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European Biopharmaceutical Review

Quality Control: Analytical Methods

Current legislation requires that raw materials used for the production of biopharmaceutical products are regularly tested to verify that they meet a number of stringent safety specifications. Conventional laboratory chemical testing methods are time-consuming and difficult to implement, requiring samples to be transported from the field to the laboratory for analysis. Near-infrared (NIR) spectroscopy has emerged as a competent technology to provide simple, accurate and fast in-line analysis of raw materials. This article discusses the importance of raw material quality, testing method requirements and the benefits of NIR spectroscopy. A real-life case study demonstrates the capabilities of NIR spectroscopy as a powerful method for identifying and quantifying phosphate salts used for the manufacture of ultra-high purity cell culture buffers.

The Importance of Raw Material Quality

Good quality raw materials are essential to ensure that effective and safe biopharmaceutical products can be manufactured. The physical properties of raw materials directly affect the quality of the final products. For example, changes in particle size or polymorphism can influence the flow properties and moisture uptake of the materials, negatively impacting their blend and compression behaviour and resulting in poor content uniformity. Low quality raw materials have been repeatedly associated with final products failing and being recalled, damaging brand reputation and incurring considerable costs.

In October 2005, regulation 2004/27/ EC was passed in the EU, requiring that active pharmaceutical ingredients (APIs) used as starting materials in dose form pharmaceutical production must have been manufactured in compliance with good manufacturing practice (GMP) basic requirements for active substances (1). The legislation applies to all registered APIs. In addition, the regulation mandates that marketing authorisation applications and variations to change the source of the active substances used must be supported by a declaration of GMP compliance. This must be provided by the active substance manufacturer by a Qualified Person (QP) of the dosage form manufacturer.

In order to comply with the legislation and produce safe products, biopharmaceutical manufacturers must perform regular analysis of their raw materials to confirm manufacturing suitability.

Raw Material Testing Methods

According to GMP basic requirements, biopharmaceutical manufacturers must implement scientifically sound procedures to ensure that raw materials conform to established standards of quality and purity (2). Analytical methods must be validated, unless the method employed is included in the relevant pharmacopoeia or other recognised standard reference. The suitability of all testing methods used must nonetheless be verified under actual conditions of use and fully documented.

Traditionally, laboratory chemical testing has been used to analyse biopharmaceutical raw materials to confirm their quality and suitability for use. Such methods require that samples are taken from the field and transported to a laboratory for testing. However, as the pharmaceutical industry moves towards 100 per cent inspection of incoming raw materials, it can be extremely time-consuming and expensive to collect samples from all containers of raw materials and send them for laboratory analysis. In addition, laboratory chemical testing involves the use of complex, laborious and lengthy protocols.

NIR Spectroscopy

NIR spectroscopy has been recognised as a first-line, go-to analytical technique for testing and characterising biopharmaceutical raw materials for quality purposes, in compliance with current legislation. This powerful technique provides simple, precise and rapid composition and contamination analysis of raw materials in the laboratory, at production point-of-use, or in realtime processes. Analysis can even be performed while the material is still packaged, thus reducing the risk of cross-contamination and saving valuable time.

A wide range of samples can be analysed using NIR spectroscopy, including solids, powders, gels, pastes, grains, films and liquids. A further significant advantage of the technique is that it can generate high-quality reflectance spectral information for both active ingredients and excipients, while also facilitating the analysis of both the chemical and physical properties of a given material. Additionally, NIR spectroscopy is non-contact, non-destructive, highly reproducible and often does not require any sample preparation.

Until recently, there has been a misconception that NIR spectroscopy is not capable of analysing inorganic materials such as salts. Some inorganic salts may not generate strong NIR spectra; however, many inorganic salts exhibit vibrations that are sufficiently active in the NIR region to allow for reliable analysis. In particular, hydrated inorganic salts can be easily analysed using NIR spectroscopy. The technique can also be used to identify different polyatomic ions and separate them from each other.

Case Study: Real-Life Application

A bioprocessing company was provided with sub-quality raw materials for preparing complex cell culture buffers. These inferior raw materials were undetectable using the standard quality assurance methods and eventually resulted in over $3 million in lost finished product, investigations and recovery, as well as a damaged reputation. Complex analysis using inductively coupled plasma mass spectrometry (ICP-MS) on the finished cell culture media traced the sub-quality raw material to a batch of poorly refined sodium phosphate dibasic (Na2HPO4), which contained significant quantities of pyrophosphate ion (P2O7)-4. The presence of the pyrophosphate ion led to incorrectly formulated synthetic cell culture media, thus damaging, destroying and invalidating healthy bioreactor runs. Subsequent NIR analysis proved that the inferior raw materials would have been quickly and positively identified and quantified at the receiving dock, if NIR spectroscopy had been used.

Analyses
An NIR spectroscopy analyser was used to identify various phosphate powders and quantitatively determine the mass per cent of pyrophosphate in sodium phosphate dibasic raw material. Two separate studies were performed to achieve these two goals.


Qualitative Identification
Six different classes of phosphate materials were selected based on their use and potential for use as incoming raw materials (see Table 1).

