Biopharmaceutical proteins concentration

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. 

Cassette Cassette products are widely used in the biopharmaceutical industry and specifically dominate the protein concentration purification market due to their compactness, which provides excellent product recovery capabUity. Cassette modules contain presealed flat sheet membranes separated by feed and filtrate spacers. This is an improved design compared to earher plate and frame configurations, making installation and replacement much easier and more reliable for the end user (shown in Figure 14.5). 

Spectrophotometric analyses are the most common method to characterize proteins. TTie use of ultraviolet-visible (UV-VIS) spectroscopy is t rpically used for the determination of protein concentration by using either a dye-binding assay (e.g., the Bradford or Lowry method) or by determining the absorption of a solution of protein at one or more wavelengths in the near UVregion (260-280 nm). Another spectroscopic method used in the early-phase characterization of biopharmaceuticals is CD. 

Interfaces between two separate phases, such as air/water, oil/water, and solid/ water, are potential adsorption sites of biopharmaceuticals. Adsorption often involves simple diffusion of surface-active solute molecules in the bulk to the interface, and hence the rate of adsorption is generally dependent on the solute concentration. At saturation, a close packed monolayer of protein molecules corresponding to 0.1 to 0.5pg/cm is normally formed at the interface [122], and this adsorption behavior is of particular concern for high-potency therapeutic proteins. However, certain proteins do not conform to such saturation-limited adsorption behavior and tend to show increased adsorption with increasing protein concentration, attaining a local protein concentration at the interface 1000 times higher than the initial concentration in the bulk solution [123]. 

Membrane-retained components are collectively called concentrate or retentate. Materials permeating the membrane are called filtrate, ultrafiltrate, or permeate. It is the objective of ultrafiltration to recover or concentrate particular species in the retentate (eg, latex concentration, pigment recovery, protein recovery from cheese and casein wheys, and concentration of proteins for biopharmaceuticals) or to produce a purified permeate (eg, sewage treatment, production of sterile water or antibiotics, etc). Diafiltration is a specific ultrafiltration process in which the retentate is further purified or the permeable sohds are extracted further by the addition of water or, in the case of proteins, buffer to the retentate. 

The freeze-drying process is initiated by the freezing of the biopharmaceutical product in its final product containers. As the temperature is decreased, ice crystals begin to form and grow. This results in an effective concentration of all the solutes present in the remaining liquid phase, including the protein and all added excipients. For example, the concentration of salts may increase to... 

The study of reaction rates or kinetics of a particular denaturation process of a protein therapeutic can provide valuable information about the mechanism, i.e., the sequence of steps that occur in the transformation of the protein to chemically or conformationally denatured products. The kinetics tell something about the manner in which the rate is influenced by such factors as concentration, temperature, excipients, and the nature of the solvent as it pertains to properties of protein stability. The principal application of this information in the biopharmaceutical setting is to predict how long a given biologic will remain adequately stable. 

Many biopharmaceutical preparations are heterogeneous and may be difficult to fully characterise. Certain fractions of a preparation may have different biological activity or kinetics than the intended product. It is important that such fractions are appropriately qualified. The proportions of these fractions may be altered when production changes are made or they may be different between similar products produced by different manufacturers. Because of their proteinaceous nature and their novel mechanisms of action, all preclinical and clinical development steps must be re-evaluated. For pharmacokinetic studies, blood concentrations should be measured by specific analytical techniques (most often ELISA), which quantify the active protein and not one of its fragments or inactive forms, such as antigen-antibody complexes. For PK-PD studies of monoclonal antibodies, relevant biomarkers are most often circu-... 

Because most proteins are susceptible to protease degradation and denaturation in biologic fluids, most biopharmaceuticals must be administered by intravenous, intramuscular, or subcutaneous injection (see Table 5.5). High concentrations of proteases are found in the gastrointestinal tract, nasal mucosa, bronchioles, and alveoli, which severely limit the bioavailability of protein pharmaceuticals after oral, intranasal, and inhalation administration. Diffusional barriers to the passage of relatively large macromolecules preclude transdermal and mucosal administration of protein pharmaceuticals. Research is under way to develop methods that will protect protein drugs from proteolysis and improve transmembrane diffusion. 

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. 

Biopharmaceuticals may be subjected to heat treatment as a means of viral inactivation. Protein damage is prevented by the addition of high concentrations of a thermostabilizing excipient such as sugars or polyhyd-ric alcohols. The non-reducing polyhydric additives increase thermostability of the protein by the formation of a hydrogen-bonded solvent shell. The typical treatment for factor VIII consists of heat treatment at 60°C for... 

In tangential filtration, membranes are used as filter media. Membranes are defined as barriers of reduced thickness, across which physical and/or chemical gradients are established to facilitate the preferential migration of one or more components from a given mixture, promoting their separation (Klein, 1991). They are usually made of polymers or inorganic materials, such as ceramic or sintered steel. In the biopharmaceutical industry, membranes find various applications, such as production of water for injection (WFI), sterilization of culture media, buffer solutions and gases, separation of cells and cell debris, and purification and concentration of proteins. 

Protein engineering can also be used for creating fusion proteins or hybrid biopharmaceuticals. An example of this type of product is Enbrel (Etanercept), which consists of the extracellular ligand-binding domain of the 75-kDa receptor for tumor necrosis factor-alpha (TNFa) and the Fc portion of human IgGl. TNF is a proinflammatory cytokine present at high concentrations in patients suffering from rheumatoid arthritis. Enbrel acts as competitive inhibitor of TNF, as it competes with the receptor molecules (TNFR) present in the cell. The antibody portion of the fusion protein increases the half-life in blood (Walsh, 2004). 

ADME Since metabolism and formation of active metabolites are not a concern for unmodified biopharmaceuticals, mass balance studies are uninformative. Tissue concentration of radioactivity using radioactive proteins is also difficult to interpret due to unstable radiolabel linkage, rapid in vivo catabolism, and recycling of radiolabeled amino acids into non-drug-related proteins/peptides. 

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