The purpose of this work was to develop equipment and procedures for large‐scale aseptic production of injectable microsphere (MS) drug conjugates. The two major challenges were (a) to prepare sufficient amounts of MSs for clinical trials, and (b) to prepare the MS‐drug product under aseptic conditions. The approach was to prepare the MS‐drug conjugate in two stages. Stage 1 was the preparation of monodisperse tetra‐PEG amine derivatized MSs (amino‐MS) from two soluble PEG prepolymers under low to no bioburden conditions. To accomplish this, custom‐engineered equipment compatible with both aqueous and organic solvents was fabricated for parallel microfluidic preparation of amino‐MS. The system was capable of preparing up to ∼2 L of high quality 50 μm diameter amino‐MS per day. Stage 2 was the sterilization of the starting amino‐MS and aseptic production of the MS‐drug conjugate. The amino‐MS were first sterilized by autoclaving then transferred to a custom‐engineered autoclave‐sterilized washer‐reactor. This apparatus allowed for activation of the amino‐MS and attachment of a linker‐drug under aseptic conditions to give the sterile MS‐drug conjugate drug substance. The final drug product was produced by addition of excipients to form a homogeneous suspension. The entire process is exemplified by an engineering production run of a sterile MS‐peptide drug product.

Sterilization of degradable polymeric biomaterials intended for injection presents a formidable challenge. Often, either the polymer backbone or labile crosslinks controlling degradation are adversely affected by commonly used sterilization methods. The purpose of this work was to develop an approach to sterilize tetra-polyethylene glycol hydrogel microspheres (MSs) with -eliminative crosslinks that are destined to be carriers for drug delivery. The approach taken was to acidify the medium to compensate for the base-catalyzed cleavage of linkers at high temperatures. We determined that rates of linker cleavage at pH 4 or below were sufficiently slow as to allow autoclaving and showed that precursor amine-derivatized MSs could withstand autoclaving at pH 4 for at least four cycles of 20 minutes each at 121 • C. Thus, amine-MSs need not be prepared aseptically, but instead can be prepared in a low bioburden environment, and then sterilized by autoclaving before drug attachment. K E Y W O R D Sbiodegradable hydrogel, drug delivery, half-life extension, microspheres, tetra-polyethylene glycol INTRODUCTIONWe have developed a general approach for half-life extension of therapeutics, in which a drug is covalently tethered to a long-lived carrier by a linker that slowly cleaves by -elimination to release the native drug (Scheme 1). 1 The cleavage rate of the linker is controlled by the nature of an electron-withdrawing "modulator" (Mod) attached to a carbon containing an acidic C-H bond. After rate-determining proton removal, the intermediate rapidly collapses to provide the free drug. These linkers are not affected by enzymes and are stable for years when stored at low pH and temperature. One carrier we use is a large-pore tetra-polyethylene glycol (PEG) hydrogel polymer. 2-4 These hydrogels-fabricated as uniform 50-μm microspheres (MSs)-are injected subcutaneously (SC) or locally through a small-bore needle where they serve as a depot to slowly release the drug. Importantly, a slower cleaving -eliminative linker is incorporated in crosslinks of these polymers, so gel degradation occurs after drug release. 3,5 Fabrication of tetra-PEG hydrogel MS-drug conjugates is achieved in two stages (Scheme 2). 3,4 Stage 1 involves preparation of amine-derivatized MSs. Here, equimolar amounts of an 8-arm PEG containing 4 amine-and 4 azido-linker end groups and a 4-arm PEG containing cyclooctyne end groups are mixed in a droplet forming device. The azide (A) and cyclooctyne (B) end groups of the PEG prepolymers react within the droplets by strain-promoted alkyne-azide cycloadditions (SPAAC) to form 1,2,3-triazoles and provide homogeneous amine-derivatized tetra-PEG

The purpose of this work was to develop technology for facile, large‐scale production of tetra‐polyethylene glycol (PEG) hydrogel amino‐microspheres to serve as intermediates for injectable microsphere‐drug conjugates produced under cGMP guidelines. Here we developed equipment and procedures utilizing tubular cross‐flow membrane emulsification to produce the amino‐microspheres. The equipment comprised a polyether ether ketone tube containing 1000 evenly spaced pores encased in a cylindrical stainless steel jacket. The dispersed phase – an aqueous solution of two polymerizable tetra‐PEG prepolymers – is delivered into the outer jacket of the assembly by a pulse dampened pump, and the continuous phase – decane and surfactant – is delivered into the bore of the microporous tube from a pressurized tank. As the dispersed phase is pressed through the pores, the continuous phase induces the detachment of small, uniform droplets of ˜60 μm diameter at the mouths of the pores. After collection of the emulsified droplets, they are allowed to self‐polymerize and are then sieved to remove aberrantly small and large particles. The polymerized amino‐microspheres – obtained in ˜70% yield – are well within acceptance specifications, and show excellent injectability. Using this approach, we could prepare about 30 L of swollen amino‐microspheres per day which could be forwarded to production of microsphere‐drug substance under cGMP guidelines.

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