High-resolution mass spectrometric approaches to study protein structure and environment in lyophilized solids

Lavanya K Iyer, Purdue University


Proteins comprise a growing class of therapeutics that is used to treat various diseases such as diabetes and cancer. However, intrinsic structural features such as the primary sequence and extrinsic factors such as pH, temperature, agitation and metal ions can promote instability that manifests as chemical degradation (e.g. oxidation, deamidation, hydrolysis) and/or physical degradation (aggregation, phase separation). Since several degradation pathways are accelerated by diffusion in solution, proteins are lyophilized to improve stability. The lyophilized formulation may still undergo degradation during manufacture and/or storage. The mechanism of protein aggregation in lyophilized solids is not well understood or predictable by conventional analytical methods such as solid-state Fourier-transform infrared spectroscopy (ssFTIR) and differential scanning calorimetry (DSC) and this poses challenges in rational formulation design. ^ This dissertation is aimed at understanding local protein structure and environment in the solid state using high-resolution mass spectrometric methods. Chapter 2 examines protein side-chain matrix accessibility using solid-state photolytic labeling- mass spectrometry (ssPL-MS). The use of a photoactive probe, photo-leucine (pLeu) enabled side-chain labeling in lyophilized formulations, reported by our group for the first time. High-resolution information at the peptide level was obtained using bottom-up tandem mass spectrometry. Differences in labeling patterns and side-chain matrix accessibility were observed when sucrose or guanidine hydrochloride was used as an excipient. This work also used a photoactive probe incorporated within the amino acid sequence of a glucagon-derived peptide to detect interactions with excipients and peptides in the solid state. Residue-level information about the preferred site of peptide-peptide crosslinking was obtained using tandem mass spectrometry. ^ Although peptide-matrix interactions could be visualized using a photoactive amino acid (PAA) derivative within the primary sequence, incorporating an unnatural amino acid into larger proteins is fairly difficult and may alter higher order structure by disturbing intra-protein contacts. Therefore, a novel photo-crosslinking method was developed to further examine the solid-state environment of lyophilized proteins, described in Chapter 3. A heterobifunctional crosslinking reagent was used to crosslink the protein with the matrix in the solid state. Some loop regions showed increased peptide-peptide adducts, while helix E showed more hydration compared to other regions. In the presence of raffinose, water replacement was not detected in the solid state; instead there was some evidence of micro-phase separation without crystallization in the solid state. Thus local protein environment in the solid state could be probed without the need for PAA incorporation within the protein sequence. ^ Lyophilization is an effective, yet expensive stabilization strategy, since conservative freeze-drying cycles often require long hours of drying. The stochastic nature of ice nucleation and lack of control over freezing can result in vial-to-vial heterogeneity due to differences in the degree of supercooling and ice crystal size. The research described in Chapter 4 focuses on using a variety of analytical methods to characterize lyophilized protein formulations to determine the effect of excipient and freezing step on protein structure. Myoglobin in the presence or absence of sucrose was lyophilized with or without controlled ice nucleation in a pilot-scale LyoStar freeze dryer. Ice nucleation occurred over a range of temperatures and times with uncontrolled nucleation, while controlled ice nucleation with rapid depressurization resulted in near-simultaneous ice nucleation. The sucrose-containing formulation showed greater retention of protein structure by ssFTIR and solid-state hydrogen-deuterium exchange mass spectrometry (ssHDX-MS). Greater conformational homogeneity was observed in the sucrose-containing formulation by ssHDX-MS peak width analysis. No significant differences in secondary structure were detected between controlled and uncontrolled nucleation using ssFTIR and ssHDX-MS. Myoglobin lyophilized with controlled nucleation in the presence of sucrose showed the greatest side-chain labeling, as determined by ssPL-MS. The results show that high-resolution mass spectrometric methods can be used to study process- and excipient effects on protein structure. ^ This thesis addresses limitations in current analytical methods used to characterize protein structure in the solid state. Whereas ssFTIR and DSC have lower sensitivity and provide information averaged over the entire sample, mass spectrometric methods can provide peptide-level information about conformational changes occurring in a small subpopulation of protein. High-resolution mass spectrometric methods have the potential to provide reliable and predictable protein formulation screening and facilitate rational drug design.^




Elizabeth M. Topp, Purdue University.

Subject Area

Pharmaceutical sciences

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