Multi-angle light scattering as a process analytical technology measuring real-time molecular weight for downstream process control

Abstract

For many protein therapeutics including monoclonal antibodies, aggregate removal process can be complex and challenging. We evaluated two different process analytical technology (PAT) applications that couple a purification unit performing preparative hydrophobic interaction chromatography (HIC) to a multi-angle light scattering (MALS) system. Using first principle measurements, the MALS detector calculates weight-average molar mass, Mw and can control aggregate levels in purification. The first application uses an in-line MALS to send start/stop fractionation trigger signals directly to the purification unit when preset Mw criteria are met or unmet. This occurs in real-time and eliminates the need for analysis after purification. The second application uses on-line ultra-high performance size-exclusion liquid chromatography to sample from the purification stream, separating the mAb species and confirming their Mw using a µMALS detector. The percent dimer (1.5%) determined by the on-line method is in agreement with the data from the in-line application (Mw increase of approximately 2750 Da). The novel HIC-MALS systems demonstrated here can be used as a powerful tool for real-time aggregate monitoring and control during biologics purification enabling future real time release of biotherapeutics.

A critical review of recent trends, and a future perspective of optical spectroscopy as PAT in biopharmaceutical downstream processing

“…Even for measuring one quality attribute, the combination of multiple sensors might be necessary. For example, the real-time monitoring of the mean molecular weight during a flow-through hydrophobic interaction chromatography (HIC) step for a mAb has been realized by static light scattering and concentration measurements by UV spectroscopy [74]. Because the scattered-light intensity is not only influenced by the molecular weight but by the concentration as well, a concentration measurement is necessary to calculate the molecular weight.…”

“…In recent publications, implemented in-line soft sensors (spectroscopic methods in combination with chemometrics) achieved limits of detection for aggregate and fragment levels below the concentration limits set by the regulatory agencies for drug products [15,45,74]. On a lab scale, Fig.…”

Technology outlook for real‐time quality attribute and process parameter monitoring in biopharmaceutical development—A review

“…Multi‐angle light scattering (MALS), refractive index, capacitance, and in‐line UV spectroscopy sensors such as FlowVPE® are some of the key technologies. Patel et al (2018) reported for the first time the use of an in‐line MALS sensor for real‐time monitoring of protein aggregates during downstream purification. The authors demonstrated that automated‐feedback control applied to the purification unit enabled precise control of protein purity in real‐time (Patel et al, 2018).…”

“…Patel et al (2018) reported for the first time the use of an in‐line MALS sensor for real‐time monitoring of protein aggregates during downstream purification. The authors demonstrated that automated‐feedback control applied to the purification unit enabled precise control of protein purity in real‐time (Patel et al, 2018). However, the technology may be challenging to implement in unit operations with significant differences in buffer conductivities and matrices, such as bind‐and‐elute mode purifications.…”

Monitoring of product variants in biopharmaceutical downstream processing : Mechanistic and data-driven modeling approaches

“…Fluorescence spectroscopy has been applied for at-line monitoring of a hydrophobic interaction chromatography step to take pooling decisions and separate correctly folded from misfolded protein [64]. Light sca ering has been employed to monitor protein refolding and aggregation of a fusion protein [65] and to take pooling decisions and separate aggregates from the mAb product [66].…”