High-performance liquid chromatography (HPLC) has provided analytical support for biopharmaceutical research, development, and manufacturing for as long as biotech has existed. Only recently, though, have HPLC, its methods and associated instrumentation achieved the speed, reliability, and robustness sufficient to analyze process streams in real time or near-real time.
If process analytic technology (PAT) is not at the tip of your tongue, it should be. This FDA-inspired initiative for real-time, in-process analytics, has been slowly gaining traction thanks to steadfast efforts by instrument vendors and best-in-class biomanufacturers. HPLC will be part of PAT moving forward.
Another important factor in the emergence of process HPLC is quality by design (QbD), a concept, like PAT, borrowed from nondrug process and manufacturing industries. Under QbD, quality is designed-in as the process moves forward, rather than tested-in afterward by quality groups.
From Lab to Manufacturing Floor
The complex nature of biomolecules in their raw form and the realities of the manufacturing floor have both contributed to holding process HPLC back. At-line or in-line analytics must be rugged, close to plug and play, and utilize methods suitable for production settings. Seemingly, every technical paper published on bioprocess PAT notes the difficulties in transferring otherwise robust analytical platforms and methods from the ideal conditions of back-room laboratories to the center stage of biomanufacturing. “Production environments are different from analytical labs,” notes John Waraska, marketing manager at ESA Laboratories(www.esalabs.co.uk).
Problems arise from every angle. Conventional columns and pressures are too slow for some processors’ tastes, while methods that serve central laboratories or benchtop analysis are unsuitable for real-time analysis. Some detection methods, such as ultraviolet, may not provide the detail required for analyzing complex process streams; others, like mass detectors, are still too complex for general use at-line or in-line. “You don’t want a mass spec on the production floor at this point in time. They’re still too skill-intensive,” Waraska adds.
The idea of a universal detector therefore holds great appeal. The closest thing on the market is ESA’s Corona CAD® (charged aerosol detector), an HPLC detector that uses evaporative techniques and light scattering to quantify almost any analyte, including proteins, small molecules, nutrients, and counter-ions—every relevant component of a fermentation sample. During method development, the CAD may be configured to split the sample stream and send half to the CAD and half to a mass detector to validate the in-process CAD method.
Recently, scientists from Discovery Laboratories (www.discoverylabs.com) presented data on the analysis of complex mixtures of lipids and peptides using CAD. Discovery Labs’ Surfaxin® pulmonary surfactant, which is currently under FDA review, consists of a peptide, KL4, two phospholipids, and palmitic acid. Where currently marketed surfactants are animal-derived—and therefore complex both medically and compositionally—Surfaxin is synthetic.
Senior director of analytical services at Discovery Labs, Michelle DeCrosta, Ph.D., says that the synthetic approach leads to tighter control over product quality. “It’s more simplistic, the impurity profiles are predictable, and it’s reproducible.”
Simplicity notwithstanding, analyzing Surfaxin conventionally requires that one detector quantify the four active ingredients that belong to three distinct chemical classes. Ultraviolet detection is not quantitative for lipids, while fluorescence requires conjugation to a fluorophore and may require calculating response corrections. Consequently, at one time Discovery Laboratories used six separate HPLC methods to quantify the actives and key impurities in Surfaxin.
ESA’s CAD detector reduces analysis to a single injection and elution in about 35 minutes. Not ultrarapid by any stretch, but “not bad by biotech standards,” Dr. DeCrosta points out. The CAD analysis is sensitive, with a limit of detection at less than half a microgram per milliliter plus good linearity for actives and impurities. “CAD allows us to increase specificity, to where we can see all actives and impurities.”
Because it relies on evaporation of sample droplets, CAD works independently of UV wavelength and does not require chromophores. According to Discovery Labs, CAD substantially reduces sample-preparation complexity and overall cycle time. The company now employs the CAD method for stability and release testing.