WO2019152672A1 - Aseptic method and apparatus to prepare sterile formulations of drug-linked hydrogel microspheres - Google Patents

Abstract

An aseptic system is designed to make possible an aseptic production method for sterile drug-linked hydrogel microspheres.

Description

ASEPTIC METHOD AND APPARATUS TO PREPARE STERILE

FORMULATIONS OF DRUG-LINKED HYDROGEL MICROSPHERES

Acknowledgement of Government Support

[0001] This invention was supported in part by a grant from the National Science Foundation, Grant no. 1429972.

Related Application

[0002] This application claims benefit of U.S. application Serial Number 62/624,633 filed 31 January 2018 which is incorporated herein by reference in its entirety.

Technical Field

[0003] The invention is in the field of drug-delivery system preparation. In particular, it is directed to an aseptic system and method adapted for preparation of sterile formulations of drug-linked hydrogel microspheres.

Background Art

[0004] Preparation of sterile formulations which comprise hydrogel microspheres (in particular systems designed for“drug” delivery wherein the microspheres are coupled to therapeutic or diagnostic agents through linkers that effect release of the therapeutic or diagnostic agent in vivo ) presents problems not encountered in preparing sterile formulations of small molecule drugs. In particular, because the hydrogel microspheres are themselves in the micron range, use of 0.2 micron filters, a traditional method for sterilization of solutions or suspensions of relatively small molecules cannot be used. In addition, because typically hydrogel microspheres are composed of polymers that are sensitive to g-radiation, radiation sterilization is not acceptable. Neither is autoclaving which would essentially deplete the integrity of the composition.

[0005] A gamma radiation method for sterilizing an insoluble biodegradable

poly(ethylene glycol) gel is described in US patent 8,986,609 that requires solvating in a protective solvent prior to irradiation. The method appears limited to this context, and is not of general applicability to microspheres useful in drug or diagnostic delivery. [0006] W02003/035244 and its US counterpart, US 7,468,151 describe a generic alternative device for aseptic particle formation. In addition, a system for parallel microfluidic processes, called Telos© is marketed by Dolomite, Charlestown, MA.

[0007] The problem of providing sterile microspheres is solved by the present invention wherein aseptic microfluidic systems designed to accommodate the challenges posed by insuring sterility of drug-linked hydrogel microspheres have been designed such that the preparation of the microspheres and the linkage of said microspheres to the drug is itself accomplished under aseptic conditions and wherein the systems permit size sorting of the microspheres to ensure a desirable predetermined size range. Other methods of preparing hydrogel microspheres, though not under sterile conditions, are known in the art. See, for example, Liu, E.Y, et al. , Lab Chip (20l7)DOI: 10.1039/C7LC01088E.

Disclosure of the Invention

[0008] Thus, in one aspect the invention is directed to aseptic methods and systems to prepare a sterile composition comprising drug-linked hydrogel microspheres. The microspheres may be formed, for example from at least two prepolymers, or may originate from a single polymer. The method of the invention comprises conducting the process in a sealed and sterilized system comprising tanks and microfluidic chips fabricated from inert materials and maintained in aseptic condition. The steps of the method are as follows:

[0009] First, effecting the formation of microspheres by creating an emulsion by introducing into a manifold under pressure optionally through filtered tubing a solution of polymer or of each prepolymer from a supply tank for polymer or each prepolymer along with introducing an antisolvent. As used herein“antisolvenf’ refers to a liquid medium that effects formation of the microspheres from the components supplied and is immiscible with the solutions of the components. When the components are aqueous prepolymers, this is typically a hydrophobic solvent and surfactant. If the components are supplied in a nonpolar solvent, the antisolvent would typically be polar. A surfactant may be included, as noted above, as required to stabilize the microemulsion. The antisolvent is introduced also through filtered tubing from a supply tank for said antisolvent. The manifold contains at least one microfluidic chip said chip containing a multiplicity of channels. The polymer or prepolymer solutions are introduced into the channels of the microfluidic chip(s) where they are mixed and emulsified with the antisolvent to form a microemulsion comprising microspheres. [0010] Then, visualizing the microemulsion from each channel of said microfluidic chip(s) to determine size distribution of the microspheres in the emulsion and retaining flow from channels meeting the criterion of containing a sufficient percentage of microspheres within a predetermined size range and discarding flow from channels that do not meet this criterion.

[0011] Then, optionally introducing said retained microemulsion flows into two or more tanks, one comprising a sieve to discard any microspheres that are too large and another comprising a sieve to discard any microspheres that are too small, and, in the case of prepolymers, providing appropriate conditions to complete polymerization to obtain stable hydrogel microspheres, and in all cases removing antisolvent. If linkage to a drug or diagnostic agent is desired, also introducing any reagents needed to link the therapeutic or diagnostic agent to the particles, so that said drug-linked hydrogel microspheres are formed.

