Recombinant Drug Products

Human Insulin, Recombinant Human insulin was the first pharmacologically active biological macromol-eculc lo be produced through genetic engineering. The FDA approved the drug in 1982 for the treatment of type I (insulin-dependent) diabetes (.see Chapter 2S). The insulin protein is a two-chain polypeptide containing SI amino acid residues. Chain A is composed of 21 amino acid.s. and chain B con- 

Insulin rDNA has been very successful. The only problem has arisen in patients who have been using porcine or bovine insulin for a long lime. Some patients who are switched lo rDNA human insulin repon difficulty in feeling their glucose level. and these patients require extra counseling in the use of the recombinani hormone. 

The first use of recombinani hGH (rtiGH) was repn. in 1982. rhGH preparations were first produced itiEr -These preparations contained a terminal methionine amino acids. Natural sequence rhGH has since beenfr duced in mammalian (mouse) cell culiure. 

Somatrein, the flrsi recombinant preparation, introduced in 1985. contains the natural 191-amino acid primary sequence plus one ntethionyl residue on the N-lerminal end. Hie sontatnipin products all contain the 191 -amino acid sequence and are identical with the hGH produced by the piiui-luiy gland. The thiee-dimen.sional cry.stal structure shows ihjl the protein is oblate, with most of its nonpolar amino acid side chains projecting toward the interior of the molecule. This rhGH is pharmacologically identical with natural hGH. 

Most current fomiulalions of rhGH are supplied in lyophi-lircd form and must be leconsliluled prior to injection. Typi-calty, 5 to 10 mg of protein are supplied in a powdered glycine and/or inannilol phosphate buffer. The preparation tsreconsliluied with. sterile water for injection, and the stability of the product is quite good. If. stored at 2 to 8°C, rhGH uill remain stable for 2 years. 

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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. 

A simple example of mapping is shown in Figure 4A for a performance qualification run for a recombinant drug product filled and released by mass. Each vial contains only a few micrograms of active protein, and therefore it is... 

Product-related substances are molecular variants of the desired product formed during manufacture and storage, which are active and have no deleterious effect on the safety and efficacy of the drug product.2 Many recombinant protein products are inherently heterogeneous, mixtures of closely related structures or product variants. These variants possess properties comparable to the desired product and are not considered impurities. It is only when they do not have properties of the desired product that their presence is problematic. 

Recombinant DNA drug production has been a success story, but the technology does have its drawbacks and disadvantages. For example, the use of bacterial plasmids and, less commonly, viruses, as vectors for the insertion of genes into host cells has its limitations. While such methods work well for smaller drug molecules, such as insulin, they are much less effective in the production of larger, more complex proteins that constitute many of the drugs that chemists would like to manufacture synthetically. 

In some ways, the use of animals (almost always mammals) as substitutes for bacteria in the recombinant DNA production of drugs is a natural and obvious extension of the techniques originally developed for the manufacture of insulin, human growth hormone, and other pharmaceuticals. Live animals have a built-in production... 

BOX 2.1. FDA PLANS TO SHIFT REVIEW AND APPROVAL OF WELL-CHARACTERIZED AND RECOMBINANT BIOTECHNOLOGY PRODUCTS FROM ITS BIOLOGICS DIVISION TO ITS DRUG DIVISION... 

Active and excipient chemical ingredients used in drug products may therefore be considered as BPCs. These materials can be made by chemical synthesis, fermentation, enzymatic reactions, recombinant DNA, recovery from natural materials, or a combination of the above. 

CE has many separation modes that are beneficial to protein impurity analysis. Within the many thousands of potential protein impurities in a recombinant product there will be several that have only minor physicochemical differences from the drug product. The application of different CE modes can potentially resolve these impurities. CE methods can be divided into four principle modes that are applicable to recombinant protein impurity analysis capillary zone electrophoresis, capillary isoelectric focusing, capillary gel electrophoresis, and micellar electrokinetic capillary chromatography. Each mode will be discussed briefly. Since the technology is so young and still very exploratory, CE methods are developed empirically for specific separations. It is difficult to provide standard protocols for CE impurity analysis. Instead, protocols that can be used as a starting point for impurity analysis will be provided as well as the citation of examples of impurity analyses from the literature to provide additional sources of protocols for interested readers. 

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