White Paper: 1.5

Key Words

• Raw Material Identification

• Raman Spectroscopy

• Regulatory Compliance

Handheld Raman Instruments for Pharmaceutical Raw Material Inspection: From the Lab to the Loading Dock

The purpose of this paper is to discuss the application of Raman spectroscopy to the unique requirements of raw material testing. In order to do so we will answer the following questions:

1. What is and why perform identity testing?2. What are the incentives to speed up testing?3. Should we consider modifying the testing process?4. How does the Thermo Scientific TruScan handheld

Raman instrument perform ID testing?5. How well does TruScan® work for common raw

materials?6. Can this be deployed in a regulated pharmaceutical

environment?

Raw MaterialsPharmaceutical raw materials are ingredients or substances in a form that are intended for use in a finished drug product. These can be liquids, solids or gases or any of the intermediate states such as aerosols, colloids or gels/pastes. There are two basic types of raw materials: active pharmaceutical ingredients (APIs) and excipients. APIs, according the FDA Q7A definition1, are substances that have a “direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease.” APIs can be derived from plant, animal, or other biological sources as well as those purified and made from inanimate sources such as minerals or substances manufactured through a controlled chemical reaction.

Excipients are defined by the FDA Office of Generic Management2 as any “inactive ingredient” or “any component other than an ‘active ingredient.’” Excipients encompass water, lubricants, fillers, disintegrants, binders, coatings, sweeteners, preservatives, flavors, colors, etc. The United States Pharmacopeia3 (USP) lists more than 40 functional purposes for excipients. The excipients group includes materials involved with drug delivery, consistency, form, dissolution/bioavailability, stability, lubrication, feel, taste, and appearance. Some might argue that these compounds are anything but “inactive,” but the FDA defines API as being disease or condition ameliorating, therefore substances that aid the API are considered excipients. In general, excipients are commonly used among many manufacturers while the APIs are often limited, at least under the patent period, to a single or small group of suppliers. Due to the availability of most excipients and over the counter actives, this discussion will focus on these compounds.

Within the excipients and API types are two classes: compendial and non-compendial and these differ simply based on whether an independent pharmacopeia organization, such as USP, has defined the purity and

identity of the compound in question. Many proprietary APIs are not listed by the USP and must be defined by the manufacturer; these comprise the noncompendial group. There are currently more than 350 excipients listed by the USP and most of these require identity testing. Amongst seventeen approved USP methods for identity testing is Raman spectroscopy4, the underlying technology in TruScan®.

Industry PressuresOver the last several decades, the pharmaceutical industry has seen a remarkable growth in terms of demand and ability to supply the demand for life sustaining drugs. Most recently, however, the industry as a whole has seen some tougher times. Insurance and government price controls have limited existing product growth while patents have expired and provided generic competition to core business. Blockbuster drugs seem to be few in the pipeline and successful new drug applications (NDAs) have declined. In this limited growth state, the industry is seeking to reduce costs to increase shareholder value. The primary means to do this is through lean manufacturing initiatives. In order for existing manufacturing facilities to compete with low cost regions (LCRs) and the ever increasing contract manufacturing sector they must do things better, faster and cheaper. Among the manufacturing measurables targeted for efficiency gains are lead times, warehouse space, quarantine times, inventory, and turn rate. “Do more with less” is not just a catch phrase for people involved with these processes. Identity testing is one of the contributing factors in all these areas.

Economic reasons are not the only pressures felt by pharmaceutical manufacturers. The FDA has been promoting its 21st century initiatives5 with vigor and as PAT6 has gotten traction, the concept of 100 percent raw material inspection is also being pronounced as one of the top manufacturing goals. Add to this industry and internationally driven quality programs such as Six Sigma7, ISO and ICH Q8 QbD objectives and what we have can be summarized in two statements “change happens” and “do it better.” The quality initiatives themselves are positive, however an industry renowned for its conservative and “rather be safe than sorry” approach now must adapt to some serious competitive pressures. Numerous sites have reported that they have been tasked with how they will inspect more of their materials in the same time frame and without additional labor resource. Actively considering alternatives to current testing schemes is no longer a luxurious option but a necessary means of survival. Some are turning to handheld Raman ID testing with TruScan to help.

