raman microscopictechnique could also be used for in situ detection of particlesinside a glass test tube.

Identification of White Particle Inside a Glass VialContaining Aqueous Solution. Glass vials are another type ofprimary container used for filling and storing liquid formula-tions in the biopharmaceutical industry. During our stabilitystudy and inspection of a batch of filled vials, it was observedthat one of the vials contained a white visible particle insidethe liquid formulation. The white particle inside the vial (15mm diameter, Fig. 5a) was seen on the upper inner surface ofthe glass wall when the vial was placed in a horizontalposition, as shown in Fig. 5b. The magnified view of the whiteparticle under the optical microscope is shown as an inset inFig. 5b, demonstrating that it was approximately 0.3 mm inlength. Due to the small size and location of the particle, the insitu Raman detecting method was again selected for thisparticle identification.

For the spectral measurement, the glass vial was carefullypositioned on the sample stage, under the objective of the

FIG. 5. The glass vial (15 mm diameter) with the white particle inside; (a) digitalphoto of the glass vial filled with drug solution; (b) micrograph of the whiteparticle inside the vial. The expanded view of the particle is shown as an inset.

FIG. 6. The Raman spectra of (top) the white particle inside the glass vial and (bottom) the polypropylene reference.

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Raman microscope, so that the white particle remained on theupper inner surface of the glass wall. With this specificorientation, the particle was able to be kept within the workingdistance of the selected objective. Then the laser beam wasfocused and the Raman spectrum of the particle was obtainedreadily in just a few minutes, shown as the top trace in Fig. 6.The bottom trace in Fig. 6 is the reference spectrum ofpolypropylene (PP) from the KnowItAllt databases. Bycomparison, the Raman spectra of the white particle and thepolypropylene reference are essentially identical. This wasdemonstrated by the peak-to-peak matches between the twospectra, including several evident peaks: strong C–H stretchingbands at 2954, 2904, and 2841 cm�1, and fingerprint regionbands at 1460, 1331, 1153, 843, 801, and 399 cm�1. The broadbump around 400 cm�1 in the particle Raman spectrum wasdue to the scattering from the glass wall through which thelaser beam passed. Therefore, the white particle inside the glassvial was identified as polypropylene based on its excellentspectral agreement with the reference.

CONCLUSION

In this paper, we demonstrated three in situ applications ofRaman microscopy in micro-particle identifications encoun-tered in a biopharmaceutical context. All these applicationsinvolved in situ detection of micro-particles that were inaqueous formulated solutions inside glass containers. Theseglass containers were syringes, test tubes, or small vials, all ofwhich are commonly used as test, storage, and shippingdevices in the biopharmaceutical industry. In the procedures forconventional analysis, these micro-particles would have to beisolated from the glass containers first, which is not only time-consuming but also carries risks of secondary contaminationand/or particle loss during manipulation. In particular, theidentification of the droplet-like particle inside the syringeposed extra challenges as the tiny ‘‘droplet’’ would not beretained through filtration or other processes. Furthermore,other commonly used analytical techniques, such as infraredmicroscopy, were not capable of detecting this droplet-likeparticle in situ due to the interference from the glass of thedevice and the aqueous formulation. However, it is shown herethat Raman microscopy was perfectly suited for this type ofanalysis due to its advantages of in situ detection and minimalinterference from the glass wall. This was also true for the othertwo case studies in which the Raman in situ detectionsignificantly accelerated the particle analysis and avoidedpotential sample contamination or losses.

The above three case studies demonstrate that the Raman

microscopic technique can be very useful in the identification ofmicro-particles that are inside a glass container. With the Ramantechnique, detected particles remained intact during analysisand potential secondary contamination or loss of the particleduring manipulation could be avoided. The confocal ability ofthe Raman microscope provided axial and lateral resolution andfacilitated the particle detection. The selection of an appropriateobjective was required for the microscope to ‘‘view’’ theparticles (within working distance) and to collect detectableRaman scattering signal. However, it should also be pointed outthat the Raman technique was not ubiquitous in particleidentification due to its limitations. For example, the impurityfluorescence and weak Raman signal are still challengingproblems encountered during the detection. Nevertheless, theRaman in situ technique provided a complementary andsometimes unique method in particle identification, particularlyfor the particles inside a glass container and behind a glass wall.

ACKNOWLEDGMENTS

Drs. Linda Narhi, Joseph Phillips, David Brems, and Ron Foster are greatlyappreciated for their helpful comments and support of the projects.

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