The In Vitro Biotransformation of the Fusion Protein Tetranectin-Apolipoprotein A1

As more and more protein biotherapeutics enter the drug discovery pipelines, there is an increasing interest in tools for mechanistic drug metabolism investigations of biologics in order to identify and prioritize the most promising candidates. Understanding or even predicting the in vivo clearance of biologics and to support translational pharmacokinetic modeling activities is essential, however there is a lack of effective and validated in vitro cellular tools. Although different mechanisms have to be adressed in the context of biologics disposition, the scope is not comparable to the nowadays widely established tools for early characterization of small molecule disposition. Here, we describe a biotransformation study of the fusion protein tetranectin apolipoprotein A1 by cellular systems. The in vivo biotransformation of tetranectin apolipoprotein A1 has been described previously, and the same major biotransformation product could also be detected in vitro, by a targeted and highly sensitive detection method based on chymotrypsin digest. In addition, the protease responsible for the formation of this biotransformation product could be elucidated to be DPP4. To our knowledge, this is one of the first reports of an in vitro biotransformation study by cells of a therapeutic protein.

G418, with medium being exchanged every two days. Confluent cultures were washed twice with sterile PBS without Ca 2 and Mg 2 and incubated for 5 min at 37ºC with 2mLof Accutase cell detachment solution. Cell detachment was facilitated by repeated pipette aspirations. As to stop Accutase solution activity, trypsin inhibitor solution was added.
The addition of the trypsin inhibitor solution was needed due to the fact that the medium used for this cell line does not contain FBS. The cells plus medium were centrifuged for 5 min at 1200 rpm and the supernatant was discarded. The cells were seeded at a density between 4x10 4 and 6x10 4 cells/mL and incubated. 10 mL of fresh, pre-warmed medium was added to the cells, the content was transferred to a t25-flask which was then returned to the incubator.

Thawing of cells
The frozen primary HUVEC Lonza pooled donor were thawed slowly by adding medium into the vial and transferring the thawed content into a tube with 9 mL of EBM-2 Basal medium supplemented with EGM-2 SingleQuot Kit. The cells were then centrifuged for 5 minutes at 1200 rpm. The supernatant was discarded and the cells were transferred to a t25-flask containing 10 mL of fresh medium.

Culturing and subculturing
The cells were culture at 37ºC, 5% CO 2 and 85% humidity, exchanging media until confluence was reached. Medium exchange was performed by warming up fresh culture medium at 37°C in water bath for at least 30 min. The used medium was carefully poured from the t-flask into a waste pot. Immediately, 10 mL of fresh pre-warmed culture medium was added to the t-flask which was then returned to the incubator. Confluent cultures were washed twice with sterile PBS without Ca 2 and Mg 2 and incubated for 5 minutes at 37ºC with 2 mL of Accutase cell detachment solution. Cell detachment was facilitated by repeated pipette aspirations. As to stop Accutase solution activity, the cells were transferred to 10 mL of fresh medium and centrifuged for 5 min at 1200 rpm. The supernatant was discarded and fresh medium was added. Cells were seeded at a density between 4x10 5 and 8x10 5 cells/mL and incubated.

Preparation of cell lysates
As to measure the DPP-IV levels in RPTEC/TERT1 and HUVEC cells, 1 mL of each which contained 10 6 cells was collected from the culture medium and centrifuged at 13000 rpm for 5 min. The medium was discarded and the cells were washed twice by adding 1mL of PBS to the cell pellet, mixing thoroughly and centrifuging at 13000 rpm for 5 min. The solution was discarded and replaced with 1 mL of 50 mM Tris-HCl buffer pH 8.3 containing 0.1% TritonX. The cells were lysed which was performed by vortexing and keeping the cells 1 h on ice or at 4°C to ensure the cells were properly lysed they were vortext in between as well. Until use the cell lysates were keept at -20ºC.

Enzymatic digest with Chymotrypsin
Enzymatic digest with chymotrypsin was conducted with the SMART Digest Kit Chymotrypsin according to the user manual. In short, 50µl sample in Tris Buffer was transferred to a PCR tube containing the SMART Digest standard resin slurry and 150 μL digest buffer. Digestion was conducted in a heated shaker at 1100 rpm at 70°C for 40 min. To stop the digestion, the tubes were centrifuged and 5 μL of the supernatant were directly injected into the HPLC system.