Current PracticeRaw material inspections are dictated by cGMP guidelines8: The container(s) are received; samples are collected; containers are quarantined; testing is conducted to prove identity; data are reviewed; QC releases the product to production; and all must be documented. The following process flow diagrams can be useful in understanding the industry conundrum.

It can be quickly seen that even with additional resources, including labor, hardware and lab space, the process will still take longer. The quarantine period will stretch to at least two shifts for every shift of the previous practice and this assumes some remarkable efficiency gains under the current testing scheme. In practice many sites actually quarantine for much longer periods as conditions such as remote labs, more involved testing procedures, lab congestion, or contract lab utilization result in quarantines measured in days or even weeks. This does not achieve the goals of lean manufacturing and requires a paradigm change to address.

The TruScan Raman spectrometer has several sampling advantages that allow it to positively impact the above process. First, the testing can be done in the receiving area. There is literally no advantage to take the sample back to the lab to test with the TruScan. Second, TruScan can sample through packaging such as double poly-bags, removing the need to open packaging and reducing the need of isolation rooms for sampling. Third, TruScan’s compendially-approved

Visual inspection and receipt of 100 �ber drum containers 2 Hours

Isolation Room Sampling 11 of 100 Drums 1 Hour

Transport Samples to Laboratory 1 Hour

Log in and Test 11 samples per compendia requirements 3 Hours

Review data and release product to production 1 hour

Q uarantine for one shift

Current Practice of Partial Raw Material Sampling and ID Testing

Diagram 1: Raw material inspection process currently in practiceIf the site is given a directive to inspect 100 percent of the raw materials coming in and we extrapolate from the current practice, assuming some efficiency gains are likely, the extrapolated practice in Diagram 2 unfolds.

Diagram 2: Modifying current practice to inspect 100 percent of incoming raw materials

Review data and release product to production 2 Hours

Visual inspection and receipt of 100 �ber drum containers 2 Hours

Isolation Room Sampling 100 of 100 Drums 5 Hours

Transport Samples to Laboratory 1.5 Hours

Extrapolated Practice for 100 Percent Raw Material Sampling and ID Testing

Log in and Test 100 samples per compendia requirements 6 Hours

Assumes less than 3 min per drum

Assumes additional hardware acquisition and lab space utilization

technology typically takes less than one minute per sample test, making it less time consuming than the common HPLC or wet chemical practices. Fourth, the PASS/FAIL declaration of the TruScan decision making engine permits the receiving area user to make an accurate release judgment. Lastly, the 21 CFR Part 11-supported test data can be sent via a network sync process for QC supervisor approval. The improved process flow is pictured below.

Essentially the implementation of TruScan technology can facilitate achievement of the 100 percent inspection goal in the same time currently doing partial testing. In cases where the quarantine period is days or weeks, TruScan’s impact can be even more dramatic, supporting full inspection and saving time over the current process. To understand whether these principles would apply to a particular situation involves a greater understanding of TruScan and Raman’s advantages and limitations.

Raman SpectroscopyFor raw materials inspection in the warehouse, Raman spectroscopy is now becoming the identification/confirmation technique of choice. Fast, non-destructive, portable, simple to use and deploy – the benefits are often stated and easy to understand. However, the theory of Raman is not as well known – and a short introduction to Raman is necessary. By understanding how a Raman spectrum is made, and how it can be used to provide an unambiguous analysis, it is easy to see how the benefits stated above come about, and why Raman is a technology well-suited for the pharmaceutical industry.

Basic Raman TheoryIn contrast to the commonly used mid-infrared (mid-IR) and near-infrared (NIR) spectroscopies, which are based upon the absorption of light, Raman is a scattering technique. The sample of interest is illuminated with an intense single wavelength light source, typically a laser. Light scattered from the sample, without any change in wavelength, is called elastic or Rayleigh scatter. A very small proportion of the light is inelastically, or Raman scattered. This Raman scatter has its frequency shifted from the original wavelength, with the difference in frequency corresponding to the vibration frequency of bonds within the molecules. Typically one photon in 106 – 108 is Raman scattered; the rest are Rayleigh scattered as shown in Figure 1.

