(W1130-09-56) Investigating Stability of Trastuzumab (Herzuma^®) Undergoing Lyophilization and Freeze-Thaw Stresses

Purpose: Recombinant therapeutic proteins have become essential treatments for life-threatening diseases. Around 50% of newly approved drugs by the FDA belong to this protein-based therapeutic class, with many more in development. Despite this growth, developing a high quality, stable protein-based therapeutic product is challenging because proteins are prone to degradation and many process steps in development and manufacturing of protein therapeutics can cause one or more types of degradation. Among various degradation pathways, protein aggregation is a primary concern. Aggregation can elicit immune responses and lead to acute reactions such as anaphylaxis or more long-term immunogenicity. Therefore, aggregation is considered a critical quality attribute during biopharmaceutical development and current guidelines recommend employing a range of analytical techniques to characterize aggregate species qualitatively and quantitatively. Freezing and thawing (F/T) and lyophilization are processes encountered in the long-term storage, development and manufacture of protein therapeutics and have been shown to impact the quality depending on the characteristics of the molecule and formulation. This study focuses on Herzuma^®, a trastuzumab biosimilar product, and utilizes various analytical tools to investigate its stability under stresses occurring during lyophilization and freeze/thaw processes.

Methods: Trastuzumab (TmAb) was initially extracted from Herzuma^® in two steps: removal of polysorbates and exchange of buffer. This yielded TmAb in histidine buffer. A portion of the extracted TmAb was then kept in histidine buffer and the other was formulated according to the composition of Herzuma^®. Five freeze/thaw cycles (-20°C/25°C) were performed on both formulated and extracted TmAb formulations. In addition, TmAbs were lyophilized and packaged in vials resembling the process of the marketed product. Reconstituted lyophilized TmAb, formulated TmAb and extracted TmAb (non-formulated) were analyzed using size exclusion chromatography (SEC), dynamic light scattering (DLS), flow imaging microscopy (FlowCam^®) and UV-Vis spectroscopy.

Results: Freeze/thaw study: No high molecular weight species (HMWs) were detected, and monomer peaks were similar in SEC analysis for both Herzuma and buffer only formulations before and after F/T cycles. DLS measurements showed that the hydrodynamic diameter and PDI was not affected by freeze/thaw stress up to 5 cycles for any of the formulations (figure 1). FlowCam did not show any signs of aggregation due to F/T stress as there was no statistically significant difference between the number of micrometer sized particles before and after F/T cycles (see figure 2). Lyophilization: SEC analysis of lyophilized vs control trastuzumab (prior to lyophilization) showed a statistically significant increase in the percentage of HMW species (figure 3). Similarly, DLS showed a statistically significant increase in the diameter of the trastuzumab after lyophilization and reconstitution compared to control samples (figure 1). UV-Vis spectroscopy data, however, showed that the average of OD from 340 nm to 360 was below the limit of detection which indicates that samples do not contain a high concentration of larger particles (aggregates).

Conclusion: Lyophilization process appeared to induce significant aggregation in trastuzumab formulated as Herzuma while aggregation studies indicate no significant effect of F/T stress up to 5 cycles for both histidine buffer and Herzuma^® formulation.

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Acknowledgements: This project is supported by the Food and Drug Administration (FDA) of the U.S. Department of Health and Human Services (HHS) as part of a financial assistance award U01FD007758 to the National Institute for Pharmaceutical Technology and education (NIPTE) totaling $1,976,692 with 100 percent funded by FDA/HHS. The contents are those of the author(s) and do not necessarily represent the official views of, nor an endorsement, by FDA/HHS, or the U.S. Government.

Figure 1: The average hydrodynamic diameter of particles in the samples (TmAb) measured by DLS after freeze/thaw cycles and lyophilization.

Figure 2: Particle count per ml for TmAb in Histidine buffer and Herzuma formulation in control and freeze/thaw samples.

figure 3: High molecular weight (HMW) species in lyophilized reformulated TmAb formulation and control TmAb in His buffer.