Biopharmaceutical proteins methods

As well as overcoming many of the inherent problems associated with agriculture, plant tissue culture also offers a number of advantages over conventional animal cell culture methods currently being applied to produce biopharmaceutical proteins commercially [8], As plant culture media are relatively simple in composition and do not contain proteins, the cost of the process raw materials is reduced and protein recovery from the medium is easier and cheaper compared with animal cell culture. In addition, as most plant pathogens are unable to infect humans, the risk of pathogenic infections being transferred from the cell culture via the product is also substantially reduced. 

Pharmaceutical companies are increasingly interested in developing products based on proteins, enzymes, and peptides. With the development of such products comes the need for methods to evaluate the purity and structural nature of these biopharmaceuticals. Proteins, unlike traditional pharmaceutical entities, rely on a specific secondary structure for efficacy. Methods to monitor the secondary structure of pharmaceutically active proteins, thus, is necessary. Infrared spectroscopy provides a way to study these compounds quickly and easily. Byler et al. (65) used second-derivative IR to assess the purity and structural integrity of porcine pancreatic elastase. Seven different lyophilized samples of porcine pancreatic elastase were dissolved in D20, placed in demountable cells with CaF2 windows, and IR spectra obtained. The second derivatives of the spectra were calculated and the spectral features due to residual water vapor and D20 removed. 

Dictyostelium discoideum cells can be transformed with plasmid DNA by convenient methods. The first attempts at this, using the calcium phosphate method, were reported in the early 1980s [17, 18]. Protocols were subsequently optimized by Firtel and coworkers [19, 20] and modified in many laboratories (e.g. Ref [21]). Up to 2000 transformants can be recovered from 10 D. discoideum cells [20], with electroporation protocols having been reported [22, 23]. It has also been noted that the choice of transformation method can strongly influence the yield of heterolo-gously expressed proteins [24]. Hence, both calcium co-precipitation and electroporation should be compared to generate optimized expression strains for the production of biopharmaceutical proteins in D. discoideum. 

Many potential degradation products are not observed in protein pharmaceuticals, primarily because much care is taken in the choice of formulations, lyophilization, and storage conditions in order to maintain protein stability. Thus, degradation is minimized and usable shelf lives are on the order of years. In order to study the degradation pathways of a biopharmaceutical protein, and to evaluate the stability-indicating ability of the analytical methods, it is sometimes necessary to perform forced degradation studies, where the biopharmaceutical protein is subjected to a variety of stress conditions, such as varying pH, elevated temperature, or the addition of oxidants. 

Recent developments in drug discovery and drug development spurred the need for novel analytical techniques and methods. In the last decade, the biopharmaceutical industry set the pace for this demand. The nature of the industry required that novel techniques should be simple, easily applicable, and of high resolution and sensitivity. It was also required that the techniques give information about the composition, structure, purity, and stability of drug candidates. Biopharmaceuticals represent a wide variety of chemically different compounds, including small organic molecules, nucleic acids and their derivatives, and peptides and proteins. 

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. 

Table 7.2 Methods used to characterize (protein-based) finished product biopharmaceuticals. An overview of most of these methods is presented over the next several sections of this chapter...

Tim Wehr is Staff Scientist at Bio-Rad Laboratories in Hercules, California. He has more than 20 years of experience in biomolecule separations, including development of HPLC and capillary electrophoresis methods and instrumentation for separation of proteins, peptides, amino acids, and nucleic acids. He has also worked on development and validation of LC-MS methods for small molecules and biopharmaceuticals. He holds a B.S. degree from Whitman College, Walla Walla, Washington, and earned his Ph.D. from Oregon State University in Corvallis. 

The objective of this book is to provide both an overview and practical uses of the techniques available to analytical scientists involved in the development and application of methods for protein-based biopharmaceutical drugs. The emphasis is on considering the analytical method in terms of the stage of the development process and its appropriateness for the intended application. The availability of techniques will reveal whether or not the analytical problem has a potential solution. Then will come the question of whether or not the technique is a truly appropriate solution. The theoretical considerations behind choosing the technique may be solid. However, the practicality of the method may not hold up to inspection. 

If analytical methods are at the heart of biopharmaceutical development and manufacturing, then protein concentration methods are the workhorse assays. A time and motion study of the discovery, development, and manufacture of a protein-based product would probably confirm the most frequently performed assay to be protein concentration. In the 1940s Oliver H. Lowry developed the Lowry method while attempting to detect miniscule amounts of substances in blood. In 1951 his method was published in the Journal of Biological Chemistry. In 1996 the Institute for Scientific Information (ISI) reported that this article had been cited almost a quarter of a million times, making it the most cited research article in history. This statistic reveals the ubiquity of protein measurement assays and the resilience of an assay developed over 60 years ago. The Lowry method remains one of the most popular colorimetric protein assays in biopharmaceutical development, although many alternative assays now exist. 

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... 

Fortunately, protein concentration methods are relatively simple (low-tech) and inexpensive. The simplest assays require only a spectrophotometer calibrated for wavelength and absorbance accuracy, basic laboratory supplies, and good pipetting techniques. Protein concentration assays are quite sensitive, especially given the typical detection limits required for most biopharmaceuticals. 

Is there a component in your protein solution that will interfere with your chosen protein quantitation method Many methods will provide misleading results in the presence of standard biopharmaceutical reagents such as detergents, chao-tropes, bases, or reductants. It is imperative to determine if excipients present in the sample interfere with the chemistry of the protein quantitation method. 

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. 

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