Visual inspection and receipt of 100 �ber drum containers 2 Hours

Loading Dock Sampling, Testing and Release 100 of 100 drums 8 Hours

Retains Collection and QC Supervisor Post-Release Review 1 Hour

Q uarantine for one shift

TruScan Practice for 100 Percent Raw Material Sampling and ID Testing

Assumes labor re-allocation and additional hardware resource (TruScan)

Diagram 3: Practice for 100 percent inspection involving the TruScan handheld Raman system

Figure 1: Visual Depiction of Raman and Rayleigh scattering

The mechanism that causes Raman scatter is different to that of mid-infrared or NIR absorption. Mid-infrared and NIR require a change in dipole moment in the vibrating bond for absorption to occur; Raman requires a change in polarizability in the vibrating bond. Polarizability can be described as the distortion of its ‘dipole’ or ‘separation of charge’ of a molecule when exposed to an electric field. Mid-IR activity may or may not result in the change in polarizability required to be Raman active, however, if a vibration is both Raman and mid-IR active, it will be seen at the same position in wavenumbers (cm-1) in both spectra, although with different intensity. Many vibrations which are weak in the mid-IR are strong in Raman and vice versa. In molecules with a center of symmetry the techniques are mutually exclusive, mid-IR and Raman are therefore said to be complementary. Symmetric vibrations give rise to intense Raman lines; non-symmetric ones are usually weak and sometimes unobservable.

In Raman, as in mid-IR, fundamental vibrational modes are interrogated resulting in outstanding molecular selectivity. Both show sharp, discrete bands, very well suited for identification.

Spectral InterpretationThe act of interpreting a Raman spectrum has been made much simpler by the advent of automatic search and match algorithms. There are a number of approaches to matching spectra. Some methods are extremely complex taking into account both spectral and physical interferences, and optimizing the search and match in certain regions of the spectrum. This allows a non-expert user to setup and operate very powerful searching techniques. Probabilistic decision algorithms, such as those used by TruScan, can provide excellent identification capability far beyond that of a simple search algorithm9. For instance, the TruScan probabilistic decision algorithm correctly reports an only 1 in 70,000 chance of a match, comparing deliberately tainted acetaminophen raw material to a reference, although the conventional search method reported a correlation value of 0.942. These chemometrically sophisticated comparisons are much more likely to avoid false positive and negative results than traditional spectroscopic searches.

Reference LibraryFor traditional benchtop Raman systems, a sample is compared to a library of reference spectra to determine if a match exists. TruScan differs from this traditional approach in that a sample is compared to a pre-selected method to determine probabilistically whether the substance is correctly identified.

TruScan also differs from traditional reference libraries in that users are not required to make a decision regarding the quality of the reference spectra. TruScan’s intelligent optimization determines this automatically. The scan time, for instance, is adjusted according to the spectrum quality to optimize the signal/noise ratio and improve spectral matching. Background lighting and material affects, such as fluorescence, are optimized to give the best possible reference spectra.Since the physical properties of the sample do not greatly impact the Raman spectrum, it is possible to build a Raman library using only a single reference for each sample. Obviously this means that Raman is not a suitable technique

Figure 2: TruScan analysis report and spectra

to differentiate between materials which differ only physically, for instance powder versus tablet form. However this characteristic of Raman does simplify the library building process substantially and makes searching against that library much easier. It also is tolerant of manufacturing variations in granulation and powder size. Raman focuses on the chemical present, not its physical form.

Raman SpecificityWhereas Raman is transparent to physical form, chemical form is a completely different issue. Raman’s resolving power can easily differentiate polymorphs, isomers, and similar chemical compounds. TruScan can easily tell the difference between anhydrates and hydrates, monohydrates and polyhydrates, L and D forms, and Iso, meta, and para configurations, as well as moiety substitutions and mono, di and tri forms. Examples of differentiated spectra are found in Figures 3 and 4.

TNP (picric acid, 2,4,6-trinitrophenol)

2,4-dinitrophenol

500 1000 1500 2000 2500 3000

2-nitrophenol

3-nitrophenol

4-nitrophenol

Raman shift (∆ cm-1)

NO2O2N

NO2

OH

OH

NO2

OH

NO2

OH

NO2NO2

NO2

OH

500 1000 1500 2000 2500 3000

ephedrine

pseudoephedrine

N H C H 3

C H 3

O H

N H C H

3

O H

C H 3

Raman shift (∆ cm-1)

Figure 3: Selectivity based on functional group position and absence.

