Biopharmaceutical proteins assays

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... 

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

Tissue culture cells are being used for target identification and validation, for primary and secondary screening, and also to produce biopharmaceutical proteins (see Part IV, Chapter 1 and 3). To handle all currently used cellular assays is beyond the scope of this chapter, and we will focus here on cell-based assays that are used as cancer models, or which are relevant for cellular transformation or cell migration and invasiveness. We will also discuss their suitabil-... 

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. 

Currently, many biopharmaceuticals, which are proteins in many cases, are produced in many bioindustry fields, and the measuring of the concentrations and bioactivities of these products is thus becoming essential in bioindustry. We have added a new section for Biorecognition assay in Chapter 11, and we explain the fundamental aspects of biorecognition and its application for the measurement of bioproducts at low concentrations. In this edition, we have included some examples and some new problems to assist in the progress with learning how to solve problem. 

Assay performance criteria for biopharmaceuticals are often highly variable therefore strict statistical criteria that attempt to rigorously establish traditional in vivo bioequivalence may not always be appropriate. In some cases an assessment of rate and extent of absorption as indicated by the maximum concentration (Cmax), time of maximum concentration (Tmax) and area under the curve (AUC) may be needed. In other cases complicating factors related to binding proteins, endogenous concentration, and unusual concentration-time profiles may need to be considered [15]. In cases where complications may arise from immune response to heterologous proteins, cross-over designs are inappropriate. 

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. 

The homology of the macaque s (cynomolgus and rhesus) protein sequences to the human sequence was over 90%. In contrast, dog, rat, and mouse sequences shared low homology with the human FceRI-alpha protein. Based on these results, the receptor of nonhuman primates would be more likely to bind a biopharmaceutical designed to interact with the human FceRI-alpha chain. This conclusion, however, would have to be confirmed in additional in vitro binding assays. 

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