Identification of Acyl-Protein Thioesterase-1 as a Polysorbate-Degrading Host Cell Protein in a Monoclonal Antibody Formulation Using Activity-Based Protein Profiling

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Author: Vašl, Jožica ; Šprager, Ernest ; Lunder, Mojca ; Bratkovič, Tomaž ; Reisinger, Veronika ; Möller, Jens ; Lin, Yuhsien ; Krajnc, Aleksander

Polysorbate (PS) degradation in monoclonal antibody (mAb) formulations poses a significant challenge in the biopharmaceutical industry. PS maintains protein stability during drug product's shelf life but is vulnerable to breakdown by low-abundance residual host cell proteins (HCPs), particularly hydrolytic enzymes such as lipases and esterases. In this study, we used activity-based protein profiling (ABPP) coupled with mass spectrometry to identify acyl-protein thioesterase-1 (APT-1) as a polysorbate-degrading HCP in one case of mAb formulation with stability problems. We validated the role of APT1 by matching the polysorbate degradation fingerprint in the mAb formulation with that of a recombinant APT1 protein. Furthermore, we found an agreement between APT1 levels and PS degradation rates in the mAb formulation, and we successfully halted PS degradation using APT1-specific inhibitors ML348 and ML211. APT1 was found to co-purify with a specific mAb via hitchhiking mechanism. Our work provides a streamlined approach to identifying critical HCPs in PS degradation, supporting quality-by-design principles in pharmaceutical development.

16 Dec 2016·ACS Chemical BiologyQ2 · BIOLOGY

Molecular Mechanism for Isoform-Selective Inhibition of Acyl Protein Thioesterases 1 and 2 (APT1 and APT2)

Q2 · BIOLOGY

Article

Author: Brooks, Charles L. ; Hwang, Sin Ye ; Torossian, Kristopher A. ; Stuckey, Jeanne A. ; Pricer, Rachel ; Majmudar, Jaimeen D. ; Chong, Fei San ; Davda, Dahvid ; Palakurthi, Jasmine ; Won, Sang Joon ; Hur, Edward S. ; Labby, Kristin J. ; Meagher, Jennifer L. ; Armacost, Kira A. ; Rodriguez, Laura A. ; Rodriguez, Christina L. ; Martin, Brent R.

Post-translational S-palmitoylation directs the trafficking and membrane localization of hundreds of cellular proteins, often involving a coordinated palmitoylation cycle that requires both protein acyl transferases (PATs) and acyl protein thioesterases (APTs) to actively redistribute S-palmitoylated proteins toward different cellular membrane compartments. This process is necessary for the trafficking and oncogenic signaling of S-palmitoylated Ras isoforms, and potentially many peripheral membrane proteins. The depalmitoylating enzymes APT1 and APT2 are separately conserved in all vertebrates, suggesting unique functional roles for each enzyme. The recent discovery of the APT isoform-selective inhibitors ML348 and ML349 has opened new possibilities to probe the function of each enzyme, yet it remains unclear how each inhibitor achieves orthogonal inhibition. Herein, we report the high-resolution structure of human APT2 in complex with ML349 (1.64 Å), as well as the complementary structure of human APT1 bound to ML348 (1.55 Å). Although the overall peptide backbone structures are nearly identical, each inhibitor adopts a distinct conformation within each active site. In APT1, the trifluoromethyl group of ML348 is positioned above the catalytic triad, but in APT2, the sulfonyl group of ML349 forms hydrogen bonds with active site resident waters to indirectly engage the catalytic triad and oxyanion hole. Reciprocal mutagenesis and activity profiling revealed several differing residues surrounding the active site that serve as critical gatekeepers for isoform accessibility and dynamics. Structural and biochemical analysis suggests the inhibitors occupy a putative acyl-binding region, establishing the mechanism for isoform-specific inhibition, hydrolysis of acyl substrates, and structural orthogonality important for future probe development.

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Indication	Highest Phase	Country/Location	Organization	Date
Neoplasms	Preclinical	US	The Scripps Research Institute	13 Jan 2014

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