Mass spectrometry study of a transferrin-based protein drug reveals the key role of protein aggregation for successful oral delivery

Abstract

A recently designed human growth hormone/transferrin fusion protein (GHT) remains one of the very few examples of a protein capable of eliciting measurable therapeutic response after oral administration. To better understand the underlying factors that resulted in this rare success of nonparenteral protein drug delivery, we analyzed proteolytic stability and receptor binding properties of this protein, the key factors in overcoming the primary barriers to successful oral delivery. Analysis of GHT by a combination of size exclusion chromatography and mass spectrometry revealed that a significant protein population exists in an oligomeric (GHTx) state in addition to the anticipated monomer (GHT1). These states of GHT were evaluated for their survivability in stomach-like conditions, as well as their ability to bind transferrin receptor (TfR). Our results reveal an exceptional stability of GHTx, as well as the preserved ability to bind TfR, a critical first step in crossing the epithelial-intestinal barrier through receptor-mediated transcytosis.

Fig. 1.

(A) SEC chromatograms of GHT (black trace) and Tf standard (gray trace). (B) ESI MS of SEC fraction B. (C) Dynamic light scattering analysis of SEC fraction A (black trace) and a reference Tf sample (gray).

Fig. 2.

Progress of TF (A) and GHTx (B) hydrolysis with pepsin at pH 3.5 monitored by SEC and kinetics of TF, GHT1, and GHTx hydrolysis (C).

Fig. 3.

GHT1 binding to TfR monitored by SEC (A) and ESI MS (B).

Fig. 4.

SEC of GHTx, TfR, and their mixture (A), and total ion chromatograms (B) of tryptic digests of SEC fractions of TfR (blue trace) and GHTx/TfR mixture (red trace) highlighted in A. (C and D) Detection of TfR in the early-eluting SEC fraction of GHTx/TfR mixture (highlighted with red in A): single-scan MS/MS spectrum (C) and extracted ion chromatogram (D) of a tryptic peptide eluting at 25 min (marked with asterisk in B). The blue trace in C shows the reference MS/MS spectrum of a tryptic fragment T71 (LTTDFNAEK) of TfR, and the blue trace in D corresponds to this peptide derived from TfR in the absence of GHTx (SEC fraction highlighted with blue in A).