Product-related impurities

Product-related impurities are molecular variants of the desired product (e.g., precursors, certain degradation products arising during manufacture and storage) which do not have properties comparable to those of the desired product with respect to activity, efficacy, and safety.2 Variants may exert different biological effects (potentially uncontrolled or hazardous) and specifically lead to antibody formation in the patient.3... 

As outlined above displacement chromatography may find its most important uses in the analytical area. The ability to enrich trace levels of components is ideally suited to the proteomics where more powerful tools are desperately needed to address the vast concentration ranges present in order to identify trace components. The technique also offers a way to isolate large quantities of protein variants which is important for the identification and characterization of minor product-related impurities commonly associated with therapeutic proteins. 

Most recombinant biopharmaceuticals are produced in microbial or mammalian cell lines. Thus, although the product is derived from a human gene, all product-unrelated contaminants will be derived from the producer organism. These non-self-proteins are likely to be highly immunogenic in humans, rendering their removal from the product stream especially important. Immunoassays may be conveniently used to detect and quantify non-product-related impurities... 

Bulk and Intermediate Purification primarily for removal of process-related impurities, e.g. reagents, host cell proteins, DNA, endotoxins some product-related impurities common methods ... 

Polishing final purification step (invariably using chromatography) to remove dose product-related impurities, residual of host cell proteins (HCPs) and endotoxins. 

Protein impurities are either process- or product-related impurities. Process-related impurities include proteins added to the culture medium, proteins used during purification, such as nucleases and chromatography ligands, and proteins from the host organism. Product-related impurities include degra-dates, aggregates, and conformational isomers of the recombinant drug product. 

Protocol 7 for CZE was adapted from Gordon et al.77 These authors used this protocol to analyze a variety of proteins including a complex protein mixture. It may provide a good starting point for protein impurity analysis. If insufficient separation is achieved then the analyst is advised to change the pH by using one of the other buffers discussed above. Such an approach showed markedly different separations for a number of recombinant proteins and product-related impurities.39,78... 

Capillary isoelectric focusing (CIEF) separates proteins based on differences in their isoelectric points. CIEF has been used to separate product-related impurities of recombinant proteins, mainly deamidated species, such as those of human growth hormone.71... 

Coupling CE with electrospray ionization mass spectrometry (MS) can potentially be a very powerful tool for detecting and identifying product-related impurities in recombinant pharmaceuticals. Proteins can be detected in the femtomole range with this mode. Conceivably, the bioanalyst could perform a peptide map with CZE-MS and detect, identify, and sequence aberrant peptides derived from degradates. 

The problem of N-terminal variants in recombinant proteins is not uncommon. E. coli synthesizes proteins with a formylated methionine at the N terminus. In vivo, E. coli often removes N-formyl methionine with the action of a deformylase followed by methionine amino peptidase. This removal is not always exact and neighboring amino acids in the peptide chain influence the removal.130 This can yield recombinant products lacking a number of encoded amino acids at the N terminus. For smaller proteins, these product-related impurities generally are detected and quantitated by RP-HPLC. However, large proteins differing by only one or two N-terminal amino acids may be difficult to resolve by RP-HPLC. In these instances, peptide mapping by RP-HPLC is a valuable tool. 

Cell substrate derived Cell culture derived Downstream derived Product-related impurities Truncated forms Other modified forms (i.e., deamidated, isomerized) Aggregates gel electrophoresis, size exclusion chromatography... 

The complexity of biological macromolecules when compared with small molecule therapeutics, differences in manufacturing, and the broad variety of potential degradation pathways lead to special requirements in quality assurance and analytical testing of pharmaceutical proteins. The product-related impurities are molecular variants formed during manufacture, storage, or use, and their properties are different from the desired product with respect to activity, efficacy, and safety. 

The question of how much degradation is sufficient to meet the objectives of stress studies is widely discussed, especially with respect to conventional therapeutics. A degradation level of 10-15% is considered adequate for validation of a chromatographic purity assay. Chromatographic methods for product-related impurities (including degradants) should be validated by spiking experiments... 

Critical quality attributes may include items, such as potency, the nature and quantity of product-related substances, product-related impurities, and process-related impurities. Such attributes can be assessed by multiple analytical procedures, each yielding different results. In the course of product development, it is not tmusual for the analytical technology to evolve in parallel with the product. Therefore, it is important to confirm that data generated during development correlate with those generated at the time the marketing application is filed. 

Product-Related Impurities Including Degradation Products... 

Process- and product-related impurities, as well as possible impurities originating from the manufacturing of the final dosage form. 

For product-related impurities, all types of chromatographic and/or electrophoretic methods (as well as peptide mapping)... 

For biopharmaceuticals, the complexity of analytical test approaches is also reflected in guidelines concerning reference standards and materials (see ICH Q6B [22]). In principle, it is desirable to use the same reference material for both biophar-maceutical assays and physico-chemical testing, but in some cases a separate reference material may be necessary. It may also be necessary to establish distinct reference materials for product-related substances, product-related impurities and process-related impurities. 

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