Host-cell proteins biopharmaceutical

Several key issues have to be addressed in the downstream processing of biopharmaceuticals regardless of the expression system. The removal of host cell proteins and nucleic acids, as well as other product- or process-related or adventitious contaminants, is laid down in the regulations and will not differ between the individual expression hosts. The identity, activity and stability of the end product has to be demonstrated regardless of the production system. The need for pharmaceutical quality assurance, validation of processes, analytical methods and cleaning procedures are essentially the same. 

This part will mainly focus on host cell proteins (HCPs) quantification. Process-specific HCP assays are in general targeted to be in place prior to the initiation of phase III clinical trials. Immunoassays are the most specific and sensitive techniques available for detecting and quantifying protein impurities. There are two methods commonly employed to quantify protein impurities in biopharmaceuticals enzyme-linked immunosorbent assays (ELISA) and immunoligand assays (ILA). Both methods are able to detect very low ppm level of impurities. ELISA have been developed to measure host protein impurities in a number of recombinant proteins including human growth hormone (Anicetti et ah, 1986), insulin (Baker et al, 1981) and staphylokinase (Wan et ah, 2002). ILA assays have been used to detect protein impurities in recombinant bovine somatotropin (Whitmire and Eaton,... 

Shukla, A. A., Jiang, C., Ma, J., Rubacha, M., Flansburg, L. and Lee, S. S. Demonstration of robust host-cell protein clearance in biopharmaceutical downstream process. Biotechnol. Prog. 24 615-622, 2008. 

Of all the possible contaminants and impurities of a biopharmaceutical product, organisms (bacteria, virus, mycoplasma) and their products (DNA, endotoxin, host protein), media components, and raw materials, it is most appropriate to use an ELISA for the HCP impurities and some of the process residuals (media components and raw materials). Impurities from media components are known or expected unlike those from the host cell. 

Eaton, L.C. (1995). Host cell contaminant protein assay development for recombinant biopharmaceuticals. J Chromatogr A 705 105-114. 

Most smaU-molecule drugs are produced by direct cheanical synthesis. Multiple steps can be involved as various intermediate molecules are created on the pathway to eventually synthesizing the desired molecnle. In contrast, larger-scale manufacture of biopharmaceuticals involves the employment of host cells to produce proteins that are safe and effective (Ho and Gibaldi 2003). The choice to use prokaryote host cells (prokaryotes are microorganisms with no distinct membrane-bound nucleus), lower eukaryote cells, or higher eukaryote (mammalian) cells, which becomes progressively more expensive in the order just listed, is predicated on what kind of host cell is needed to carry out necessary posttranslational modifications. 

Although numerous cell lines have been screened for their efficiency as a host system for recombinant protein production, only a few have shown favorable properties for the expression of biopharmaceuticals (Hauser, 1997). Regulatory and economic issues for large-scale production and the intended application of the recombinant protein (diagnosis, therapy, etc.) have to be carefully considered (Makrides and Prentice, 2003). Three mammalian cell lines are now commonly used by the pharmaceutical industry Chinese hamster ovary (CHO) cells, the murine myeloma SP2/0 and the NS0 cell line (see Table 3.1). These cell lines have been used to produce 11 of 21 therapeutic products approved from 1996 to 2000 (Chu and Robinson, 2001). 

Transgenic plants might also be used for production of recombinant proteins. While many of these systems are still early in the development stage, plants offer very robust and high-capacity system for biopharmaceutical production. Since plants cannot always properly modify proteins as mammalian cells, their utility may be limited. However, early studies suggest that plants may serve as useful hosts for production of vaccines and antibodies. For example, HepB surface antigen has been successfully expressed in potatoes [11], and clinical trials are underway with secretory antibodies (SIgAs), such as CaroRx , developed in plants [12]. 

Today, recombinant protein production involves many options. In addition to E. coli, several yeast systems (see Part IV, Chapter 13), insect cells (see Part IV, Chapter 14), different mammalian expression systems (CHO, BHK, NSO, HKBll, PER.C6) (see Part II, Chapter 3 and Part IV, Chapters 1 and 3) other alternative expression systems are currently under development for the production of biopharmaceuticals. These include transgenic animals or plants, and will be discussed in Part IV, Sub-Part 2 of this book. This chapter will focus on E. coli, a still-modern secretory Saccharomyces ccrevisiac system, and the recently developed mammalian HKBll expression system. An E. coli host/vector system is described that was originally developed for the efficient production of an interleukin-4 variant Later, it transpired that this system is ideally suited to the expression of other proteins and Fab fragments. The secretory... 

In the next contribution we learn about hands-on experience and recent improvements with different production systems for biopharmaceuticals at Bayer Health-Care. As previously also published in Nature by Heiner Apeler, Head of Expression, an E. coli host/vector system was originally developed for the efficient production of an interleukin-4 variant, but afterwards it was optimized for the expression of other proteins and even Fab fragments. Process development and optimization of the yeast secretory Saccharomyces cerevisiae for expression of a protease inhibitor will also be presented. The focus, however, is on the use of a recently developed mammalian HKBll (hybrid clone of human kidney and B cells) expression system for recombinant human glycoprotein biopharmaceuticals. HKBll is a favorable cell host for the production of human proteins, because it dehvers biopharmaceuticals that are structurally identical to the natural product. The host/vector system supports the production of gram quantities of proteins in a large-scale transient transfection format as well as the development of stable cell fines. These systems together... 

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