[0012] The drug-linked hydrogel microspheres are then washed to remove excess reagents and a suitable dosing formulation buffer is supplied.

[0013] The sterile composition comprising said optionally drug-linked hydrogel microspheres is then recovered, optionally directly into a syringe.

[0014] Temperature, pressure, flow rates, and visualization of the microemulsion formation and the retaining and discarding of channel flows are monitored and controlled by a computer operated supervisory control and data acquisition (SCAD A) system.

[0015] In another aspect, the invention is directed to the apparatus/system itself. The components of the system can be sterilized by autoclaving or chemical means and are composed of inert materials. The system is monitored while in use for integrity of the components.

[0016] In still another aspect, the invention is directed to a sterile composition of microspheres prepared by the invention method.

Brief Description of the Drawings

[0017] Figure 1 shows a schematic of the overall process of the invention when prepolymers are employed.

[0018] Figure 2 shows a more detailed schematic of the physical elements of the system of Figure 1. [0019] Figure 3 shows the results of typical size sorting conducted in a two-tank system.

[0020] Figure 4 shows a diagram of one embodiment of a sieved tank.

[0021] Figures 5A-5G show a block diagram of one embodiment of the invention.

Modes of Carrying Out the Invention

[0022] The invention is directed to systems and methods for preparing a sterile composition of microspheres of uniform size distribution and to the compositions thus obtained.

[0023] One important element of the invention is the use of microfluidic chips with multiple channels for formation of microemulsions comprising the desired microspheres for example composed of hydrogel, thus permitting assessment of the size range of the microspheres formed in the context of the process in order to ensure an appropriate size distribution, wherein a desirable percentage of the microspheres in each microfluidic chip channel is within a predetermined size range. It is particularly advantageous to use multiple such microfluidic chip channels so that channels that do not contain a satisfactory percentage of microspheres within the predetermined size range can be discarded and the operation continued. This is in contrast to the use of a single vessel where unsatisfactory results would mandate abandoning the process and beginning again.

[0024] The invention system may provide several opportunities for ensuring appropriate particle size— both the sorting of channels to retain only those with an acceptable percentage of appropriate sized microspheres and in some embodiments the provision of sieves downstream in the system to remove particles that are too large or too small.

[0025] By providing filtration systems using 0.2 micron filters to ensure sterile conditions for the fluids entering the system, the entire process of preparing the drug delivery

formulation wherein microspheres are optionally coupled to therapeutic or diagnostic agents through linkers is aseptic ab initio without the need for further sterilization. This requires suitable design of components of the system such that contamination is not introduced in any step in the process. In addition, the process can be monitored for integrity by tapping samples of the fluids passing through the system as the process is conducted and assessing these for contamination. One method for assessing contamination is mass spectrophotometric analysis for detection of amino acids or proteins associated with contaminating

microorganisms. [0026] While the system and method of the invention is illustrated using at least two types of prepolymers, a single prepolymer or a single preformed polymer could also be used.

[0027] The system and process of the invention can be explained by the depictions shown in the drawings. These are intended as schematics to be generalized and are not intended to limit the invention.

[0028] Figure 1 shows an embodiment wherein two prepolymers, A and B that form a hydrogel, are employed and the microspheres are ultimately linked to a drug. The starting materials which are of appropriate pharmaceutical grade are subjected to sterilizing filtration and injected into sterile equipment where, after a subsequent sterilizing filtration, solutions of the prepolymers A and B are mixed with an antisolvent and optionally a surfactant (not shown) to obtain a microemulsion. When the prepolymers are hydrophilic, for example polyethylene glycols, they may be provided as aqueous solutions and the antisolvent is water- immiscible, for example a hydrocarbon. Alternatively, hydrophilic prepolymers may be provided as solutions in a polar organic solvent such as acetonitrile or dimethylformamide and the antisolvent is again an immiscible hydrocarbon. When the prepolymers are hydrophobic, they may be provided in an organic solvent and the antisolvent is organic- immisicible, for example water. The microemulsion is formed at the inlet of a microfluidic channel system and adjusted for flow rate and pressure through a quality control imaging and processing system as shown on the dotted line. Temperature may also be controlled. The microemulsion then flows through channels of microfluidic chips to effect particle size fractionation by retaining channels with appropriate size distributions and discarding channels that do not meet this criterion. The flow from the retained channels comprising the microemulsion is then subjected to chemical derivitization to add the drug or diagnostic reagents which are introduced, as were the prepolymers, through sterilizing filtration and using appropriate grade reagents. This may be preceded by introducing reagents (not shown) to complete polymerization and stabilize the particles if necessary. The derivatized particles are sorted for size at this point through sieving (not shown) and concentrated after washing and then introduced (in this illustration) into a syringe.

