(W1030-09-55) Aggregation of a Trastuzumab Radioimmunoconjugate under Handling Stress

Purpose: Monoclonal antibodies (mAbs) have emerged as a leading class of protein pharmaceuticals and are used to treat numerous disease conditions like cancer and autoimmune disorders. The high specificity and affinity of mAbs makes them compelling candidates for delivering radionuclides to targeted tumor tissues. As a result, radioimmunoconjugates (RICs) have been developed wherein different nuclides are conjugated to mAbs for diagnostic and therapeutic applications [1]. However, aggregation and instability present challenges in formulation and administration of these complex molecules. RICs are particularly prone to aggregation due to partial unfolding and destabilization of tertiary structure of mAbs due to bioconjugation reaction conditions and radiochemical synthesis [1, 2]. Improper handling during storage, transport and administration is one of the major sources of instability in formulations containing mAbs. Different processes like incorrect method of injecting mAbs into infusion bags, vigorous manual agitation of vials containing liquid mAb preparations and repeated movements of the syringe plunger, often result in aggregation [3]. Therapeutic handling of mAbs using syringes has been found to induce aggregation because of their adsorption on the inner surface of the syringe barrel. Additionally, movement of plunger head within the syringe releases micron sized aggregates into the mAb solutions [4]. While typically, care is taken to prepare RIC formulations with high quality that are free of aggregates and impurities, little to no information is available regarding the effects of handling on RICs. The combination of mechanical stress due to syringe handling and inherent sensitivity to aggregation makes the instability of RICs a critical concern. Therefore, this study aims to investigate the impact of syringe handling and transfer on aggregation behavior of RICs.

Methods: Trastuzumab (TmAb) was extracted and purified from a marketed biosimilar Herzuma® through polysorbate removal and buffer exchange into ultrapure water to yield 10 mg/ml stock solution. This stock solution was subsequently diluted and filtered to obtain 1 mg/ml TmAb solution in 0.9% sodium chloride (NaCl). Similarly, non-radioactive model systems, that are analogous in structure to TmAb RICs: Zirconium-desferrioxamine B -TmAb (Zr-DFO-TmAb) were prepared as described previously [5, 6]. This solution underwent buffer exchange and was filtered into 0.9% NaCl to obtain a final concentration of 1 mg/ml. A volume of 2.0 ml of both unconjugated TmAb and Zr-DFO-TmAb solutions was drawn into a 3.0 ml disposable plastic syringe attached to an 18G x 1-1/2” (1.27 mm x 38 mm) hypodermic needle. Mechanical stress was applied manually to the solutions by pulling and then pushing the plunger to the original position inside the syringe. This process was repeated 15 times, and the final stressed solution was transferred into a glass vial. A second type of stress included drawing the solutions into the syringe, pulling, pushing the plunger and dispensing the solution into a glass vial. The same solution was drawn into the syringe again and the cycle of applying stress was repeated. This cycle of drawing, stressing and collecting the solution was repeated 15 times to get a final stressed solution which was transferred into a glass vial for further analysis. Unstressed solutions of both unconjugated TmAb and Zr-DFO-TmAb were used as controls. The impact of stress and aggregation propensity of unconjugated TmAb and Zr-DFO-TmAb solutions were subsequently analyzed using various analytical techniques including dynamic light scattering (DLS) and flow imaging microscopy (FIM).

Results: DLS results showed a considerably increase in hydrodynamic diameter in both unconjugated TmAb and Zr-DFO-TmAb solutions when subjected to the second type of syringe stress as compared to their unstressed controls and first type of syringe stress. The unconjugated TmAb and Zr-DFO-TmAb solutions exhibited a hydrodynamic diameter of 336.80 ± 326.73 nm and 1003.63 ± 1383.91 nm respectively while their corresponding control solutions had a diameter of 10.83 ± 0.06 nm and 10.97 ± 0.06 nm. FIM analysis revealed that Zr-DFO-TmAb solutions had statistically significant higher particle counts under both types of syringe stress conditions compared to the unconjugated TmAb solutions. Following the first type of syringe stress, for particles large than 2 µm, unconjugated TmAb solution had 3166 ± 1546 particles/ml, whereas Zr-DFO-TmAb solution displayed 4070± 3115 particles/ml. On applying the second type of syringe stress, the particle counts in unconjugated TmAb solutions increased to 48486 ± 16497 particles/ml while the Zr-DFO-TmAb solutions showed an even higher particle count of 303676 ± 147574 particles/ml.

Conclusion: The results suggest that the Zr-DFO-TmAb is more susceptible to aggregation upon exposure to handling stress in a syringe compared to the unconjugated TmAb. The process of conjugation considerably affects the aggregation behavior and overall stability of TmAb. This underscores the importance of developing appropriate handling and delivery techniques for RIC formulations.

References: 1. Vivier, D.A.-O., S.A.-O. Sharma, and B.A.-O.X. Zeglis, Understanding the in vivo fate of radioimmunoconjugates for nuclear imaging. (1099-1344 (Electronic)).
2. Donnelly, P., et al., A Desferrioxamine B squaramide ester for the incorporation of zirconium-89 into antibodies. Chem. Commun., 2016. 52.
3. Nejadnik, M.R., et al., Postproduction Handling and Administration of Protein Pharmaceuticals and Potential Instability Issues. Journal of Pharmaceutical Sciences, 2018. 107(8): p. 2013-2019.
4. Maruno, T., et al., Sweeping of Adsorbed Therapeutic Protein on Prefillable Syringes Promotes Micron Aggregate Generation. Journal of Pharmaceutical Sciences, 2018. 107(6): p. 1521-1529.
5. Bhatt, N.B., et al., A comprehensively revised strategy that improves the specific activity and long-term stability of clinically relevant (89)Zr-immuno-PET agents. (1477-9234 (Electronic)).
6. Pandya, D.N., et al., (89)Zr-Chloride Can Be Used for Immuno-PET Radiochemistry Without Loss of Antigen Reactivity In Vivo. (1535-5667 (Electronic)).

Acknowledgements: This study is supported by the University of Iowa Research Development Office (RDO) through the OVPR Interdisciplinary Scholars Program.