Peptide Affinity Chromatography Applied to Therapeutic Antibodies Purification

Abstract

The interest in therapeutic monoclonal antibodies (mAbs) has significantly grown in the pharmaceutical industry, exceeding 100 FDA mAbs approved. Although the upstream processing of their industrial production has been significantly improved in the last years, the downstream processing still depends on immobilized protein A affinity chromatography. The high cost, low capacity and short half-life of immobilized protein A chromatography matrices, encouraged the design of alternative short-peptide ligands for mAb purification. Most of these peptides have been obtained by screening combinatorial peptide libraries. These low-cost ligands can be easily produced by solid-phase peptide synthesis and can be immobilized on chromatographic supports, thus obtaining matrices with high capacity and selectivity. Furthermore, matrices with immobilized peptide ligands have longer half-life than those with protein A due to the higher stability of the peptides. In this review the design and synthesis of peptide ligands, their immobilization on chromatographic supports and the evaluation of the affinity supports for their application in mAb purification is described.

Keywords: Biopharmaceuticals; Mass spectrometry; Monoclonal antibodies; Solid-phase peptide synthesis.

Fig. 1

Number of therapeutic monoclonal antibodies (mAbs) approved by the FDA each year (bars) and total approved (line) (updated until September 30th, 2021)

Fig. 2

Biopharmaceuticals recovery and purification steps (downstream processing) (SLS: solid–liquid separation such as filtration or centrifugation)

Fig. 3

Solid-phase peptide synthesis: N-α-protected (Fmoc in Fmoc/tBu chemistry) and side chain protected amino acid is coupled to a solid phase through a linker. After removing the N-α-protected group, the second N-α-protected amino acid is coupled. Coupling (a) and deprotection (b) steps are repeated until the desired amino acid sequence has been elongated. Finally, side chain protecting groups (stars) are removed and the peptide is cleaved from the solid support by a global deprotection step (c)

Fig. 4

Phage-display library screening: Phage particles with peptides displayed on their surface are incubated with the immobilized antibody. After washing the non-interacting phages, the ones adsorbed are eluted, isolated, and amplified in E. coli. Screening is repeated many times to obtain high affinity ligands

Fig. 5

One bead-one peptide library constructed by split and mix or divide, couple and recombine method. (a) the resin is divided into equal portions; (b) in each portion a different amino acid is coupled (c) after coupling and washing, the resin is recombined. The process is repeated until the desired length of the peptide is reached (X = variable positions). (d) Finally, all side chains are deprotected, leaving the peptides anchored to the resin beads for subsequent solid-phase analysis

Fig. 6

Parallel synthesis of candidate peptides for their analysis. (a) Evaluation of antibody binding to peptidyl-resin candidates: (a₁) antibody labeled with a reporter group such as a fluorescent dye is incubated with each peptidyl-resin. (a₂) Color fluorescence beads are observed under a microscope. (b) Peptide stability assessment: (b₁) peptidyl-resin beads are incubated in solutions with proteases or in cell culture broth. (b₂) whole peptide or C-terminal degradation products are separated from the solid support with ammonia vapor. (b₃) Peptide and degradation products are analyzed by MS. (c) SPR affinity analysis: (c₁) peptide is separated from the solid support with ammonia vapor. (c₂) interactions between peptides and antibodies can be studied in real time by SPR without labeling the analytes

Fig. 7

Peptide ligand site directed immobilization. a Lys is added at the C or N terminus allowing peptide immobilization on a N-hydroxysuccinimidyl (NHS) activated matrix through the Lys ε-amino group. b Cys is added at the C or N terminus allowing peptide site directed immobilization through its sulfhydryl group on an iodoacetyl activated matrix in those cases where the peptide has Lys in its sequence

Fig. 8

Affinity chromatographic performance evaluation. a Equilibrium adsorption isotherm measurement. b Breakthrough curve measurement