Analytical Tools for Process and Product Characterization - Select the best approach to determine critical quality attributes. - BioPharm International


Analytical Tools for Process and Product Characterization
Select the best approach to determine critical quality attributes.

BioPharm International
Volume 22, Issue 8


Chromatography columns and ultrafiltration membranes are commonly used during the downstream purification of therapeutic proteins or MAbs. Column or membrane cleaning is typically performed to remove any protein or impurity carryover from previous runs and to restore performance between campaigns.31,32 The effectiveness of cleaning is evaluated by measuring the low protein concentration (in μg/mL range) in a mock run following cleaning using the same elution or ultrafiltration buffers and conditions.

The most commonly used methods are classic colorimetry-based assays such as Bradford, BCA (bicinchoninic acid), and Lowry methods for total protein assay. However, a variety of buffer systems with different pH levels, components, and salt strengths are used in purification processes, and many of them are not compatible with these colorimetric assays. For example, BCA assays are not compatible with reducing sugars or chelating agents, and Bradford is not compatible with detergents above certain levels. Furthermore, the high-salt strengths and buffer capacity of certain in-process pools (e.g., ion exchange elution buffer) may precipitate assay reagents or dominate the pH of an assay mixture, making these pools incompatible with the assays. Interfering substances can be removed by sample preparation steps such as buffer exchange, blocking reagents for reducing agents, or protein precipitation with organic solvents. These sample preparation steps, however, can potentially introduce assay artifacts, especially at a very low level of protein concentrations. In addition, the response factor for the same protein in different buffer systems can vary significantly because of these interfering factors, making it necessary to make protein standards in different buffers for different column pools. This in turn makes the assay more time-consuming and labor-intensive. A simple, sensitive, and robust method that does not need sample preparation steps would be beneficial for the analysis of column or membrane cleaning samples.

We have developed a flow injection protein assay (FIPA) using a regular HPLC system with a fluorescence detector. The intrinsic fluorescence of tryptophan is used to monitor and quantitate protein concentration against a standard curve obtained from the same protein of interest spiked into Dulbecco's phosphate buffered saline (DPBS) containing 0.005% polysorbate solution. Samples are directly transferred into glass HPLC vials pre-coated with Sigmacote to prevent protein loss to the glass surface. Samples are then injected onto an HPLC system with no column connected, using DPBS as the isocratic mobile phase, which effectively prevents nonspecific interaction of low concentration protein analytes with instrument surfaces and minimizes run-to-run carryover to <0.5%. The flowthrough peak is monitored by a fluorescence detector (exc 280 nm, emi 335 nm) and integrated to obtain the peak area, which is compared against a standard curve to obtain assay results. The method has been shown to be linear in the range of 1 to 16 μg/mL of protein and no interference was observed from chelating reagents, reducing sugar, or detergents because of the selectivity of fluorescence detection. The limit of detection is observed to be about 0.5–1 μg/mL (depending on the sample matrix) and is equivalent to traditional colorimetric methods. Response factors of the same protein in different commonly used inorganic buffers are relatively constant. Intermediate precision of FIPA across laboratories was determined to be around 10% at the 6 μg/mL protein level and is comparable to traditional colorimetric methods.

FIPA can be used specifically to quantify the protein of interest. It is not a total protein assay and it requires that the protein of interest have tryptophan residues in its sequence. Wavelengths that are specific to tyrosine or phenylalanine also can be used for this purpose with decreased sensitivity because of the lower molar absorptivity and quantum yield of these two amino acids.

Table 2. Comparison of the flow injection protein assay with colorimetric methods commonly used to evaluate column and membrane cleaning
FIPA provides a simple, sensitive, and robust protein quantitation method to demonstrate the effectiveness of column or membrane cleaning. Compared with other methods (Table 2), FIPA is quicker and has the least matrix interference. Another significant operational advantage of FIPA is that a large set of samples can be analyzed quickly by HPLC without as much analyst hands-on time as needed with the other methods.

Brent S. Kendrick, PhD, is scientific director, Greg Chrimes is associate scientist, Steven L. Cockrill, PhD, is principal scientist, John P. Gabrielson, PhD, is senior scientist, Kelly K. Arthur is associate scientist, Brad D. Prater is senior associate scientist, Qiang Qin, PhD, is senior scientist, and Bing Zhang is senior associate scientist of Analytical Sciences, all at Amgen, Inc., Longmont, CO.

Anurag S. Rathore, PhD, is biotech CMC consultant and a faculty member at the Indian Institute of Delhi, India, 805.744.8986,
Rathore is also a member of BioPharm International's editorial advisory board.

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