Determining Protein Part 1: Methodology

Determining Protein Part 1: Methodology Determining protein Nadine Ritter and John McEntire concentration is one of the most important analytical methods hether a product consists of crude nucleic acids, cell culture media containing dyes used in the discovery, development, and tissue homogenates, semipurified or surfactants, and formulations with high con- manufacturing of biological fluids, or highly purified centrations of reducing agents, detergents, or salts protein products. recombinant proteins, the amount may each require different types of protein con- Part 1 discusses a of protein in a given amount of centration analysis to obtain accurate results in number of methods for sample is valuable information. the presence of interfering matrix components. analyzing samples Protein concentration is analyzed to The amount of sample available for testing ranging from complex Wmonitor the amount of material recovered at each (both volume and concentration) is also a driving mixtures to highly step in a purification process and to determine the purified proteins. factor in selecting the appropriate method. Pro- appropriate quantity of material to use in subse- tein assays can vary significantly in their mini- quent process steps. In biopharmaceutical pro- mum required volume, their linear working duction, determining protein concentration is one range, and their ability to measure highly diluted way to measure the manufacturing consistency of samples accurately. There is often a trade-off be- each batch or the stability of a protein solution tween sensitivity and accuracy, although vendors over time. Protein concentration values are also of commercial protein assay kits are working to needed when preparing test samples for analytical improve both. methods to assess product purity, identity, and Matrix components can significantly affect potency. Because of its wide-ranging use in oper- protein concentration determination. Therefore, ations from discovery and development through with most protein concentration assays you commercial manufacturing, the measurement of should analyze the sample buffer alone to evalu- protein concentration is one of the most useful ate and correct for background signals. If the and significant test methods performed on interference is too great, then either remove or di- biotechnology products. In Part 1 of this discus- lute the matrix or select another assay. Even sion, we look at a number of methods for deter- when no individual components interfere, the mining protein concentration. synergistic effect of some substance combina- The nature of the protein preparation to be tions can create assay artifacts. When in doubt, it tested is a critical factor in the selection of an ap- is always wise to confirm the effects of the spe- propriate concentration determination method. cific buffer component or components at the con- Some methods, such as colorimetric assays, can centrations expected for the product with the pro- be used with both crude and purified protein solu- tein concentration assay chosen. tions to determine a total protein concentration. Others, such as UV absorbance, require solutions COLORIMETRIC METHODS of relatively pure protein for accurate results. Colorimetric assays are commonly used in bio- Unless methods are chosen that can isolate the molecular laboratories for determining protein signal specific for a single protein, heterogeneous concentration because the procedures and their in- protein mixtures will generate a combined re- strumentation requirements are simple. In such as- sponse from the proteins present. Solutions says, the protein is reacted with one or more chro- containing host cell elements such as lipids or mogenic substances to yield a detectable signal. 12 BioPharm APRIL 2002 Two basic forms of colorimetric assays are used. in data from a standard curve. Generally, a stock The first form involves reactions between the pro- solution of the standard protein is prepared and tein and chemicals in solution that yield colored, its concentration accurately determined (by fluorescent, or chemiluminescent products. In the amino acid analysis or microanalytical total second form, a colored dye is bound to the protein nitrogen, for example). and an absorbance shift is generated. Within the For long-term use (and when validating a col- linear working range of both types of assays, the orimetric assay), it is important to consider the intensity of the color quantitatively correlates to stability of the standards under their intended the amount of protein present. storage conditions. If a stock solution of the stan- These methods are well established, proven dard shows precipitation or microbial growth with many different protein preparations, and rel- upon storage, its protein concentration will no atively easy to use. When optimized for specific longer be as expected. Assays performed with the applications, most are precise and reproducible. solution will not be accurate. To increase confi- However, their disadvantages include limited dence in the stock standard solution concentration sensitivity at very low ranges (for example, accuracy over time, perform stability tests and es- below 1 µg/mL), interferences from buffer com- tablish appropriate storage conditions. ponents, unstable chromogenic reaction products, and potential inaccuracies if a protein standard does not react similarly to the test protein. Although the method Standards. The quantitation of these assays is instructions or based on the preparation of a standard curve with known concentrations of standards. Standards can literature references be nonproduct surrogate proteins (such as bovine Buffers and reagents. In addition to stable stan- may give the linear serum albumin, BSA) or a well-characterized dard solutions, accurate assay results use the reference standard of the product itself. A set of same type of buffer in preparing the standard working range of x,y data points is generated from the known stan- curve dilutions as in the test sample. If the con- dard concentrations and their corresponding centration of buffer components affects a colori- most colorimetric absorbance readings, and a best-fit regression line metric assay, adjust the standards to contain the assays, it should be is drawn through them. The test protein same concentration of buffer constituents across absorbance readings are plotted against this best- the dilution range of the standard curve. For some verified for specific fit line, and the concentration results are calcu- colorimetric assays (such as the Lowry method), applications. lated by linear regression analysis. chromogenic reactions are a function of time. Because protein standards drive the quantita- Therefore, the timing of the reaction incubations tive results, the accuracy of colorimetric assays for individual assay tubes can be critical to depends on the validity of the standard curve. obtaining accurate and precise protein concentra- Key elements in preparing accurate and precise tion results. You may have to coordinate the ad- standard curves include the protein chosen as the dition of reagents to ensure that each vial is incu- known standard (either a surrogate or a reference bated for the same length of time at the same protein), the preparation of the standard curve di- temperature before measuring its absorbance lutions, establishment of the assay’s linear work- reading. ing range, and the stability of the stock standard The effect of reagents on spectrophotometric solution. cuvettes used for holding assay solutions can af- Not all proteins react similarly with the vari- fect precision. Reagents such as colloidal gold ous chromogenic reagents used in colorimetric (Aurodye) or Coomassie blue dye can bind to assays. For the most accurate results, use a some cuvettes, causing a drift in absorbance read- protein standard that will react as closely as pos- ings from the first to the last sample. Also, the sible to the protein or proteins being tested. use of glass rather than plastic cuvettes can affect Ideally, a highly purified solution of the protein assay results. Procedures for cleaning quartz or being tested will provide the most accurate stan- glass cuvettes between samples must be adequate dard curve values because it will react in the to prevent sample carry over. When using dispos- assay exactly as will the test article. When a well- able plastic cuvettes, manufacturing quality and characterized product reference standard is consistency from lot to lot are critical for obtain- unavailable, a representative protein such as BSA ing reliable data. can be used as a surrogate standard. Procedures. Although the method instructions In either case, you must know the exact or literature references may give the linear work- concentration of the standard to have confidence ing range of most colorimetric assays, it should BioPharm APRIL 2002 13 Colorimetric Assays We describe here six colorimetric protein assay’s working range is 0.01–1 mg/mL. chloroauric acid (Aurodye), which is highly concentration assays that may be used with Color reaction development is time- sensitive to protein. The working range of biopharmaceutical products. For additional dependent and light-sensitive and can vary this assay is 20–200 ng/mL. The reaction colorimetric assays, refer to Thorne (1). Many significantly from protein to protein. In occurs in 30 minutes, and the color is stable of these assays are available as commercial addition, numerous buffer components can for up to 24 hours.

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