This installment of "Biotechnology Today" focuses on developing a rapid method for reversed-phase high performance liquid
chromatography (HPLC) analysis of granulocyte colony stimulating factor (GCSF) purity for process analytical technology (PAT)
applications. It also highlights an approach that one could take to achieve faster HPLC analysis.
Progress in recombinant techniques, from many years of successful research, has led to the production of substantial amounts
of novel protein- and peptide-based therapeutics. The evaluation of product quality (that is, identity, content, and purity)
has a major role in the manufacturing process of biopharmaceuticals (1). The biotech production process itself usually shows
high variability, which results in variability in product quality and, at times, lot failure. The heterogeneity that is typical
in biotech therapeutics requires thorough characterization using multiple orthogonal techniques (2,3). This thorough characterization
is a requirement for receiving regulatory approval to commercialize the product.
Of the many tools that are used, high performance liquid chromatography (HPLC) is the workhorse for analysis of biopharmaceutical
proteins. The significant advantages that HPLC offers include high reproducibility, high sample throughput because of autosampling
capabilities, high resolution of separation, and easy quantitation, high precision, and high robustness. Typical HPLC methods
are 30–60 min long and are thus not amenable to high-throughput analysis. The focus of HPLC development in recent decades
has been on reducing the time of analysis. Chromatographers have applied many different approaches to achieve this goal. Short
columns with high flow velocities (4), high temperature (5–8), reduced particle size of packing (9), and ultrahigh pressure
(10–13) are some of the approaches that have been used in both academic and industrial attempts to improve the speed of separation.
The challenge is to reduce the separation time without significantly sacrificing the column efficiency or resolution. Simply
shortening the column or increasing the mobile-phase velocity can result in shorter analysis time, but at the cost of reduced
analyte retention time and lower efficiency.
Monolithic columns offer fast separations without compromising efficiency or resolution (14,15). The high permeability and
large number of theoretical plates per unit pressure drop associated with monoliths are because of their most important and
distinguishing physical features such as large through-pore size and skeleton ratios and high porosities (13). The short diffusion
path length and high porosity supplied by the large through-pores not only lower the plate height, but also decrease the hydraulic
resistance of the mobile-phase flow, thus reducing the pressure drop. The lower pressure drop permits operation at high flow
rates on relatively long columns using a conventional HPLC system, but is not achievable with conventional packed columns.
The efficiency of these monolithic columns to perform faster analysis is a necessary step in using HPLC as a process analytical
technology (PAT) tool (16–19).
Lately, PAT has been receiving a lot of interest within the biopharmaceutical community because of the potential for continuous
real-time quality assurance, resulting in improved operational control and compliance. It has been defined as a system for
designing, analyzing, and controlling manufacturing through timely measurements (that is, during processing) of critical quality
and performance attributes of raw and in-process materials and processes, with the goal of ensuring final product quality
(20,21). The concept involves using on-line, at-line, or off-line analysis of critical quality attributes and process parameters
to facilitate real-time decision making based on the measurement of critical quality attributes rather than surrogate measurements
(22). The objective for PAT implementation could thus be one or more of the following (2,23):
- Better process understanding
- Improved yield because of prevention of the scrap, rejects, and reprocessing
- Reduction in the production cycle time by using on-line, at-line, or off-line measurements and control
- Decrease in the energy consumption and improved efficiency from conversion of the batch process into a continuous process
- Cost reduction because of reduced waste and reduced energy consumption
- Real-time release of the batches
A series of case studies has recently been published examining the use of on-line HPLC as a PAT tool, at pilot and manufacturing
scales, for performing analyses to facilitate real-time decisions for column pooling based on product quality attributes (16).
HPLC has also been proposed for the monitoring of protein purity in a refolding step to enable the timely ending of the step
on the basis of product quality data (24). This column installment focuses on developing a rapid method for reversed-phase
HPLC analysis of granulocyte colony stimulating factor (GCSF) purity for such PAT applications. It also highlights the approach
that one could take to achieve faster HPLC analysis.