As promised, I am highlighting another tool for bringing CROs and CMOs and, of course, generic houses into the 21st century. For decades, Raman spectroscopy was an expensive, lab-based research tool. Mainly used for structural elucidation and identification of materials in mixtures, it was hardly ubiquitous in Pharma labs; certainly not contract labs. It was not a simple tool because the LASERs used, mostly in the visible wavelength range, cause major fluorescence interference, often overwhelming the “pure” Raman spectrum. Because of this problem, Raman isn’t a common tool in QC labs or production facilities.

Fortunately for serious analysts, the hardware and software have become better, easier to use, and safer. The LASERs have become smaller, safer, more dependable, and less expensive. And computers, with more power, speed, and capacity, have become major reasons that complicated algorithms may be used in almost instantaneous generation of a usable spectrum.

While there are any number of valid reasons why Raman has been slower to become a mainstay in the Pharma industry, but none as strong as tradition: since the mid-1970s, if you wanted to assay something, you forced it through an HPLC. Techniques such as near-infrared and Raman were very slow to be tried. Newness, to Pharma executives, means “lack of regulatory experience” which, in turn, means fear of being the first to submit an application for fear of delay. Fortunately, attitudes are changing for the better. In this column, we examine two very different pieces of equipment: both new, yet each leaning towards different application.

The first instrument addresses one of the most infuriating annoyances and impediment to clean spectra associated with Raman spectroscopy: a massive fluorescence background with popular LASERs. I will not to go into detailed Raman theory, but simply examine some solutions to existing problems. Fluorescence background steadily diminishes as you increase the wavelength of the incident LASER—from 532 nm to 1064 nm—so a number of companies have simply moved to longer wavelengths. Unfortunately, the Raman signal, itself, diminishes by the fourth power of the incident wavelength, making ID or analysis of lower concentrations difficult, if not impossible as the units move to longer wavelengths.

For decades, the best method to discriminate against fluorescence was to “gate” the light emitted by the sample. That is, a very quick LASER pulse strikes the sample and only the first 100 picoseconds of the emitted light is collected. The Raman spectrum emerges first, but is quickly overwhelmed by the fluorescence signal (see Figure 1). A gated or time-resolved signal is principally the Raman spectrum, with any residual fluorescence easily suppressed digitally.

While a number of experimenters built such equipment (including the supplier of the second unit discussed), Timegate Instruments¹ has successfully produced a commercial version. Their “electronic shutter” also allows the analyst to use whichever wavelength is best for the analysis, not relegated having to choose the wavelength with the least background interference. This is important when looking for low concentrations or materials with a weak Raman signal.

Perhaps a more informative view of the time-gated spectra is a 3-D graph of the Raman shift (cm-1) vs. time delay vs. counts, available in the software. This visualization aid is helpful for selecting wavelengths to monitor in an experiment or process. While the unit used for small molecule applications, an excellent application is in biological synthesis such as fermentation production.

Since the Raman activity is strongest for linear molecules with a center(s) of symmetry, capable of having an induced dipole (not getting technical here; however, you could take a course in Group Theory, if you desire a better understanding), water, being both non-linear and containing a permanent dipole is nearly invisible in Raman. Unlike IR and NIR, where, in an aqueous solution, it is, respectively, nearly impossible to measure and a nuisance, Raman excels in water-based applications.

So, for a typical fermentation broth, the time-gated unit may be used to examine both the cells and the surrounding medium (see Figure 2). Such information allows the production team to monitor the growth of cells, remaining nutrients, and potential harmful byproducts, simultaneously. While excellent results have been seen with NIR, this specificity of cells versus ambient solution is far superior. This application would allow for the natural differences in fermentation rates and may be used, if for nothing else, to know when to add nutrients or harvest the crop.

While a number of portable and hand-held Raman instruments are built by several reputable instrument companies, the second, smaller, yet quite rugged and accurate instrument I wish to mention is built by Real-Time Analyzers and was originally designed to be used on the International Space Station. It was thought that medicines might degrade faster or differently in orbit, due to the extra radiation, thus, the “normal” expiry dates may not be valid. So, a simple, light and accurate unit was commissioned by NASA for use by the astronauts. Actual spectra are not available. It is also considered being employed to control diversion and of controlled drugs in hospitals and clinics.

The ability to perform quantitative measurements (to assure proper dosage form) is critical in clinical investigations, where the company’s new drug is being compared with a look-alike in a manner designed to be double-blind. That is, neither the doctor nor patient knows which drug, or placebo, he/she is taking. Clearly, if they are that similar, the operators making the blister packs for the study might well make errors in tablet/capsule placement, especially considering a major study may well use thousands of cards. If there are poor results, there is no evidence whether it was the drug’s performance or improper dosing that might have caused them. Non-destructive, through-the-plastic Raman can be used to spot-check as many cards as QA feels is necessary for statistical surety.

Some other applications
Any number of Raman applications would give a competitive edge, some are:

1. Rapid ID of incoming raw materials. Chemical ID, crystallinity, and polymorphic form are just some of the things able to be checked.
2. Rapid check of finished materials arriving from production to the packaging area, assuring no errors in packaging. This is even more critical when a contract company produces numerous products for more than one client. Expecting packaging staff to visually distinguish products may a recipe for disaster.
3. Non-destructive check for content uniformity, as well as proper drug, polymorph, and amorphous vs. crystalline form in final dosage form. Unless a company has a PAT program, this is a good intermediate step for safety and goodness.
4. A future application is rapid, non-destructive screening for potential breakdown products in stability studies, allowing samples to be checked in between set sampling points. This is similar to the NASA study, not published, in the Space Station.
5. Pre-formulation (non-destructive) screening of complex—via Design of Experiment—samples to aid in formulation studies; eventually speeding up time to market. 

References

1. Timegate Instruments Oy, Teknologiantie 5B, 90590 Oulu, Finland
2. Real-Time Analyzers, 352 Industrial Park Rd., Middletown, CT 06457, USA

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Emil W. Ciurczak
DoraMaxx Consulting

Emil W. Ciurczak has worked in the pharmaceutical industry since 1970 for companies that include Ciba-Geigy, Sandoz, Berlex, Merck, and Purdue Pharma, where he specialized in performing method development on most types of analytical equipment. In 1983, he introduced NIR spectroscopy to pharmaceutical applications, and is generally credited as one of the first to use process analytical technologies (PAT) in drug manufacturing and development.