Recombinant manufacturing of multispecies biolubricants

Abstract

Lubricin, a lubricating glycoprotein abundant in synovial fluid, forms a low-friction brush polymer interface in tissues exposed to sliding motion including joints, tendon sheaths, and the surface of the eye. Despite its therapeutic potential in diseases such as osteoarthritis and dry eye disease, there are few sources available. Through rational design, we developed a series of recombinant lubricin analogs that utilize the species-specific tissue-binding domains at the N- and C-termini to increase biocompatibility while replacing the central mucin domain with an engineered variant that retains the lubricating properties of native lubricin. In this study, we demonstrate the tissue binding capacity of our engineered lubricin product and its retention in the joint space of rats. Next, we present a new bioprocess chain that utilizes a human-derived cell line to produce O-glycosylation consistent with that of native lubricin and a purification strategy that capitalizes on the positively charged, hydrophobic N- and C-terminal domains. The bioprocess chain is demonstrated at 10 L scale in industry-standard equipment utilizing commonly available ion exchange, hydrophobic interaction and size exclusion chromatography resins. Finally, we confirmed the purity and lubricating properties of the recombinant biolubricant. The biomolecular engineering and bioprocessing strategies presented here are an effective means of lubricin production and could have broad applications to the study of mucins in general.

Keywords: biomanufacturing; lubrication; lubricin; mucin.

Figure 1:. Generation of multispecies recombinant lubricins (rLub).

(A) Upper: Schematic of an engineered rLub with 59 synthetic KEPAPTTP repeats flanked by the native N- and C- globular domains of native human (Hn), equine (Eq), or canine (Ca) PRG4; threonine residues in the repeats are post-translationally modified with O-GalNAc glycans that can be further extended at the 3’ and 6’ hydroxyls indicated in red. Lower: constructs for the recombinant human (rHnLub), equine (rEqLub), and canine (rCaLub) lubricin-like glycoproteins. (B) Western blot showing rHnLub, rEqLub, and rCaLub produced in HEK 293-F cells. (C) Liquid chromatography, mass spectroscopy analysis (LC-MS) of O-glycans released from rHnLub through β-elimination. (D) Photographic and fluorescent images with fluorescent quantification of bovine cartilage explants following a short incubation with either unlabeled or fluorescently labelled rHnLub. (E) Representative confocal images of the surface of the cartilage explants in D additionally labelled with Hoescht 33342 to stain the chondrocyte nuclei.

Figure 2:. Retention kinetics of recombinant human-like lubricin following intra-articular injection.

(A) Schedule for IVIS imaging of sulfo-Cy7.5 labeled rHnLub (rHnLub-Cy7.5), sulfo-Cy7.5 labelled 500 kDa dextran (Dextran-Cy7.5), and unconjugated sulfo-Cy7.5 carboxylic acid (free dye) following intra-articular injection into the knees of Sprague-Dawley rats. (B) Dual-mode micro-CT/IVIS imaging of the rat knee joint following intra-articular injection of rHnLub-Cy7.5. (C) Representative IVIS images of the rat knee at the indicated time points following intra-articular injection with free dye, dextran-Cy7.5, and rHnLub-Cy7.5. (D) Normalized total fluorescence in the rodent knee at the indicated time points following injection of free dye, dextran-Cy7.5, and rHnLub-Cy7.5. Inset shows the same curves, expanded to visualize the rapid decay in the first 5 days. (E) Model and fitted parameters from the curves in (D).

Figure 3:. A scalable bioprocess chain to produce recombinant lubricin.

(A) Schematic diagram of the complete bioprocess chain showing the minimum set of operations. (B) Cell viability (squares) and viable cell density (circles) of a 1 L rEqLub production perfusion culture. (C) Harvested volume (squares) and cumulative rEqLub production (circles) from the perfusion culture in (B).

Figure 4:. Cation exchange chromatography is a high-resolution capture operation.

(A) Plot of the predicted local charge (100 AA window) along the rLub polypeptide backbone without glycosylation showing the net positive charge in the terminal, globular domains. (B) Product concentration by collected fraction during a linear gradient elution with NaCl from a cation exchange column at pH 5.5 (solid bars) or pH 6.8 (open bars). Concentration of rLub was determined by dot blot relative to the feed concentration. (C) Silver-stained gel image of the pH 5.5 separation shown in (B). The heavy bands above 460 kDa correspond to rLub. (D) Same as (C), but for the pH 6.8 separation. (E) UV absorbance (solid line) and conductivity (dashed line) chromatograms of the cation exchange step elution protocol. (F) Silver-stained gel image of the capture by step elution shown in (E). Lanes: F – feed, T – flow-through, W – column wash, E1 – 400 mM elution, E2 – 800 mM elution.

Figure 5:. Intermediate purification with hydrophobic interaction chromatography removes most residual impurities.

(A) UV absorbance (solid line) and conductivity (dashed line) chromatograms from a 1.0 M to 0 M sodium sulfate gradient elution. The peak in the shaded box corresponds to elution of the majority of rLub. The asterisk denotes the major impurity peak. (B) Silver-stained gel image of fractions from (A). Lanes: F – feed, T – flow-through. The rLub fractions are from the shaded area and asterisk from the corresponding peak in (A).

Figure 6:. Polishing by size exclusion chromatography results in a high-purity product.

(A) UV absorbance chromatogram of an SEC polishing operation on a Sephacryl S-400 column. The single and double asterisk denote peaks containing rLub. (B) Silver-stained gel image of the collected fraction from (A). Lanes: F – feed, followed by the first five fractions of the elution (shaded region in (A) with * and ** marking the lubricin containing peaks in (A)), 2 – rLub product from a 2-step process omitting intermediate purification by HIC.

Figure 7:. Recombinant lubricin is an effective cartilage lubricant.

(A) Coefficient of friction as a function of sliding speed. Cartilage explants were maintained in a bath of either PBS, 0.2 mg/mL rEqLub, 1 mg/mL rEqLub or bovine synovial fluid (BSF) (n= 5). (B) Coefficients of friction at 0.1, 1, and 10 mm/s as a function of lubricant.