[0029] It is again emphasized that this schematic is itself simply an illustration and, for example, the sterile composition may be recovered in ways other than filling a syringe, such as by placing the sterile composition into disposable plastic ware, wherein further

concentration can be accomplished prior to shipping the microspheres themselves. The sieving size differentiation not shown in the figure, but mentioned in the description above is also, for example, optional.

[0030] Figure 2 is another, alternative illustrative depiction of the embodiment of the invention shown in Figure 1. As shown in this example, pressure for introduction of the particle formation reagents— prepolymers A and B and the hydrophobic

anti sol vent/ surfactant— is supplied by nitrogen. In Figure 2, separate tanks are shown for each component (A, B and antisolvent) but separate compartments of a single tank could be used. Thus in designating a number of tanks, it is intended simply that these reagents be kept separate until they are mixed upstream of the channels of a microfluidic chip as described below. As shown, these are supplied upstream of channels of a microfluidic chip wherein the top and bottom arrows show the flow of the solvent/surfactant and the two intermediate arrows show the flow of the respective prepolymers. These are mixed as shown when the solvent/surfactant encounters the mixture with a flow perpendicular to the flow of the mixture resulting in the formation of droplet particles from the prepolymers in the microemulsion which emulsion containing microspheres then flows into the multiplicity of channels of the microfluidic chip (only one channel of which is shown). Appropriate conditions for this step are critical in terms of control of the flow rate, and pressure which need to be adjusted in such a manner that appropriate size microspheres are obtained.

[0031] The flow rate of mixtures must be fast enough to permit the particles to form, but not of sufficient velocity that the channel becomes plugged. In a preferred embodiment, the channel is coated with a hydrophobic coating, to keep the dispersed phase from wetting the channel as this would result in failure to form drops, as well as additional polymerization and formation of satellite drops that are smaller or larger than desired. As shown, a computer operated system supervises and controls these factors and functions as a data acquisition system that monitors this process.

[0032] This supervisory control and data acquisition (SCADA) system also analyzes each channel for size distribution and sends to waste the flow from channels wherein too many microspheres fall outside a predetermined size range. Typical predetermined size ranges are between 10 and 100 mih, more typically between 40 and 80 mih preferably with an average diameter of 50 mih. The percentage of particles with the predetermined size range to provide a desired size distribution depends on the nature of the composition itself, but is typically at least 50% of the particle content of the channel more preferably 60% or 70% and more preferably more than 90% or 98%. Only a single channel is shown in this detail of the particle analysis component, but typically, the number of channels is at least 7, although as few as 2 channels are theoretically possible. More commonly, the number of channels is 10, 20, 40, 70, 100, and intermediate values. Microfluidic chips comprising 7 channels are readily obtainable so multiples of 7 are convenient, such as 7, 14, 21 ... etc.

[0033] As also shown in Figure 2, the flow from channels containing appropriate concentrations of particles of predetermined size /. e. , a desired size distribution are then passed into a first tank to which is added reagents to complete polymerization, including, optionally crosslinking the hydrogel, and then contacted with reagents for attaching the therapeutic or diagnostic agent through a linker to the hydrogel microspheres. (Both types of such agents are referred to herein as“drugs” for brevity.) The first tank contains a sieve that retains particles whose size is too large which are then sent to waste and those particles passing through the sieve enter into the second tank which introduces reagents for washing and substituting buffer as excipient. An outlet shown by an arrow allows harvest of the product while a sieve at the bottom of the tank admits and discards particles that are too small. The product is then recovered, for example, by filling a syringe, or container.

[0034] The reagents shown are for illustration only and depend on the nature of the hydrogel components and on whether additional modification is to be made, such as linking to a drug. In any case, the downstream vessels sequentially remove, by sieving,

microparticles that are too large or too small.

[0035] Figure 3 upper left shows a typical distribution of particles from the first sieved tank of Step B of Figure 2 that are passed to a second sieved tank, while those that are retained by the sieve and sent to waste are shown in Figure 3 lower left. The flow through the sieve in the second sieved tank, which is discarded, as shown in Figure 3 upper right and the retained product is shown in Figure 3 lower right.

[0036] It is thus seen that the method of the invention provides an efficient aseptic method and apparatus/system to prepare drug release compositions based on hydrogel microspheres wherein therapeutic and diagnostic agents are coupled to said microspheres typically through a linker or wherein sterile microspheres per se are simply formed.