Figure 4: Diastereomer differentiation based on position of hydroxyl group at top of molecule.

Raw Materials SamplingPerhaps the greatest benefit Raman has over other techniques providing similar identity information is its extremely simple sampling. There is no requirement for any accessories or sample preparation; sampling can be taken directly through packaging including glass sample vials or plastic bags, as shown in Figure 5.The contribution to the Raman signal from transparent container materials is negligible, and a library spectrum obtained with one container material can be challenged by samples in other container materials without degrading the quality of the search results. This has been experimentally verified in the following example.

Excipients StudyThree TruScan handheld verification systems built at different times from different runs of parts were utilized to measure 32 randomly chosen pharmaceutical excipients and over the counter APIs to evaluate the applicability of the Raman units for use in incoming inspection. Table 1 is a list of the compounds investigated.

Table 1: Materials Tested • Sodium L-ascorbate • Hydroxypropyl cellulose • Acetaminophen • Magnesium stearate • D-Lactose monohydrate • Polyvinylpyrrolidone • Calcium carbonate • Potassium bicarbonate • Calcium phosphate dibasic • Potassium citrate tribasic monohydrate • Calcium stearate • Sodium bicarbonate • Calcium sulfate • sodium carboxy methyl cellulose • Calcium sulfate dihydrate • Sodium citrate • Cellulose • Sodium phosphate dibasic • Citric acid • Sodium phosphate monobasic • Colloidal silica • Stearic Acid • Dextrin from corn • Sulfanilamide • Dextrose anhydrous • Talc • Dextrose monohydrate • Titanium (IV) oxide, anatase • Ethyl cellulose • Trimagnesium phosphate • hydroxy propyl methyl cellulose • Zinc sulfate

Methods were created by simple association of a reference spectrum with an appropriately titled method. For each material, one reference signature was acquired by each one of three non-test handheld Raman instruments. The reference samples were in a borosilicate glass vial (VWR, West Chester, PA) and the data collected through the wall of the vial. The reference acquisition process was initiated and simply allowed to continue until terminated by the TruScan unit’s software after a high-quality reference spectrum was acquired. It was quickly determined that colloidal silica, talc, sodium carboxy methyl cellulose, and hydroxy propyl methyl cellulose did not provide an adequate signal to be measured with a TruScan instrument. The methods were then transferred to the three test instruments.

Challenge samples of the 28 remaining compounds were prepared consisting of approximately 2g sealed in 2-mil-thick polyethylene bags. Three measurements were made of each sample through the bag, one measurement with each of the three TruScan instruments. Each test measurement was automatic with the TruScan unit controlling laser power and exposure time to achieve the necessary spectral signal/noise ratio in the shortest measurement time possible. This calculation yielded a p-value for each comparison. The p-values are plotted in Graph 1.

Figure 5: Sample presentation to the instrument through bag

Graph 1: Probabilistic decision making graph

Results DiscussionTo interpret the results, it is easier to think of the numbers at the top of the chart as methods and on the left side of the chart as compounds. In fact, the method number equals the compound number, but for our discussion let us keep them separate for clarity. Probabilistic values (p-values) are indicated in the bar below the graph. P-values less than 0.01 are considered unambiguous non-matches and therefore colored red to indicate the probabilistic absolute that they will not be falsely indicated. The green colored boxes are for p-values greater than 0.1 and are considered positive matches. These values are certain matches and will return a sure positive in the probabilistic approach. The yellow boxes are the ones in which the values are between 0.01 and 0.1 which bracket the TruScan decision making cutoff of 0.05. To TruScan, anything p > 0.05 is a match within the preset 95 percent confidence limit.

In the case of test compounds 10 and 11, yellow indicates that TruScan could report a false negative. This is primarily due to the influence of the packaging in these cases since the Raman signal is weak for these compounds and the polyethylene bags begin to influence the spectra.

In the case of compound 14 tested against method 13, indicated by a yellow box, there would be a danger of reporting a false positive. This was not due to packaging but to the similarity of the spectra. In only one combination did a definitive false positive arise and that is seen in the case of compound 13 tested against method 14. These compounds are calcium stearate and magnesium stearate, respectively. The result is understandable as the stearate part of the molecule dominates the Raman spectra and the calcium and magnesium elements are weak Raman scatterers. If there is a danger of these two compounds being present then a risk assessment would dictate the appropriate response. The situation can be ameliorated through SOPs (the sample being withdrawn from the packaging for instance), labeling changes (read calcium and/or magnesium stearate) or additional ID testing.

