BioPharm

20 BioPharm International eBook October 2018 www.biopharminternational.com they can be irreversible, or revers- ible aggregates depending on how they form and formulation con- ditions; and they can vary in size from soluble dimers all the way up to visible particles," says Kendrick. "Aggregation measurements, con- sequently, need to span a variety of analytical techniques to charac- terize them." For size variant and degree of P E G y l a t i o n c h a r a c t e r i z a t i o n , K e n d r i c k u s e s s i z e - e xc l u s i o n chromatog raphy (SEC) coupled with multi-angle light scattering (MALS) coupled with ultraviolet ( U V ) a nd ref rac t ive index (R I) detection. Analytical ultracentri- fugation (AUC) is used to confirm the size distribution profile within t he for mu lat ion prof ile, some - thing outside the SEC capabilities, he says. "We w ill use dy namic light scattering (DLS) as needed to further characterize the soluble aggregate profile," he adds. F o r s u b v i s i b l e p a r t i c l e s , Kendr ick uses microf low imag- ing to get images of each particle down to 1 μm and, based on the morphology, can classify the sol- uble aggregates or the subvisible aggregates as proteinaceous; or if the particles are in a drug-product syringe with silicon oil, can dis- tinguish the silicon oil from the protein particles. "We can also collect the pro- te i n ag g regate s dep e nd i ng on their size and characterize their morphology if they are small sol- uble aggregates, their potency, and their str uct ure. We can look at intramolecular beta sheet content with techniques like RedShiftBio's microfluidic modulation spectros- copy (MMS) infrared technology. It is pretty good at probing low levels of intramolecular beta sheet structure." F l e m i n g c o n c u r r e d w i t h Kendr ick's descr iption of tech- niques to assess protein aggrega- tion and noted that Immumogen uses differential scanning calo- rimetry (DSC), DLS, and the IR approac h f rom Re d Sh i f t Bio to look at the secondary str ucture for A DCs. "We k now f rom t he conjugation process we can have alterations in the secondary and tertiary structure, so we look at that," says Fleming. They use cap- illary electrophoresis in reduced a nd non-re duce d mo de u nde r denat u r i ng cond it ions to look at fragmentation to look at how intact conjugates are after the con- jugation process. Roberts uses a range of tech- n iques to eva luate c ha nges i n secondary and tertiary structure of proteins including UV absorp- tion, intrinsic and extrinsic fluo- rescence spectroscopy, MMS, IR, and CD spectroscopy. Other tech- niques include laser scattering and neutron scattering, calorimetry, and combination techniques. Yoder says he likes "to use as much of the characterization tool- box as possible, so anything from mass spectrometry to IR spectros- copy, to CD. Those are probably the most common methods, and of course, we use chromatographic methods to look in more detail at purity and stability." TECHNIQUE LIMITATIONS AND EVOLUTION Despite the range of techniques that are available, "The shortcom- ing that all techniques have is that you can't get everything in one measurement," says Roberts. With soft-matter physics, you may only need two techniques, or you might need different flavors of the same technique, he says. "That does not work with proteins, at least not for the kind of questions we have to answer, so we are always going to need multiple techniques." "Even with all the techniques that anyone can bring to bear, you still are missing some things, you are not getting the full 3-D structural picture you might want and the dynamics of that picture," Roberts explains. "You are always getting a piece of the puzzle. Even if you can double the number of techniques that we have right now, we still would be missing pieces." Roberts is charged with finding new technologies for characteriz- ing protein-protein interactions, protein-environment interactions, structures, and stability, or to find technologies maturing in other fields and adapt them for biopro- cessing research. "I am always interested in exploring either truly new techniques using a technology that has not been brought to bear on proteins before, or technologies from other fields that we can adapt and bring to bear on protein-based problems," he says. For example, laser scatter ing technolog y was mat u re in t he polymer and colloid sciences fields 20 years ago, he explains, but pro- tein scientists only had x-ray dif- fraction to get 3-D structures. Now, scattering techniques are a stan- dard technique in protein research laboratories. When the scattering Biopharma Laboratory Best Practices Protein Characterization Protein characterization is a crucial part of the overall quality control strategy for biopharmaceutical drugs including an understanding of the product attributes.

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