Although linkers are advantageous when active agents are included, direct coupling to the microspheres or simple entrapment of an active are alternative embodiments also suitable to the system and process of the invention. [0037] The multichannel device, in some instances a microfluidic chip, employs at least two inlets -at least one for the materials that will compose the microparticles and at least an additional juxtaposed inlet for an anti-solvent that will effect microsphere formation. The starting material for the microsphere composition may be a polymer of which the

microspheres are composed, in which case only a single inlet for supply of the starting material is required (although additional inlets could be employed if desired). Alternatively, the ultimate polymers of which the microspheres are composed may be assembled during the process of the invention and in the invention system. In this case, more than one inlet or a prior junction of tubes leading to the inlet is required for the multiple starting materials - all of which are designated“prepolymers”. For example, two such prepolymers, A and B, in one embodiment are a multivalent polymer A and a crosslinker B that is capable of intra- and/or inter- linkages of polymer A. At least two inlets or prior junction (for A and B) would then be required, along with the inlet for the anti-solvent. In another instance, two different multivalent polymers, Al and A2 are employed (along with crosslinker B), thus requiring a total of four inlets or appropriate prior junctions. In the case of prepolymers, what is referred to as a“multivalent polymer” may be comparatively small and traditionally thought of as an oligomer or even a monomer. Thus,“prepolymer” as used herein refers simply to components, including crosslinkers that ultimately compose the microsphere where chemical bonding is effected to provide the ultimate interlocking substance. “Prepolymer” thus may itself refer to a polymer.

[0038] Formation of hydrogels and materials that are suitable prepolymers, suitable linkers and suitable drugs are described, for example, in U.S. patent 9,649,285, PCT

Publication WO 2013/036847 and published U.S. applications 2015/0352246 and

2016/0271227. The contents of these documents are incorporated herein by reference.

[0039] An illustration of the general operation of the apparatus of the invention may be described as follows:

[0040] Liquid process reagents (prepolymer solutions and continuous phase) are introduced into a properly cleaned, steam-sterilized, and integrity -verified filter-tank assembly by means of 0.2 um sterilizing inlet filters of acceptable chemical compatibility. Integrity-verification consists of testing the tanks, fluidic-tubes, attached filters and all respective fittings by means of a computer controlled test system that measures filter-bubble points and apparatus leak-tightness, all in the post-sterilization - pre-use state, and records data as proof of integrity. This method of integrity testing applies to all sterilizing filters, fluidic components, reactors, and sensors subsequently mentioned.

[0041] Once filled with liquid process reagents the tanks are pressurized with inert gas (nitrogen), also introduced through integrity-verified nitrogen-inlet 0.2 um sterilizing filters. The gas pressure drives the fluids from the tanks through a set of 0.2 um sterilizing liquid- outlet filters, and into a flow-rate sensor assembly. The flow sensor assembly is attached between the filter-tank assembly and a downstream multi-chip microfluidic emulsion synthesis assembly by means allowing for sterilization of the sensor assembly by a liquid- chemical germicide and post-sterilization integrity-verification as previously described.

[0042] A computerized controller monitors the liquid flow rates from the sensors in addition to the pressure in the tanks via pressure transducers mounted to the non-sterile side of the nitrogen-inlet filters. The PID based controller maintains a constant liquid flow rate set point, using sensor liquid flow as the process variable, and output to high resolution proportional pressure regulators that adjust the pressure in the tanks as needed to maintain liquid flow. This type of closed-loop pressure-driven flow control provides stable pulse-free flow.

[0043] The flow-regulated liquid process streams leaving the sensor assembly pass to a manifold that distributes the streams to multiple emulsion-producing microfluidic chips. Each chip containing multiple emulsion producing geometries or“drop-formers” that mix the prepolymer solutions immediately before emulsifying the mixture into the continuous phase. The output emulsion of each microfluidic chip passes through a valve allowing for the output to be: collected, stopped, or diverted to waste. For collection a second manifold merges the output of all chips and feeds the combined emulsion to the washer-reactor assembly. This microfluidic chip-valve-manifold assembly being sterilized prior to use by steam or liquid chemical germicide and integrity-verified in terms of leak tightness.

[0044] During the emulsion generation process a machine-vision camera system records images of the emulsified drops within the microfluidic chips. This computer controlled system records images of every emulsion producing geometry at a frequency deemed appropriate to support acceptable product quality. The images are passed into a computerized analysis program that generates particle-size statistics that predict emulsion quality on an individual-chip and batch process basis. Individual chips producing unacceptable product can be shut off from the system without interrupting production from the remaining chips. [0045] The combined emulsion from the microfluidic chip assembly is passed to a stirred temperature-regulated sieve-bottom reactor assembly, where the product-emulsion is allowed to pool to the desired batch size and if prepolymers have been used polymerize for an appropriate time. After polymerization time is reached the polymer-microspheres (no longer an emulsified liquid) suspended in the continuous phase are passaged through a metallic sieve, capable of excluding particles of undesirably large size, into a second sieve bottom stirred sieve-bottom reactor. The second reactor has a metallic sieve capable of retaining particles of the desired size and passing process fluids and particles of undesirably small size. In this second reactor process fluids (wash solvents, reagents for chemical reactions, etc) are introduced through 0.2 um sterilizing filters of appropriate chemical compatibility. The dual washer-reactor assembly containing provisions for stirring, sieves, and sterilizing filters being sterilized by steam prior to use and integrity -verified as previously described.

