Purity analysis, traditional methods

An important fact inherent in the purity analysis of a recombinant pharmaceutical is that the absolute purity of any protein is an elusive, if not an unobtainable, measurement. For biopharmaceuticals, purity is a relative term. Protein purity is method-dependent and is defined by the shortcomings of the analytical procedure. Also, unlike small traditional drugs, proteins are highly complex molecules. For these two reasons, more than one method must be utilized to define a protein s purity. The greater the number of methods used in the purity analysis, the greater the assurance is that the product is pure. Furthermore, the purity determined by an analytical method can only be properly interpreted based on the method s validation. Analytical methods validation is critical to and inseparable from purity determinations. A detailed discussion on analytical methods validation is beyond the scope of this chapter but other sources of information are available for the interested reader.11 13... 

In conclusion, the application of a separation technique such as HPLC to the measurement of concentration in the determination of octanol-water partition coefficients greatly enhances the traditional methods. It is possible to automate the measurements and use much smaller amounts of compound without the need for very high purity. With the application of a fast generic gradient method, there is no need for method development and the analysis time can be reduced to 5-7 min per sample. 

In the manufacture of proteinaceous drugs, the purity of the final product is of paramount importance. Purity, however, is by no means an absolute term it depends on the method used for its measurement. Since traditional chromatographic methods and slab gel electrophoresis have been the main tools for biopolymer analysis, the commonly used but utterly vague terms chromatographicalfy or electrophoreticalfy pure demonstrate the role of available techniques not only in the measurement but also in the definition of purity. 

It is important to make the distinction between the determination of polymorphic identity and polymorphic purity. The former is essentially a qualitative determination, asking the question, Ts a particular polymorph present in a given sample The latter is a question of quantitative analysis, and it is generally (though not always) assumed that the sample is chemically pure, so the analytical problem to be addressed is the determination of the relative amounts of different polymorphs in the sample. Recalling that different polymorphs are for all intents and purposes different solids, the determination of polymorphic purity is then no different in principle from quantitative determination of the composition of a mixture of solids. Such quantitative determinations comprise one of the traditional activities of analytical chemistry, especially when the materials are different chemical entities. In those cases, a variety of different analytical methods may be employed. In the case of polymorphic mixtures, or the determination of polymorphic purity, the choice of analytical method is considerably more restricted, and X-ray diffraction is one of the most definitive techniques (see e.g. Stowell 2001). 

Data which establishes the compound s molecular formula is required. Traditionally an accurate combustion analysis (within 0.3 - 0.5%) has been used to determine the empirical formula of a compound, and also to justify that the compound is of high chemical purity. However, combustion analysis data will be identical for structural isomers of any type (geometrical isomers, diastereoisomers, enantiomers etc.) and other spectroscopic or chromatographic methods will therefore be required in order to determine levels of isomeric impurities. 

The most obvious way to use immobilized antibodies for analytical affinity chromatography is to simply use it in a traditional single-column method to determine an antigen s concentration and/or purity. However, there are a number of ways this technique can be advanced to more sophisticated analyses. For example, instead of immobilizing an antibody, the antigen may be immobilized to quantify the antibody as has been done with the Lewis Y antigen [3]. However, the analysis is still a single-column method. 

Cereal proteins play an important role in food functional properties and have been traditionally separated and characterized using slab-gel electrophoresis techniques. CE is emerging as a valuable new technique for the analysis of cereal proteins, particularly to bring improvements in resolution and speed of analysis. Methods based on CZE and SDS-CGE can be used for a variety of applications, including culti-var differentiation and purity screening. 

TLRC can be used for animal, human, and plant metabolism analysis radiochemical purity and stability assessment toxicology and biochemical studies and separation, detection, and quantification of separated radioactive zones of all compound classes. Traditional film autoradiography and LSC continue to be widely used, but phosphor imaging and layer scanners are being increasingly applied. The instruments for these methods are highly automated and... 

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