Biopharmaceutical proteins selection

With the advent of biopharmaceutical products, selection of container/closure systems has become even more critical. Frequently lyophilized products are more sensitive compared with traditionally derived pharmaceutical products. For instance, protein and peptide-based products may have a greater tendency to adsorb to the surface of their container/ closure system. Additionally, these products are typically smaller in final cake weight therefore, they are much more sensitive to moistiu-e. In any case, the performance of the lyophiiization process and the quality of the final product can be direetly related to the container/closure system. 

More recently, increasing research attention has focused upon the use of mucoadhe-sive delivery systems in which the biopharmaceutical is formulated with/encapsulated in molecules that interact with the intestinal mucosa membranes. The strategy is obviously to retain the drug at the absorbing surface for a prolonged period. Non-specific (charge-based) interactions can be achieved by the use of polyacrylic acid, whereas more biospecihc interactions are achieved by using selected lectins or bacterial adhesion proteins. Despite intensive efforts, however, the successful delivery of biopharmaceuticals via the oral route remains some way off. 

As described in the following chapter, there are many biopharmaceutical applications of protein assays. Assigning the protein concentration for the drug substance, drug product, or in-process sample is often the first task for subsequent analytical procedures because assays for purity, potency, or identity require that the protein concentration be known. Hence it is typical for several different methods to be employed under the umbrella of protein concentration measurement, depending on the requirements of speed, selectivity, or throughput. The protein concentration is valuable as a stand-alone measurement for QC and stability of a protein. However, protein concentration methods provide no valuable... 

The MS techniques described previously for characterization of the final recombinant protein product can be applied at all stages during process development. MS might be used upstream to define clone selection, processing format, and purification steps, and downstream to characterize the final product, ascertain lotto-lot reproducibility, determine stability, and define the formulation of biopharmaceutical molecules. Presented here are some examples found either in the literature or from our own experience in which MS has been found to be a useful or necessary tool. Potential limitations of MS methods are discussed, and when appropriate, other analytical methods are mentioned that can be alternatives to MS and are also efficient tools for biopharmaceutical development. 

The first step in biopharmaceutical development is the selection of a clone in a specific cell line. Whole-mass analysis, if possible, is a fairly simple and powerful tool at this stage to verify the successful expression and translation of the desired protein. VanAdrichem et al.65 described the use of MALDI MS to monitor protein expression in several mammalian cell lines like CHO DXB11, CHO SSF3, and hybridomas. Quantitative MALDI-TOF MS measurements of an IgG antibody and insulin during large-scale production in hybridoma cells were comparable to affinity chromatography results. 

The ELISA can be used for identification and quantitation of the protein product (biopharmaceutical) of interest throughout the development, production, and manufacturing process. For example, in the initial development phase, ELISAs can aid in the selection of the best cell line. In the early manufacturing steps, it can be used to identify the appropriate product-containing pools or fractions in process to be subjected to further purification. Because of the selectivity of ELISA, it is a suitable tool to select out the protein of interest from complex protein mixtures, such as cell culture fermentation media or product pools in early steps of protein recovery as well as downstream processing. Even complex mixtures do not require much sample preparation. It is important to determine... 

Biopharmaceuticals are becoming increasingly important. The reason is that they are more potent and specific, as they are similar to the proteins within the body and hence are more effective in treating our diseases. There are three major areas in which biopharmaceuticals are used as prophylactic (preventive, as in the case of vaccines), therapeutic (antibodies), and replacement (hormones, growth factors) therapy. Exhibit 4.1 presents selected statistics for biopharmaceuticals. 

The production of heterologous proteins for therapeutic use requires selection of the producer cell line, based on yield, monoclonality (for proteins), product quality, stability, and absence of contaminants like bacteria, molds, mycoplasmas, and viruses. Progress in the production of biopharmaceuticals by cell culture is due mainly to the use of diploid cells and continuous cell lines, together with the maintenance of cells by cryo-preservation. It is important to guarantee that the expression system chosen is able to generate the product in a consistent and economically feasible way (Levine and Castillo, 1999). 

This chapter will discuss various experimental approaches used to select the relevant species for conduct of toxicology studies for biopharmaceuticals, as well as highlight advances made in scientific approaches and technologies to facilitate this process. Methods discussed include the traditional immunohisto-chemistry and tissue cross-reactivity studies, flow cytometry, protein sequencing, and functional in vitro assays, as well as newer approaches such as utilization of microarray databases for genomic mRNA expression data and use of transcript profiling studies as an adjunct to functional assays, to understand similarity in pharmacological responsiveness between animals and humans. 

Among the most highly selective cytokine biopharmaceuticals are the interferons. Interferons are members of a large family of related proteins that may be divided into two categories type I and type II interferons. Type I interferons (i.e., a- and P-interferon) possess antiviral and anti-proliferative properties, whereas type II interferons (i.e., y-interferon) have immunostimulatory activity. Several animal species were examined for their responsiveness to interferons, and with the exception of nonhuman primates, all tested animal species were found to be unresponsive [15]. 

---