[0046] The compositions prepared by the methods and systems described are

compositions of microspheres of uniform size, optionally linked to active agents, such as drugs, which are free of microbial contamination. Typically, the microspheres have diameters of 20-200 nm or 20-80 nm or 40-70 nm and vary by no more than ±15% or ±10% or ±5%. In some embodiments, the microspheres are formed from prepolymers of PEGylated linkers comprising cognate functional groups, such as azides/cyclooctynes, amines/acyl halides or succinimidyl esters, and the like. In some embodiments, the linkers are

biodegradable, such as by beta elimination. Other biodegradable linkages may also be used, such as those susceptible to hydrolysis - e.g. esters, amides, carbonates, phosphoesters and the like.

[0047] All documents cited herein are incorporated by reference. References to“a”,“an” and the like refer to one or more than one unless otherwise clear from the context. In addition plural forms of nouns may include the singular as well as multiple embodiments of the designated subject.

[0048] The following examples are intended to illustrate but not to limit the invention. Preparation A

Preparation of Prepolymer A: N^a-Linker-Lysine-PEG wherein R = CtLSCriCtL-

Step 1

[0049] /V“ -(7 -Azido-l-methylsulfonyl-2-heptyloxycarbonyl)-lf -Boc-Lys-OSu . A suspension of H-Lys(Boc)-OH (72 mg, 0.29 mmol)

in 0.79 mL H₂0 was successively treated with 1 M aq NaOH (0.29 mL, 0.29 mmol), 1 M aq NaHC0₃ (0.27 mL, 0.27 mmol), and a 0.2 M solution of 0-(7-azido-l-methylsulfonyl-2- heptyl)-0’-succinimidyl carbonate (100 mg, 0.266 mmol, 0.1 M final concentration) in 1.35 mL of MeCN. After stirring for 1 h at ambient temperature, the reaction was judged to be complete by C18 HPLC (ELSD). The reaction mixture was partitioned between 40 mL of 1 : 1 EtOAc:KHS0₄ (5% aq). The layers were separated, and the aqueous phase was extracted with 20 mL of EtOAc. The combined organic layer was successively washed with water and brine (20 mL each). The organic phase was dried over MgS0₄, filtered, and concentrated to provide the crude intermediate carboxylic acid (124 mg, 0.244 mmol, 92% crude yield) that was used in its entirety in the next step.

Step 2

[0050] DCC (60% in xylenes, 2.6 M, 0.12 mL, 0.32 mmol) was added to a solution of A“- (7-azido- 1 -rnethylsulfonyl-2-heptyloxycarbonyl)-A^f£-Boc-Lys-OH (124 mg, 0.244 mmol, 0.1 M final concentration) and /V-hydroxysuccinimide (36 mg, 0.32 mmol) in 2.4 mL of CH2CI2. After stirring at ambient temperature for 1 h, the reaction mixture was filtered through a cotton plug, and the filtrate was loaded onto a SiliaSep 4 g column. Product was eluted with a step-wise gradient of acetone in hexane (0%, 10%, 20%, 30%, 40%, 50%, 30 mL each). Clean product-containing fractions were combined and concentrated to provide the title compound (124 mg, 0.205 mmol, 77% yield, two steps) as a white foam.

[0051] LC-MS, two diasteremoers ( m/z ): calc, 627.2; obsd, 627.1 [M+Na]⁺ and calc,

505.2; obsd, 505.0 [M-Boc+H]⁺.

Step 3

[0052] [IP -(7-Azido-l-methylsulfonyl-2-heptyloxycarbonyl)-Bt -Boc-Lys] ₄-PEG20_kDa,

105BH09. PEG₂o_kDa-( H)₄ (250 mg, 12.5 pmol, 50.0 pmol NH₂, 0.02 M NH₂ final concentration) was dissolved in 1.25 mL of MeCN. A solution of A“-(7-Azido-l- m ethyl sulfonyl-2-heptyloxycarbonyl)-AA-Boc-Lys-OSu (39 mg, 65 pmol) in 1.25 mL of MeCN was added. The reaction was stirred at ambient temperature and analyzed by Cl 8 HPLC (ELSD). The starting material was converted to a single product peak via three slower eluting intermediate peaks. After 45 min, Ac₂0 (4.7 pL, 50 pmol) was added. The reaction mixture was stirred 30 min more then concentrated to ~l mL by rotary evaporation. The product was precipitated by addition of the reaction concentrate to 25 mL of stirred MTBE. After triturating and incubating on ice for 30 min, the suspension was centrifuged (3000g,

4°C, 2 min), and the supernatant was decanted. The precipitate was washed with MTBE (2 x 25 mL) and the supernatants decanted. Residual volatiles were removed under vacuum to provide the title compound (255 mg, 11.6 pmol, 93% yield) as a white powder.

