(M1030-01-02) Using Novel Light Scattering Technology for Expedited Biologics Stability Assessment:  Implications for Successful Development and Manufacturing

Purpose: ARGEN™ is a versatile, higher throughput, batch processing static light scattering (SLS) instrument designed for in situ and real-time stability and viability assessments of all classes of biologics. ARGEN is patented on the principle of simultaneous multiple sample light scattering (SMSLS) permitting analysis of up to 8 samples in parallel. Each sample cell has dedicated optics permitting autonomous operation as well as independent experimental parameter control (temperature, stirring rate) on each sample. SLS is exquisitely sensitive to changes in molecular weight and permits temporal monitoring of lower to higher order oligomeric state transitions. aggregation, degradation, complexation, disassembly and solution behavior in situ for all classes of biologics. These applications make ARGEN ideal for rapid vetting, grid screening and ranking of formulation stability under thermal, stirring and buffer stress as well as long or short-term shelf-life determination at low temperature(s). For this work, ARGEN was utilized to assess: (1) The effect of protein concentration on aggregation propensity under stirring stress, (2) rAAV capsid stability and genome (DNA) ejection, and (3 ) Thermal behavior of RNA in a physiological buffer system.

Methods: SLS Measurements - Data was collected using ARGEN™, a higher throughput, batch processing. static light scattering (SLS) device (λ=600 nm, 90° detection) developed by Yokogawa Fluence Analytics (YFA) and patented on the principle of simultaneous multiple sample light scattering (SMSLS). The instrument utilizes 1 cm pathlength cuvettes and is equipped with eight autonomous sample cells permitting independent experimental parameter (temperature and stirring rate) adjustment. Additionally, each sample cell is engineered with a dedicated, fixed optical detector train to mitigate measurement error(s). Contact Stirring Experiments - Each sample cell is equipped with a stepper motor attached to a rotating magnet which couples to a micro stir bar at the bottom of the cuvette to simulate perturbations experienced during bioprocessing. Stirring rate (0-2000 RPM) is manually controlled for precision via the ARGEN™ intuitive control software independently for each sample cell.

Results: Characterizing the Effect of Protein Concentration on Aggregation Propensity Under Stirring Stress. This case study determined the effects of protein concentration on aggregation propensity under stirring stress using bovine serum albumin (BSA) in phosphate buffered saline, pH 7.4. At concentrations ≦2.0 mg/ml, aggregate sizes increased with increasing [BSA] as indicated by changes in M_w(t)/M_o. At higher concentrations (4 mg/ml to 10 mg/ml), aggregation proceeded in a quasi-stochastic manner with the largest aggregates forming at 6 mg/ml. Thermal stability and solution behavior of RNA in phosphate buffered saline (pH 7.4) Eight samples of the crude yeast RNA prepared in PBS, pH 7.4 and analyzed in parallel from 30°C-65°C for 2 hours to determine disparities in aggregation or degradation propensities. At 40°C, the RNA remained stable with diminutive changes in M_w/M_o throughout the duration of the 2 hour experiment. M_w/M_o changes at 30°C, 35°C and 45°C indicate aggregation and the formation of higher order species. The initial, rapid decrease in M_w/M_o at 65°C followed by an increase in M_w/M_o from 2000s to 6500s may indicate denaturing of the RNA followed by intermolecular base pairing and the formation of higher order species. Monitoring rAAV Capsid Stability and Genome (DNA) Ejection - These experiments defined temporal changes in molecular weight (average) for full and empty rAAVs at 44°C. Empty capsids rapidly formed higher order aggregates due to colloidal instability. This provides possible evidence into the role of DNA in the maintenance of capsid architecture as evidenced by a rapid increase in molecular weight (average). Analysis of full capsids demonstrated the complete ejection of DNA in ~5.6 hours (~20000s) from the outset of the experiment. Molar Mass Determination of Full and Empty rAAV capsids - Absolute molecular weights for the full and empty rAAV capsids were determined using the Zimm approximation, neglecting the interaction term (A₂). Fractional errors are due to concentration uncertainties (pooling of mixed and empty capsids), as well as neglect of the interaction term (A₂) and Mie scattering effects (angular dependence of non-Rayleigh scatterers).

Conclusion: ARGEN is a novel static light scattering instrument patented on the principle of simultaneous multiple sample light scattering (SMSLS) permitting batch monitoring of solution behavior for up to eight samples in parallel. The ability to temporally monitor changes in molecular weight (average) under different stress modalities in situ permits the definition of a comprehensive stability landscape which is the threshold for successful development. The independent operation of each sample cell enables the user to establish precise experimental parameters on each sample for grid screening and discovery of stabilizing conditions. Additionally, the instrument has a thermal range of 3⁰C-80⁰C making it ideal for optimizing shelf-life under refrigerated conditions. These case studies demonstrated the ability of ARGEN to rapidly characterize the solution behavior of multiple classes of biologics including proteins, RNA and viral vectors under stirring, buffer and thermal stress in situ and in real-time.

References: Curtis W. Jarand, Karen Baker, Matthew Petroff, Mi Jin, and Wayne F. Reed, DNA Released by Adeno-Associated Virus Strongly Alters Capsid Aggregation Kinetics in a Physiological Solution, Biomacromolecules 2024 25 (5), 2890-2901DOI: 10.1021/acs.biomac.4c00027.

Acknowledgements: This work was supported and funded by Yokogawa Fluence Analytics (YFA) headquartered in Stafford, TX. A special thanks to YFA team members for their support and contributions.


Normalized molecular weights (Mw/M0) vs. time (s) for total RNA (Candida utilis) in phosphate buffered saline (pH 7.4) from 30°C-65°C


Normalized molecular weight (Mw/M0) vs. Time (s) for full and empty AAV capsids at 44°C (Reference 1)


Normalized molecular weight (Mw/Mo) vs. time (s) for [BSA] = 0.5 mg/ml-10 mg/ml