Kevin Zen, senior director, IGM Biosciences

Manufacturers of monoclonal antibodies (mAbs) and other therapeutic proteins are heeding calls from regulatory agencies to shift from off-line to on-line and/or in-line (OL/IL) measurement of critical quality attributes and process parameters (CQAs, CPPs). But developers of cell and gene therapies (CGTs) still rely on off-line methods. Industry collaboration is necessary to develop sufficiently sophisticated technologies for monitoring of complex CGT quality attributes. Zen explored recent advances in analytical instrumentation for applications involving mAbs and gene therapies delivered by adenoassociated virus (AAV) vectors.

Zen began with discussion of process analytical technologies (PATs). He noted that upstream scientists still monitor familiar process parameters such as bioreactor pH, oxygen and nutrient consumption, and so on. But as OL/IL offerings proliferate, total- and viable-cell density (TCD, VCD) are gaining in importance. Timely monitoring of such CPPs enables upstream scientists to control process productivity in ways that are not possible with measurements of pH and dissolved-oxygen levels.

Downstream teams likewise are transitioning to OL/IL monitoring. Zen highlighted SoloVPE and FlowVPX systems (Repligen) for measurement of protein concentration — e.g., before ultrafiltration/diafiltration (UF/DF). Both platforms use variable-pathlength technology to take multiple absorbance readings per application, increasing measurement accuracy and overcoming limitations with conventional ultraviolet–visible light (UV-vis) instruments with fixed optical pathlengths. The FlowVPX system can be installed in line between chromatography and UF/DF steps or between UF/DF and bulk filling.

Next, Zen focused on product characterization. For mAbs, critical needs relate to analysis of posttranslational modifications (PTMs), charge variants, and degradation products (fragments and aggregates). For AAV gene therapies, assessment of full, partially filled, and empty capsids remains the most arduous analytical activity. That said, product-release testing also is demanding — e.g., with analysts needing to calculate genomic, infectious, and capsid titers. Those can be measured by quantitative or digital polymerase chain reaction (qPCR, dPCR); infectious-center assays; and capsid-dependent enzyme-linked immunosorbent assays (ELISAs) or sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), respectively.

Several advances are facilitating characterization. Zen highlighted that scientists at Rutgers University have developed a sequential-injection–based PAT — called the N-GLYcanyzer system — for OL monitoring of N-linked mAb glycosylation. Zen remarked that although the method is somewhat time-consuming, it facilitates glycan analysis significantly.

Contract partners are beginning to offer multiattribute monitoring (MAM) for identification and monitoring of CQAs. Peptide maps are generated by digesting protein samples into component peptides, separating those by LC, and applying high-resolution tandem mass spectroscopy (MS/MS). For routine monitoring, analysts can perform conventional MS, then compare those maps with standards. Doing so can reveal peaks that indicate product heterogeneity or instability.

Regulators recently issued guidances advising application of orthogonal methods to quantify levels of empty capsids. Zen noted that although the guidance specifies transmission-electron microscopy (TEM) and ELISAs with qPCR/dPCR as potential methods, regulators are beginning to discourage their use because they can generate variable results depending on sample type and preparation quality. The accuracy and reproducibility of analytical ultracentrifugation (AUC) make it a preferred method among regulators, but it is time-consuming, requires specialized equipment, and provides low throughput. Charge-detection mass spectrometry (CDMS) is a promising method because it provides accuracy comparable to that of AUC but exhibits higher throughput and requires smaller samples.

Imaged-capillary isoelectric focusing (icIEF) long has held promise as an orthogonal method to ion-exchange (IEX) chromatography for charge-variant analysis (CVA). Conventional icIEF systems only separate charge fractions, however; they cannot characterize charge species. Zen explained that two new technologies could enhance icIEF’s utility for CVA, especially during gene-therapy product development. Bio-Techne’s MauriceFlex platform can perform icIEF and fraction collection within easy-to-install cartridges that can be analyzed for intact MS. Sciex’s Intabio ZT system integrates icIEF charge separation with real-time UV-based quantitation and on-chip electrospray ionization. Coupling the Intabio ZT system with a Sciex ZenoTOF 7600 mass spectrometer enables separation, quantitation, and identification of charge variants within a single platform.

Zen concluded with advances in rapid microbiological methods for CGT product release, including sterility assays based on solid-phase cytometry, nucleic-acid amplification, and adenosine triphosphate (ATP) bioluminescence.

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See full presentations online at https://bioprocessintl.com/category/bpi-theater/bpi-theater-bio-2024.