To understand the removal of impurities during biopharmaceutical manufacturing processes, analytical techniques such as high performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), and real-time-polymerase chain reaction (PCR) are required. Typically, the product-related impurities are analyzed by HPLC and process-related impurities are analyzed by real time-PCR for host-cell DNA and ELISA for host-cell proteins content. Here, we present work done to enable HPLC and real time-PCR analysis for use as process analytical technology tools.
Process analytical technology (PAT) is a system used for designing, analyzing, and controlling manufacturing through timely measurement (that is, during processing) of critical quality and performance attributes of raw and in-process materials and processes, with the goal of ensuring consistent product quality (1–5). It is important to understand that the goal of PAT is not only the use of these analytical techniques for monitoring, but also to control the manufacturing process to consistently yield the desired product quality (2,3).When performing process-scale chromatography, because of the high resolution separation being performed, it is often the case that baseline separation between the product and the impurities is not achieved. Typical industry practice in such cases involves fractionation of the product peak into multiple small fractions, analysis of the various fractions for purity, and pooling of the fractions as per preset purity-based pooling criteria (6–8). This practice, however, has its own share of deficiencies. Collection of fractions, sampling of fractions, holding the fractions until analysis is complete, and, finally, pooling of fractions may necessitate open operation and increases the vulnerability toward product contamination. Product degradation may occur while it is held in storage for the analysis of the fractions to be complete (often 10–20 h). High performance liquid chromatography (HPLC) and quantitative polymerase chain reaction (qPCR) are two of the most commonly used analytical methods for monitoring product-related impurities and host-cell nucleic acids, respectively. This article addresses how to use these tools in a manner that facilitates PAT implementation and results in an efficient and robust process.
The application under consideration uses process-scale chromatography (cycle time 2 h) to separate the product from a product-related impurity. Presently, the eluate from the column is collected as fractions (every 5 min) and stored until the analytical results are declared. The analysis time is 45 min, and it takes ~10 h for analysis. This long analysis time decreases the productivity of the manufacturing plant. HPLC is used in this case for the measurement of product purity.
Host-cell nucleic acid (DNA) is a critical quality attribute for biotech therapeutics. Near complete removal of DNA is necessary for achieving product approval (9–11). For robust operations, DNA is measured at the various stages of the process: the harvested broth, clarified harvest, affinity chromatography, anion-exchange chromatography, cation-exchange chromatography, and tangential flow filtration. The currently available analytical methods such as the threshold method, slot-blot hybridization, and qPCR require high analysis times (~9 h). Furthermore, presently each sample type requires a different sample preparation protocol, which is often tedious to perform.
In the following sections, we discuss the development of these two methods to enable us to monitor these critical quality attributes (CQA) with the same accuracy and precision as the existing methods, but at a significantly smaller analysis time to facilitate PAT implementation.