Production of proteins of consistent quality in heterologous, genetically-engineered expression systems is dependent upon identifying the manufacturing process parameters which have an impact on product structure, function, or purity, validating acceptable ranges for these variables, and performing the manufacturing process as specified. One of the factors which may affect product consistency is genetic instability of the primary product sequence, as well as instability of genes which code for proteins responsible for post-translational modification of the product. Approaches have been developed for mammalian expression systems to assure that product quality is not changing through mechanisms of genetic instability. Sensitive protein analytical methods, particularly peptide mapping, are used to evaluate product structure directly, and are more sensitive in detecting genetic instability than is direct genetic analysis by nucleotide sequencing of the recombinant gene or mRNA. These methods are being employed to demonstrate that the manufacturing process consistently yields a product of defined structure from cells cultured through the range of cell ages used in the manufacturing process and well beyond the maximum cell age defined for the process. The combination of well designed validation studies which demonstrate consistent product quality as a function of cell age, and rigorous quality control of every product lot by sensitive protein analytical methods provide the necessary assurance that product structure is not being altered through mechanisms of mutation and selection.

Less than a decade ago, the use of continuous mammalian cell lines for the production of cloned proteins was considered strictly a research tool. At that time, few thought it possible to allay the many safety concerns associated with transformed cells. It soon became clear that mammalian expression systems had numerous advantages over bacteria for production of therapeutic proteins, initiating a multidisciplinary effort to address these concerns in a thorough and reliable manner. The success of these efforts is exemplified by the emergence of product molecules into the market. Today, there are seven recombinant human therapeutics that have received FDA approval. Almost half of them (OKT3, t-PA, and EPO) are produced in mammalian cells, with the remainder produced in bacteria (insulin, growth hormone, and alpha-interferon) or yeast (hepatitis vaccine). At least a dozen more recombinant cell culture products are in advanced human clinical trials. With the accumulation of data and experience, continuous mammalian cell lines will no doubt be the preferred hosts for many future products of biotechnology.

Principles of process validation are extremely powerful tools in assurance of product quality. They are especially useful for reducing those risks not easily measured routinely during production. When combined with effective process and facility design principles, characterization of cell banks and products, appropriate lot release tests, and adherence to cGMP, safe cell culture biologicals can be prepared in a reliable manner.

Recombinant chicken GH (rcGH) was produced and characterized. Comparison of protein sequence, amino acid composition, mol wt, purity, and immunocross-reactivity showed that except for the N-terminal methionyl group arising from the bacterial expression system, the recombinant and pituitary-derived cGHs were identical. When tested in a hypophysectomized rat growth assay, the recombinant and pituitary materials had the same specific bioactivity. Within 60 min after sc injection of rcGH (480-960 micrograms/kg) in chickens, plasma GH levels increased 4- to 6-fold and remained significantly elevated for at least 5 h. Thrice-daily injections from age 2-24 days had little effect on growth or feed consumption in either male or female broiler chicks. Plasma levels of insulin and triglycerides were significantly elevated by rcGH in 24-day-old females, but not in males. Injection of rcGH counteracted a reduction of tibia length observed in saline-injected controls. The rcGH had no effect on carcass protein, ash content, or nitrogen retention. It is important to note that exogenous GH can be a productivity-enhancing factor in other commercially important species. Administration of bovine GH to cows has been shown to induce a significant increase in milk production (28). This study shows that administration of rcGH to chickens can lead to some significant metabolic effects. However, it is the conclusion of this report that the level of circulating GH is not the limiting factor in the growth of this highly selected species.

A crucial aspect of hemostasis in mammals is the control of the formation and dissolution of a fibrin matrix. The enzymatic system in plasma for fibrin formation consists of more than a dozen proteases and cofactors that act in series (Jackson and Nemerson 1980). Localization of these reactions is achieved by the requirement for the phospholipid surface, which is supplied by activated platelets at the site of trauma. Regulation of fibrin formation is achieved through control of protease activation, the requirement for a phospholipid surface at certain steps of the cascade, and feedback activation and inactivation reactions involving coagulation factors, other plasma proteins, and cell-surface proteins. The resulting fibrin matrix serves to prevent unwanted blood loss at the site of an injury.

Publisher Summary This chapter discusses the development of a method for large-scale production of human immune interferon based on induction scheme with the addition of a cell fractionation process and a novel method for product recovery. A 10- to 15-fold enhancement in interferon yield and a 50-fold increase in purity of crude product were accomplished by nylon wool column fractionation of lymphocytes combined with simultaneous reduction of autologous plasma protein levels prior to induction. The production method described is suitable for large-scale preparation of natural human immune interferon. Its principal advantages include greater interferon yield and purity without substantial increase in process costs. The process provides a method for the production of both higher and more consistent yields of interferon. A major factor in the higher yield from nylon wool fractionated buffy coat cells is the prolongation of the active production period compared to unfractionated cell preparations. Higher purity without concomitant yield loss results from removal of about 70% of buffy coat plasma, a step which results in reduced yields when using unfractionated cells. This sequential application of nylon wool fractionation and protein removal steps combines to provide 50-fold improvement in initial purity, which leads to higher purification yields and a reduction in purification costs.