Crossing the hurdles of translation—a robust methodology for synthesis, characterization and GMP production of cross‐linked high molecular weight hyaluronic acid particles (cHA)

Abstract

Hyaluronic acid (HA) is one of the most widely used extracellular matrix substrate in tissue engineering, drug delivery, and other clinical applications, due to its unique physiochemical properties and ubiquitous biological presence. In the past decade, there has been a surge in research paradigms involving HA products to evaluate their commercial feasibility. Numerous papers and reviews have reported procedures for chemical modifications and cross‐linking of HA, but the intricacies of their scale‐up in the production processes are often not discussed. Protected by confidentiality agreements with industry partners, information on these procedures is exclusive and not accessible within an academic setting. Establishing translatable synthetic protocols of HA would address this significant gap in the field and facilitate their use in other applications. The current method details a reproducible and robust method for producing particles that are composed of high molecular weight hyaluronic acid (cHA) and cross‐linked via a 4‐arm polyethylene glycol amine linker using 4‐(4,6‐dimethoxy‐1,3,5‐triazin‐2‐yl)‐4‐methyl‐morpholinium chloride chemistry. A critical analysis of previously reported procedures for their advantages and limitations (reaction parameters, molar equivalents, and reagents used for cross‐linking), forms the basis for this procedure and its subsequent adaptation to good manufacturing practices requirements. As a component of a Class III medical device, the reported cHA is in the form of non‐sized particles.

A nanoparticle platform for combined mucosal healing and immunomodulation in inflammatory bowel disease treatment

“…DMTMM is a morpholine-based reagent that is water-soluble and can be used as an alternative to EDC/NHS chemistry. This type of chemistry, indeed, offers higher aqueous stability of HA over EDC/NHS; it consists of a single step and can be carried out in water at physiological pH values [ 17 ]. We conjugated KPV to the HA backbone.…”

“…Our strategy provides with increased levels of GLP-2, in addition to anti-inflammatory effects. To achieve HA-KPV conjugation, we exploited a redox responsive (disulphide bond) linker using 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM·Cl), more easily scalable [ 17 ]. We chose a high MW HA (1 MDa) that has been described as anti-inflammatory [ 18 ].…”

A high molecular weight hyaluronic acid biphasic dispersion as potential therapeutics for interstitial cystitis

“…cHA was synthesized as per our reported cross‐linking methodology developed for its GMP translation. This methodology is an improvement of our previously reported and the literature procedures for cross‐linking HA 42‐46 . A homogeneous solution of HA (2 g at 10 mg/ml) was prepared in MES buffer (pH 6.0; 0.1 M) at 25°C.…”

“…In NMR, the peak integrals were compared for quantitative estimation. Carbazole assay was carried out following previously reported procedures using samples with and without salts 42,47,48 . All the reagents were freshly prepared and no commercial kits were used.…”

“…Considering this, the manuscript describes an in vitro model of urothelium barrier damage in order to examine the effects of native high molecular weight HA solution (commercial), cross‐linked HA solution (cHA) and the combination of biphasic solution (native HA solution and cHA). HA cross‐linking was carried out as per our reported GMP translatable methodology for the synthesis of cHA 42 . The cross‐linking was performed with 4‐arm‐polyethyeleneamine (PEG) linker using 4‐(4,6‐Dimethoxy‐1,3,5‐triazin‐2‐yl)‐4‐methylmorpholinium chloride (DMTM) chemistry.…”