Step 4

[0053] [IP -( 7-Azido-l-methylsulfonyl-2-heptyloxycarbonyl)-Lys]4-PEG20kDa , 105BH10.

TFA (1.3 mL) was added to a solution of [A^a-(7-azido-l-methylsulfonyl-2- heptyloxycarbonyl)-A^/£-Boc-Lys]₄-PEG₂o_kDa (255 mg, 11_.6 pmol, 100 mg/mL final

concentration) in 1.3 mL of CH₂Cl_2. The reaction was stirred at ambient temperature and analyzed by Cl 8 HPLC (ELSD). The starting material was converted to a single product peak via three faster eluting intermediate peaks. After 45 min, the reaction mixture was concentrated to dryness, and the resulting oil was triturated with 20 mL Et₂0. After incubating on ice for 30 min, the suspension was transferred to a 50 mL Falcon tube and centrifuged (3000g, 4°C, 2 min) then decanted. The precipitate was successively washed with Et₂0 and MTBE (lx 25 mL each) and the supernatants decanted. Residual volatiles were removed under vacuum to provide the title compound (256 mg, 11.6 pmol @ 4 TFA, quantitative yield) as a white powder. Azide content of the title compound was determined by DBCO titration (101SF88): 20 pmol/mL by weight, 19.9 pmol/mL by DBCO titration. [0054] Also prepared by this procedure: prepolymers wherein the modulator is CN, PhS0₂, 4-MePhS0₂, Me₂N-S0₂, (Me0CH₂CH₂)₂N-S0₂, and wherein the 4-arm PEG-(NH₂)₄ had an average molecular weight of 5, 10, or 20 kDa. Similarly prepared were prepolymers wherein the linker is attached to the N^e-amino group of lysine and the N^a-amino group is left as the free amine, by starting with N^a-BOC-Lysine.

Preparation B

Preparation of Prepolymer B: (^'Cvclooct-4-yn- 1 -yloxycarbonyl h-

[0055] PEG_20kDa-( H)₄ (3.00 g, 149 pmol, 597 pmol NH₂, 0.02 M NH₂ final

concentration) was dissolved in 24 mL of MeCN. DIPEA (239 pL, 1.37 mmol) and a 0.12 M solution of 0-(cyclooct-4-yn-l-yl)-0’-succinimidyl carbonate (6.5 mL, 0.78 mmol) in MeCN were successively added. The reaction was stirred at ambient temperature and analyzed by Cl 8 HPLC (ELSD detection). The starting material was converted to a single product peak via three slower eluting intermediate peaks within 20 min. After 30 min, Ac₂0 (56 pL, 0.60 mmol) was added. The reaction mixture was stirred 30 min more then concentrated to -10 mL by rotary evaporation. The product was precipitated by addition of the reaction concentrate to 300 mL of stirred MTBE. After stirring at ambient temperature for 0.5 h, the supernatant was decanted. The residual solid was resuspended in 300 mL of MTBE. After stirring for 10 min, the supernatant was decanted, and the residual solid was transferred to a vacuum filter. The solid was washed with MTBE (2 x 50 mL) and dried under high vacuum for 20 min to provide the title compound (2.83 g, 137 pmol, 92% yield) as a white powder.

[0056] C18 HPLC, purity was determined by ELSD: 94.4% (RV = 8.46 mL) and 5.6% shoulder (RV = 8.24 mL) attributed to the PEG₂o_kDa(NH₂)₃ contaminant in the starting PEG. Cyclooctyne content of the title compound was determined by N₃-DBCO assay: 20 pmol/mL by weight, 19.5 ± 2 pmol/mL by N₃-DBCO assay.

Example 1

Microfluidic formation of degradable amino-PEG microspheres

[0057] A two-reagent hydrophobic flow focusing microfluidic chip with seven parallel 50 pm drop forming channels (Dolomite, Telos) was used to prepare microspheres. Fluid flow was controlled by a custom fabricated gas-pressure driven pump. The driving pressure is computer-controlled using proportional pressure regulators (Proportion Air, MPV series) to maintain a stable flow rate using a feedback loop from a liquid flow sensor (Sensirion, SLI- 0430). Flow control is scalable to deliver liquid from 0.5 mL to multi-liter reservoirs, and produces constant flow rates with ±1% SD. This system was used to deliver the two hydrogel prepolymer solutions described in Preparations A and B as well as the antisolvent (decane containing 1% w/v Abil-EM90 (Evonik) and 1% w/v PGPR 90 (Danisco)).

