Development of peptide ligands for the purification of α-1 antitrypsin from cell culture fluids

Peptide ligands for the affinity purification of adeno-associated viruses from HEK 293 cell lysates

“…The physicochemical properties of the selected sites guided the design of a peptide library for the selection of candidate ligands: (i) a chain length of 6 or 8 monomers was chosen based on the average size of the pockets (Van der Waals volume ∼ 950 - 1150 Å 3 ; projection area ∼ 150 – 250 Å 2 ), which provides an ideal balance – based on prior knowledge 50,55,58,80,81 – between the expected biorecognition activity and manufacturing cost; (ii) the combinatorial positions in the library were randomized with alanine, asparagine, glutamic acid, histidine, isoleucine, lysine, phenylalanine, serine, and tryptophan, which were adopted as the amino acids capable of forming a network of diverse non-covalent interactions with the selected binding pockets; and (iii) a Gly-Ser-Gly (GSG) tripeptide spacer was utilized to link the combinatorial segment of the library to the resin to improve peptide display, thus promoting the outcome of library screening and subsequent Edman sequencing. The peptide libraries were synthesized following the “split-couple-and-recombine” technique 82,83 on ChemMatrix beads – porous, hydrophilic, translucent particle that have proven an excellent substrate for the synthesis and selection of peptide ligands 57,84,85 .…”

“…The labeling method of viral vector and proteins was performed as reported in the previous studies 50,55 . The AAV2, AAV9 were labeled using NHS-Alexafluor 594 (NHS-AF594, red), while the host cell proteins (HCPs) contained in the HEK 293 cell culture lysate were collectively labeled with NHS-Alexafluor 488 (NHS-AF488, green).…”

“…Peptide synthesis was performed on a Syro I automated peptide synthesizer (Biotage, Uppsala, Sweden) using nine protected amino acids, namely Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Trp(Boc)-OH, Fmoc-His(Trt)-OH, Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Glu(OtBu)-OH. 50,51 Each amino acid coupling step was performed at 45°C for 20 min, using 3 equivalents (eq.) of protected amino acid at the concentration of 0.5 M, 3 eq.…”

“…sate. The screening process of AAV-targeting peptides was performed as reported in the previous studies 50,55 . A screening mix was initially prepared by spiking AF594-labeled AAV2 and AAV9 in AF488-labeled HEK 293 cell culture lysate to obtain a final AAV titer of 5•10 11 vp/mL and a HEK 293 HCP titer of ~0.5 mg/mL.…”

Simultaneous Recovery of Bovine Serum Albumin and Bovine Immunoglobulin G with Dual-Ligand Hydrophobic Charge-Induction Chromatography

“…Chromatography can be categorized based on the type of ligand employed, such as ion-exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, and mixed-mode chromatography. − The performance of chromatography is greatly affected by the type and operating mode of ligands. , Therefore, the discovery and optimization of ligands have always been a major focus of research in chromatography development. − However, the rate of discovery of new effective ligands has recently decreased. Fortunately, some studies have determined promising results by optimizing ligand operation modes. − For example, the grafting of ligands to create a three-dimensional distribution has been shown to increase chromatographic adsorption capacity. ,− Sun and colleagues , conducted a systematic study on an anion exchanger of polyethylenimine (PEI)-grafted Sepharose FF and demonstrated enhanced adsorption capacity, effective pore diffusion, and an upgraded adsorption rate of bovine serum albumin (BSA).…”