mp9b00614_si_001.pdf (1.11 MB)
Optimizing the Formulation and Lyophilization Process for a Fragment Antigen Binding (Fab) Protein Using Solid-State Hydrogen–Deuterium Exchange Mass Spectrometry (ssHDX-MS)
journal contribution
posted on 2019-10-17, 12:35 authored by Lokesh Kumar, Karthik Balakrishna Chandrababu, Shenbaga Moorthy Balakrishnan, Andrea Allmendinger, Benjamin Walters, Isidro E. Zarraga, Debby P. Chang, Purnendu Nayak, Elizabeth M. ToppSolid-state hydrogen–deuterium
exchange with mass spectrometry
(ssHDX-MS) was evaluated as an analytical method to rapidly screen
and select an optimal lyophilized fragment antigen binding protein
(Fab) formulation and the optimal lyophilization cycle. ssHDX-MS in
lyophilized Fab formulations, varying in stabilizer type and stabilizer/protein
ratio, was conducted under controlled humidity and temperature. The
extent of deuterium incorporation was measured using mass spectrometry
and correlated with solid-state stress degradation at 50 °C as
measured by size exclusion chromatography (SEC) and ion-exchange chromatography
(IEC). ssHDX-MS was also used to evaluate the impact of three different
types of lyophilization processing on storage stability: controlled
ice nucleation (CN), uncontrolled ice nucleation (UCN), and annealing
(AN). The extent of deuterium incorporation for different Fab formulations
agreed with the order of solid-state stress degradation, with formulations
having lower deuterium incorporation showing lower stress-induced
degradation (aggregation and charge modifications). For lyophilization
processing, no significant effect of ice nucleation was observed in
either solid-state stress degradation or in the extent of deuterium
incorporation for high concentration Fab formulations (25 mg/mL).
In contrast, for low concentration Fab formulations (2.5 mg/mL), solid-state
stability from different lyophilization processes correlated with
the extent of deuterium incorporation. The order of solid-state degradation
(AN < CN < UCN) was the same as the extent of deuterium incorporation
on ssHDX-MS (AN < CN < UCN). The extent of deuterium incorporation
on ssHDX-MS correlated well with the solid-state stress degradation
for different Fab formulations and lyophilization processing methods.
Thus, ssHDX-MS can be used to rapidly screen and optimize the formulation
and lyophilization process for a lyophilized Fab, reducing the need
for time-consuming stress degradation studies.