[Q058] Flow rates were 2.0 mL/h for each prepolymer solution and 14 mL/h for the antisolvent. Quality control was performed by photographing the chip at 5x magnification with a high speed camera (Uni Brain, Fire-I 580b) attached to a microscope (Nikon, EQ- 51436) equipped with an automated stage to visualize the seven channels of the chip. Images of each channel were collected every 2.5 minutes. A single 7-channel device produced 8 mL/hr of water-swollen microspheres, while a manifold comprising 5 7-channel devices produced 40 mL/h of water-swollen microspheres.

[0059] The combined output of the microfluidic devices was collected in a sealed vessel comprising a steel-mesh bottom filter of a size to retain particles significantly larger than the desired diameter and allowed to mature for 24 h. The solution was then allowed to drain through the bottom mesh filter and collected in a second vessel comprising a steel-mesh bottom filter of a size to retain particles significantly smaller in diameter than the desired particles. The liquid phase and any particles smaller than the mesh cutoff were allowed to drain through the mesh filter, and the retained microspheres were washed sequentially with heptane, ethanol, and acetonitrile to provide a washed slurry of amino-microspheres in acetonitrile.

Example 2

Conversion to cvclooctvne-PEG microspheres.

[0060] To prepare microspheres for linker-drug attachment, a suspension of the amino- microsphere slurry resulting from the microfluidic method described in Example 1 in a suitable solvent such as acetonitrile are treated with a bifunctional reagent that comprises an active ester or carbonate for attachment to the amines of the microspheres and a second functional group that is complementary to the attaching group on the linker-drug. Thus, when the linker-drug has an azide group to allow attachment, the amino-microspheres are reacted with a bifunctional reagent having an active ester/carbonate (for example, and NHS or nitrophenyl ester or carbonate) and a cyclooctyne; typical examples include DBCO-NHS ester, BCN-NHS carbonate, BCN p-nitrophenyl carbonate, or 5-hydroxycyclooctyne NHS carbonate. The derivatization is typically performed in the presence of a tertiary amine base. Using the equipment described in Example 1, a sterile-filtered solution of the bifunctional reagent in an inert solvent such as acetonitrile is introduced through a port into the slurry of amino-microspheres prepared in the second sterile vessel. After stirring and allowing for reaction, the solvent and any excess reagents are allowed to drain through the mesh filter bottom, and the sterile cyclooctyne-derivatized microspheres are washed with solvent.

Example 3

Drug loading of cvclooctvne-PEG microspheres

[0061] The sterile cyclooctyne-PEG microspheres of Example 2 are suspended in the apparatus in a suitable solvent, and a solution of the azido-linker-drug is added by sterile filtration through a reagent port in the second vessel. This mix is stirred until completion of the loading reaction, then the solvent and any excess reagents are allowed to drain through the mesh filter bottom, and the sterile linker-drug-microspheres are washed with solvent. The sterile microspheres are exchanged into the dosing buffer by a repeated sequence of buffer addition, mixing, and draining. Finally, the dosing buffer is added to an amount resulting in a concentration of suspended linker-drug microspheres ready for loading into injection syringes.

Example 4

Microfluidic formation of degradable amino-PEG microspheres using Multiplexed Array

[0062] Large-scale preparation of degradable amino-PEG microspheres was performed as described in Example 1 using an array of 5 microfluidics chips (Figure 5D), each comprising 7 microfluidics channels, that were simultaneously used to provide 35 operational microfluidics channels. All components of the system in contact with prepolymers, microemulsions, or microspheres were sterilized by autoclaving or chemical sterilization using Spor Klenz^® (Steris Life Sciences), and junctions between sterilized components were treated with Spor Klenz during reassembly.

[0063] Prepolymer A [H-Lys(NH-CO-O-L(R¹)-N3)]4-PEG20_kDa (17.5 g) was dissolved to 160 mL using 20 mM acetate buffer, pH 5; titration indicated a azide group concentration of 21.4 ± 0.9 mM. The solution was diluted to give 330 g of feedstock having a final azide group concentration of 10 mM. [0064] Prepolymer B [cyclooctyn-4-yl-oxycarbonylamino]4-PEG2_0kDa (19.0 g) was dissolved to 183 mL using 20 mM acetate buffer, pH 5; titration indicated a cyclooctyne group concentration of 22.2 ± 1.5 mM. The solution was diluted to give 381 g of feedstock having a final cyclooctyne group concentration of 10 mM.

[0065] Reagent tanks (Figure 5A) were charged through 0.2 um sterile filters with 254 g of Prepolymer A feedstock, 253 g of Prepolymer B feedstock, and 2211 g of continuous phase.