More than 80 percent of the materials investigated could be clearly measured and differentiated. This is consistent with the findings of our application department. Testing more than 4,000 chemical entities, they have found that nearly 90 percent were amenable to TruScan identification and verification. The results strongly establish TruScan as a receiving area identity verification tool.

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Data ManagementAssuming that the instrument is suitable for use in the receiving area and is suitable for compendial ID testing, the final question is: can TruScan be implemented in a regulated environment? This concern is one of data management. The desire to move toward paperless reports requires appropriate controls and assurances. TruScan was designed to facilitate 21 CFR Part 11 electronic records requirements. It contains multilevel password protection with administrator-controlled rights. Once a test is completed, it cannot be erased or modified by a user. The electronic record will only permit a note to be added explaining why the record might not be good. The records can only be removed from the TruScan instrument with a sync process that transfers the records to a sync server. The records are transferable in one or all of several formats including PDF, JPEG and TXT (see Figure 2). The text file is designed to be automatically input into 21 CFR Part 11-compliant host software.

The PDF and JPEG records contain: • instrument description • serial number • user • login date and time • test method (barcode) • sample ID (barcode) • test date and time • method reference spectra • overlay of sample spectra • probability value • PASS/FAIL result • Self test date and time, result, standard • Self check values

The interface to TruScan is through a web portal, so there is no PC software that must be purchased, installed, validated and updated. The TruScan contains its own IP address and access is controlled through the same administrative controls on the instrument. A network connection and web browser is all that is needed to “see” the instrument from a PC or laptop. Syncing is also an administrative right. One of the advantages of the web-based IP address in TruScan is the ability to administer an official set of company methods and reference spectra very easily through email or an internal network.

ConclusionUntil recently, Raman spectroscopy systems have belonged in research and development as they have been extremely complicated systems with multiple modes of operation, requiring specialized operation and maintenance. However, advances in semiconductor lasers, optical miniaturization techniques, compact data processing capabilities, fluorescence suppression and probability-based statistical analysis algorithms have combined to allow the development of compact, rugged, self-contained Raman instruments that can be reliably used in harsh, outside-the-laboratory environments.The TruScan instrument can perform well against a large portion of pharmaceutical raw materials tested and can help the quality control and plant operations team get closer to the 100 percent inspection goal. One of the biggest benefits of TruScan is its ability to analyze substances through glass and plastic. Thus in most cases, samples do not need to be removed from their glass or plastic containers. Finally, every substance has a unique Raman spectrum, making the technique ideal for identification purposes.

For raw material identity verification, TruScan—with its point-and-shoot operation and unambiguous results presentation—is an ideal tool for busy warehouse personnel, removing the need to collect samples and providing immediate PASS/FAIL results.

References

1 http://www.fda.gov/cder/guidance/4286fnl.htm#P1422_111232 , Guidance for Industry Q7A Good Manufacturing Practice Guidance for Active Pharmaceutical Ingredients

2 http://www.fda.gov/ohrms/dockets/dockets/90s0308/90s-0308-m000569-vol36.pdf and 21 CFR 210.3(8) 3 http://www.uspnf.com/ , United States Pharmacopeia - National Formulary 4 USP 30 <1120> RAMAN SPECTROPHOTOMETRY, Qualitative Purposes “qualitative Raman measurements may be used as a compendial ID test”. 5 http://www.fda.gov/oc/guidance/gmp.html , Pharmaceutical cGMPs for the 21st Century: A Risk-Based Approach 6 http://www.fda.gov/cder/guidance/6419fnl.pdf , Guidance for Industry, PAT — A Framework for Innovative Pharmaceutical Development,

Manufacturing, and Quality Assurance 7 http://www.iqpc.com/cgi-bin/templates/genevent.html?topic=237&event=10368& , Lean Six Sigma for Pharmaceutical and Biotech Manufacturing Excellence 8 Code of Federal Regulations, Title 21, Part 210.82 and Part 210.84 9 Technical Note 2.0, Characterizing and Validating Handheld Raman Instrument Discovery Library Performance

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