Pharmaceutical Technology - November 2018

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44 Pharmaceutical Technology LABORATORY BEST PRACTICES 2018 P h a r mTe c h . c o m ment," says Shah. "Once the IND is filed and prom- ising clinical data are obtained, the scale of these processes significantly changes to support clinical demand and commercial projections. Going from one CMO (contract manufacturing organization) to another, these tools become really important in es- tablishing comparability—making sure the processes are transferred for effective scale-up and the product manufactured has similar or better quality and im- purity profile." Key techniques for crucial measurements Aggregation is the most complex degradation path- way to characterize, says Kendrick. "Aggregates can form unfolded aggregates; they can form native ag- gregates; they can be irreversible, or reversible aggre- gates depending on how they form and formulation conditions; and they can vary in size from soluble dimers all the way up to visible particles," says Kend- rick. "Aggregation measurements, consequently, need to span a variety of analytical techniques to charac- terize them." For size variant and degree of PEGylation charac- terization, Kendrick uses size-exclusion chromatog- raphy (SEC) coupled with multi-angle light scattering (MALS) coupled with ultraviolet (UV) and refractive index (RI) detection. Analytical ultracentrifugation (AUC) is used to confirm the size distribution profile within the formulation profile, something outside the SEC capabilities, he says. "We will use dynamic light scattering (DLS) as needed to further characterize the soluble aggregate profile," he adds. For subvisible particles, Kendrick uses micro- flow imaging to get images of each particle down to 1 μm and, based on the morphology, can classify the soluble aggregates or the subvisible aggregates as pro- teinaceous; or if the particles are in a drug-product syringe with silicon oil, can distinguish the silicon oil from the protein particles. "We can also collect the protein aggregates depend- ing on their size and characterize their morphology if they are small soluble aggregates, their potency, and their structure. We can look at intramolecular beta sheet content with techniques like RedShiftBio's mi- crofluidic modulation spectroscopy (MMS) infrared technology. It is pretty good at probing low levels of intramolecular beta sheet structure." Fleming concurred with Kendrick's description of techniques to assess protein aggregation and noted that Immumogen uses differential scanning calo- rimetry (DSC), DLS, and the IR approach from Red- ShiftBio to look at the secondary structure for ADCs. "We know from the conjugation process we can have alterations in the secondary and tertiary structure, so we look at that," says Fleming. They use capillary elec- trophoresis in reduced and non-reduced mode under denaturing conditions to look at fragmentation to look at how intact conjugates are after the conjuga- tion process. Roberts uses a range of techniques to evaluate changes in secondary and tertiary structure of proteins including UV absorption, intrinsic and extrinsic fluorescence spectroscopy, MMS, IR, and CD spectroscopy. Other techniques include laser scattering and neutron scattering, calorimetry, and combination techniques. Yoder says he likes "to use as much of the charac- terization toolbox as possible, so anything from mass spectrometry to IR spectroscopy, to CD. Those are probably the most common methods, and of course, we use chromatographic methods to look in more de- tail at purity and stability." Protein Characterization

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