 

These materials are representative of the variety of minerals that may be refined from phosphoritic ores. The phosphates were analysed directly inside their original containers using a fibre optic probe, whereby spectra were averaged from 16 scans using 8cm-1 resolution. A total of 11 samples from each material were used in the analysis. The spectra were subjected to chemometric treatment using TQ Analyst software. A discriminant analysis algorithm was selected using multiplicative signal correction in the pathlength. The raw spectra were used without pre-processing or derivatives except for a linear removed baseline to account for baseline shifts due to differences in sample refl ection. The spectral regions used in the method ranged from 7400 to 4100cm-1. Example spectra from each of the classes are shown in Figure 1. Even though there were only subtle absorbing bands and spectral characteristics in the samples, robust calibration methods were developed.

 

Quantitative Measurement
This quantitative study used 13 samples of sodium phosphate dibasic (Na2HPO4)contaminated with various amounts of anhydrous sodium pyrophosphate (Na4P2O7) ranging from 0 to 15.4 per cent (approximately 0 to 10 per cent pyrophosphate ion). The 13 samples were collected in two dram vials which were placed on the integrating sphere module of the instrument. Spectra were averaged from 64 scans using 8cm-1 resolution. Typically, spectra from each sample were collected twice and the two examples were merged in the chemometric analysis. A partial least squares (PLS) method was developed that allowed for quantitative prediction of the weight per cent of pyrophosphate in the Na2HPO4. Most of the samples were used to build and develop the PLS chemometric calibration, while four samples were not directly used to build the model but were later used as validation standards to test its robustness and reliability. The standard spectra were subjected to a second derivative preprocessing treatment with a Norris derivative smoothing algorithm (segment 11, gap 5). The region of analysis ranged from 7400 to 4700cm-1.

Results

Qualitative Identification
The fully developed discriminant analysis method identifi ed the various raw materials successfully without any errors. There was suffi cient spectral variability between each of the classes that they were clearly separated. Mahalanobis distances were reported for the spectra, which indicated how closely each spectrum clustered around the class average. Smaller Mahalanobis distances indicated that the sample was spectrally close to the class average, while larger Mahalanobis distances indicated that the sample fell far away from a particular class average.

Ideally, each spectrum’s lowest Mahalanobis distance would be to its correctly identified class, while the next highest Mahalanobis distance would be relatively large. Mahalanobis distances can be thought of as the number of standard deviations a spectrum might fall from the class average. Distance values of less than one indicate that the spectrum is very similar to the class average. Table 2 lists the average Mahalanobis distance ratios of the closest incorrect classes to the correct classes. Ratios higher than three suggest that there is a substantial separation between the clusters of spectra assigned to each class. The ratio for the sodium pyrophosphate was extremely large, indicating that it was considerably different than the next nearest class of material.

Quantitative Measurement
The PLS method developed from the second derivative spectra provided a calibration curve with excellent correlation between the NIR predicted values and the mass percentages determined gravimetrically. A calibration curve for the pyrophosphate ion mass percentage demonstrates this high correlation (see Figure 2).

In addition to the high correlation, the PLS method provided a low level of error. Root mean square error of calibration (RMSEC) was calculated from the standards used to develop the method and root mean square error of prediction (RMSEP) was calculated from the four validation standards used to predict the robustness of the method. The RMSEC and RMSEP values were 0.46 per cent and 0.68 per cent respectively. This indicates that the method can easily predict the amount of pyrophosphate contamination in Na2HPO4 between 0 to 10 per cent and be accurate, on average, to less than 0.75 per cent.

Other than high correlation and low level of error, a predicted residual error sum of squares (PRESS) plot was used to evaluate the quality of the method. The errors of an ideal PRESS plot decrease rapidly with increasing number of factors used in the method until it reaches a minimum and remains stable. Figure 3 shows that the PRESS plot for this method follows the ideal form, indicating that the method is robust with good predictive capabilities. The method created for this chemometric analysis used four factors.

Conclusion

NIR spectroscopy has emerged as the technique of choice for monitoring the quality of biopharmaceutical raw materials. Accuracy, short time of analysis and elimination of the need for sample preparation make this an ideal technology for robust and reliable raw material characterisation. NIR spectroscopy successfully replaces time-consuming, expensive and laborious chemical testing methods,allowing for at-line and in-line analyses to be performed. Experimental results demonstrate the advanced capabilities of the technique for identifying and qualifying phosphate salts intended for use in the production of ultra-high purity cell culture buffers, contrary to the common misconception that NIR spectroscopy is not suitable for analysing inorganic materials.


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Todd Strother received his PhD in Analytical Chemistry from the University of Wisconsin-Madison, focusing on surface immobilisation of biomolecules. Todd worked in the biotechnology industry for six years prior to joining Thermo Fisher Scientific in Madison Wisconsin. At Thermo Fisher, Todd has specialised in using near infrared spectroscopy for cell culture and bioreactor applications. Currently, he is the product specialist for infrared spectroscopy accessories. He also holds a graduate degree in Science Education and has taught in both secondary and post-secondary institutions.
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Todd Strother at Thermo Fisher Scientific
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