Molecular characterization and functional activity of an IL-15 antagonist MutIL-15/Fc human fusion protein.

Fc fusion proteins are a new emerging class of molecules for immune-targeted delivery of therapeutic proteins. Biophysical and bioanalytical characterization is critical for clinical development and delivery of therapeutic proteins. Here we report molecular and functional characterization of a recombinant human fusion protein Mutant IL-15/Fc. MutIL-15/Fc has a molecular weight of ∼95 kDa as determined by multiangle laser light scattering with online size exclusion chromatography and migrated at a faster rate (lower retention time) in gel filtration column. The kinetics of binding of MutIL-15/Fc to Fcγ receptor is best fitted in a bivalent modal with K(D1) 5 µM and K(D2) 9 µM determined by surface plasmon resonance (BIAcore). N-Glycoprofiling analysis revealed extensive glycosylation of MutIL-15/Fc. The Fc and IL-15 components in the MutIL-15/Fc are detected using the dual mode ELISA. The HT-2 cell proliferation inhibition assay is qualified as a quantitative in vitro marker functional assay. Molecular state changes associated with forced stress analyzed by SEC-MALS resulted in changes in bioactivity and Fc:Fcγ receptor interaction affinity. These data provide a systematic approach to molecular and functional characterization of the MutIL-15/Fc to establish product consistency and stability monitoring during storage and under drug delivery conditions.

Detailed Protocol for the Novel and Scalable Viral Vector Upstream Process for AAV Gene Therapy Manufacturing.

Recombinant adeno-associated viral (rAAV) vector-based gene therapy has been adapted for use in more than 100 clinical trials. This is mainly because of its excellent safety profile, ability to target a wide range of tissues, stable transgene expression, and significant clinical benefit. However, the major challenge is to produce a high-titer, high-potency vector to achieve a better therapeutic effect. Even though the three plasmid-based transient transfection method is currently being used for AAV production in many clinical trials, there are complications associated with scalability and it is not cost-effective. Other methods require either large-scale production of two herpes simplex viruses, rHSV-RepCap and rHSV-GOI (gene of interest), with high titers, or a stable cell line with high titer wild-type adenovirus infection. Both of these options make the process even more complex. To address this issue, we have developed a stable cell line-based production with the use of only one rHSV-RepCap virus. Using this new methodology in small-scale production, we achieved ∼1-6 E + 04 vg/cell of AAV9 in the top producer clones. Large-scale production in 10-CS (10-Cell Stack) of one of the top producing clones resulted in ∼1-2 E + 13 vg/10-CS with 50% of full capsid ratio after purification. This method could potentially be adapted to suspension cells. The major advantage of this novel methodology is that by using the rHSV-RepCap virus, high titer AAV can be produced with any GOI containing a stable adherent or suspension producer cell line. The use of this AAV production platform could be beneficial for the treatment of many diseases.

Element of a validation method for MU-B3 monoclonal antibody using an imaging capillary isoelectric focusing system.

This paper presents an imaging capillary isoelectric focusing (CIEF) assay for the determination of the identity, stability, and isoform distribution of a murine monoclonal antibody (MU-B3). The experiments were conducted using a Convergent Bioscience iCE280 instrument. The optimum carrier ampholyte composition that gave the best peak separation was found to be 25% Pharmalyte pH 3-10 and 75% Pharmalyte pH 5-8. The antibody gave a highly reproducible CIEF profile with three major peaks having average isoelectric point (pI) values of 6.83, 6.99, and 7.11. Intraday and interday reproducibility of pI values was found to be within RSD of 0.5%. The CIEF profile was also the same, with an alternate column cartridge and alternate batches of methyl cellulose. A plot of peak areas versus MU-B3 concentration was linear (R2 = 0.995) up to a concentration of 0.5 mg/mL in the sample solution. Peak area measurements were reproducible to within 7% RSD. The CIEF profiles of two other antibodies were distinctly different from the profile of MU-B3, showing that the assay is specific. After a sample of MU-B3 was subjected to heat stress by exposure to heat at 55 degrees C for 4 h, its CIEF profile was altered with extra peaks appearing at lower pI values, indicating that the assay could be used to monitor stability. The result of the heat stress experiment was also confirmed with a parallel slab-gel IEF analysis of the antibody sample before and after application of the heat stress. The results of this work suggest that imaging CIEF can be used for product testing under a quality control environment. The assay can be used for pI profiling of proteins and for monitoring structural changes (deamidation, glycosylation, etc.) during the manufacturing process and upon storage.

Characterization and analysis of thermal denaturation of antibodies by size exclusion high-performance liquid chromatography with quadruple detection.

Size exclusion chromatography (SEC) coupled with online light scattering, viscometry, refractometry, and UV-visible spectroscopy provides a very powerful tool for studying protein size, shape, and aggregation. This technique can be used to determine the molecular weight of the component peaks independent of the retention times in the SEC column and simultaneously measure the hydrodynamic radius and polydispersity of the protein. We applied this technology by coupling an Agilent Chemstation high-performance liquid chromatography system with a diode array UV-visible detector and a Viscotek 300 EZ Pro triple detector (combination of a light scattering detector, refractometer, and differential pressure viscometer) to characterize and compare the molecular properties of a number of monoclonal antibodies. Our studies reveal that different monoclonal immunoglobulin Gs (IgGs) and chimeric IgGs show slightly different retention times and therefore different molecular weights in gel filtration analysis. However, when they are analyzed by light scattering, refractometry, and viscometry, different IgGs have comparable molecular weight, molecular homogeneity (polydispersity), and size. Gel filtration coupled with UV or refractive index detection suggests that antibodies purified and formulated for preclinical and clinical development are more than 95% monomer with little or no detectable soluble aggregates. Light scattering measurements showed the presence of trace amounts of soluble aggregate in all the IgG preparations. The different IgG molecules showed different susceptibility to heat and pH. One of the murine antibodies was considerably less stable than the others at 55 degrees C. The application of this powerful technology for the characterization of monoclonal antibodies of therapeutic potential is discussed.