Targeted Capture of Chinese Hamster Ovary Host Cell Proteins: Peptide Ligand Discovery

Abstract

The growing integration of quality-by-design (QbD) concepts in biomanufacturing calls for a detailed and quantitative knowledge of the profile of impurities and their impact on the product safety and efficacy. Particularly valuable is the determination of the residual level of host cell proteins (HCPs) secreted, together with the product of interest, by the recombinant cells utilized for production. Though often referred to as a single impurity, HCPs comprise a variety of species with diverse abundance, size, function, and composition. The clearance of these impurities is a complex issue due to their cell line to cell line, product-to-product, and batch-to-batch variations. Improvements in HCP monitoring through proteomic-based methods have led to identification of a subset of “problematic” HCPs that are particularly challenging to remove, both at the product capture and product polishing steps, and compromise product stability and safety even at trace concentrations. This paper describes the development of synthetic peptide ligands capable of capturing a broad spectrum of Chinese hamster ovary (CHO) HCPs with a combination of peptide species that allow for advanced mixed-mode binding. Solid phase peptide libraries were screened for identification and characterization of peptides that capture CHO HCPs while showing minimal binding of human IgG, utilized here as a model product. Tetrameric and hexameric ligands featuring either multipolar or hydrophobic/positive amino acid compositions were found to be the most effective. Tetrameric multipolar ligands exhibited the highest targeted binding ratio (ratio of HCP clearance over IgG loss), more than double that of commercial mixed-mode and anion exchange resins utilized by industry for IgG polishing. All peptide resins tested showed preferential binding to HCPs compared to IgG, indicating potential uses in flow-through mode or weak-partitioning-mode chromatography.

Targeted capture of Chinese hamster ovary host cell proteins: Peptide ligand binding by proteomic analysis

“…In comparison, benchmark anion exchange resin Capto Q yielded 60.7% (179 of 295) at 20 mM and 33.6% (71 of 211) at 150 mM. Lower HCPs binding by Capto Q at a high salt concentration was anticipated, given that this resin relies solely on electrostatic binding; further, increased binding of the mAb product (isoelectric point ~ 7.6) by Capto Q at pH 8 (64.8% ± 2.6% at 20 mM and 57.0% ± 2.9% at 150 mM) also reduces the number of binding sites available for HCP capture as shown in Table (Lavoie et al, ). The mixed‐mode resin Capto Adhere showed high overlap in bound HCPs (71.4%, 220 of 308) at low salt concentration; however, promiscuous binding of HCPs was also accompanied by significant loss of mAb product (>80% for all pH conditions; Lavoie et al, ).…”

“…The feed samples were prepared by diafiltration of the cell culture fluid against the different buffers, incubated for 1 hr with the equilibrated adsorbents, and the supernatants (unbound and wash fraction) were collected and pooled before analysis. The resulting performance of each resin as a function of buffer condition by total HCP and IgG bound as reported in Lavoie et al () is summarized in Table .…”

“…The synthesis procedure was performed according to a protocol adapted from Menegatti et al () and is described in detail in prior work (Lavoie et al, ). Individual ligand candidates previously identified were synthesized and pooled as described in previous work (Lavoie et al, ).…”

“…The synthesis procedure was performed according to a protocol adapted from Menegatti et al () and is described in detail in prior work (Lavoie et al, ). Individual ligand candidates previously identified were synthesized and pooled as described in previous work (Lavoie et al, ). Briefly, the peptides were individually synthesized directly on Toyopearl AF‐Amino‐650M resin, and the resulting adsorbents were pooled as follows: (a) hexameric hydrophobic positive (6HP) resin, comprising of the peptide sequences GSRYRYGSG, RYYYAIGSG, AAHIYYGSG, IYRIGRGSG, HSKIYKGSG; (b) hexameric multipolar (6MP) resin, comprising ADRYGHGSG, DRIYYYGSG, DKQRIIGSG, RYYDYGGSG, YRIDRYGSG; (c) tetrameric hydrophobic positive (4HP) resin, comprising HYAIGSG, FRYYGSG, HRRYGSG, RYFFGSG; and (d) tetrameric multipolar (4MP) resin, comprising DKSIGSG, DRNIGSG, HYFDGSG, and YRFDGSG.…”

“…Briefly, the peptides were individually synthesized directly on Toyopearl AF‐Amino‐650M resin, and the resulting adsorbents were pooled as follows: (a) hexameric hydrophobic positive (6HP) resin, comprising of the peptide sequences GSRYRYGSG, RYYYAIGSG, AAHIYYGSG, IYRIGRGSG, HSKIYKGSG; (b) hexameric multipolar (6MP) resin, comprising ADRYGHGSG, DRIYYYGSG, DKQRIIGSG, RYYDYGGSG, YRIDRYGSG; (c) tetrameric hydrophobic positive (4HP) resin, comprising HYAIGSG, FRYYGSG, HRRYGSG, RYFFGSG; and (d) tetrameric multipolar (4MP) resin, comprising DKSIGSG, DRNIGSG, HYFDGSG, and YRFDGSG. All ligands were synthesized using standard Fmoc‐amino acid coupling chemistry and deprotection procedures as described in prior work (Lavoie et al, ). Resins were washed three to five times first with DMF then 20% methanol and stored in 20% methanol at 2–8°C.…”