[0066] Using N₂ pressure from Pressure pump assembly (Figure 5B), the prepolymer feedstocks were fed into the microfluidics chip bank (Figure 5D) comprising 5 7-channel chips at a flow rate of 10 mL/h; the continuous phase was fed at a flow rate of 65 mL/h. The resulting microemulsion was collected in the first washer/reactor vessel in Figure 5E shown as WR1 preheated to 50 °C and allowed to mature for 24 h. This was drained through the large-gauge sieve of WR1 into the second washer/reactor vessel shown as WR2. The continuous phase was drained through the small-gauge sieve of WR2, and the retained microspheres were washed successively with heptane, ethanol, and acetonitrile to provide 480 g of sterile microsphere slurry.

[0067] Optical microscopy indicated that the microsphere preparation consisted of a highly-uniform suspension of 67.3 ± 6.0 um-sized particles.

Claims

1. An apparatus for the production of sterile microparticles comprising

a) two or more pressurized reagent delivery tanks one tank comprising at least first outlet for a first polymer or prepolymer and a second tank comprising a second outlet for an antisolvent, said outlets in fluid communication with a multichannel device for formation of a microemulsion comprising said microparticles;

b) wherein each channel of said multichannel device has at least a first inlet in fluid communication with said first outlet of a) and a second inlet in fluid communication with said second outlet of a), said inlets positioned to effect formation of said microemulsion of said microparticles and wherein each channel further comprises an outlet for said microemulsion;

c) wherein each channel is equipped with a sensor for imaging said

microparticles; and

d) a computerized supervisory control and data acquisition system for control of components a)-c),

wherein the apparatus comprises a sealed system fabricated from inert materials and maintained under sterile conditions.

2. The apparatus of claim 1 which further comprises downstream of said multichannel device at least two collection vessels comprising sieves to remove microparticles that are too large and too small in fluid communication with said channels.

3. The apparatus of Claim 1 wherein the reagent delivery tanks comprise sterilizing filters for the introduction of liquid components.

4. The apparatus of Claim 1 wherein the multichannel device for formation of a microemulsion is a microfluidics chip.

5. The apparatus of Claim 1 wherein the sensor for imaging said microparticles is a photomicrographic camera.

6. An aseptic system for preparation of a sterile composition comprising hydrogel microspheres formed from components that comprise a polymer or at least two prepolymers, which system comprises a sealed and sterilized system comprising at least one microfluidic chip with a multiplicity of channels, all elements of said system being fabricated from inert materials and maintained in sterile condition, wherein said system comprises:

a) said at least one microfluidic chip comprising a multiplicity of channels, each channel having at least two inlets and an outlet;

b) wherein said inlets comprise immediately upstream of said channel, a first tube or a confluence of first tubes for supplying a solution of a polymer or prepolymer and a second tube for supplying antisolvent, wherein said first tube is arranged in parallel to the channel and the second tube is at an angle essentially perpendicular thereto;

c) a sensor for determining the size distribution of particles in each channel and controls for directing the outlet flow from channels having an undesired size distribution to waste and for directing the flow from channels having a desired size distribution to downstream elements of said system;

wherein pressure and flow rates through said system are monitored and controlled by a computer operated supervisory control and data acquisition (SCAD A) system.

7. The system of claim 6 wherein the system further comprises downstream elements that comprise at least a first collection vessel with one or more inlets for washing and medium-exchange fluids, an outlet for a composition comprising said microspheres, and a sieve for removal of microparticles that are too small, and upstream of said first vessel, a second vessel having one or more inlets for any desired reagents and a sieve for removal of microparticles that are too large.

8. The system of claim 6 which further comprises a pump for introducing under pressure said polymer or prepolymers and antisolvent.

9. The system of claim 8 wherein the pressure is exerted by an inert gas.

10. The system of claim 6 wherein said sensor comprises a microphotographic equipment.

11. The system of claim 6 which comprises 0.2 micron filters for removing contaminants from introduced fluids.

12. The system of claim 7 wherein the tanks and vessels in said system are made of stainless steel and USP VI polymers.

13. The system of claim 6 wherein said multiplicity of channels is in the range of 2-100.

14. An aseptic method to prepare a sterile composition comprising hydrogel microspheres formed from one or more components that are polymers or prepolymers, which method comprises conducting the following steps in a sealed and sterilized system comprising tanks and microfluidic chips fabricated from inert materials and maintained in aseptic condition, which steps comprise:

a) introducing under pressure through filtered tubing a solution of each polymer or prepolymer in solvent from a supply tank along with introducing an antisolvent through filtered tubing from a supply tank for said antisolvent into a manifold containing at least one microfluidic chip said chip containing a multiplicity of channels, wherein the prepolymer or polymer solution(s) is/are mixed and emulsified with the antisolvent to form a microemulsion of microspheres that is introduced into said channels;

b) visualizing the microemulsion from each of channel to determine microsphere size distribution;

c) retaining flow from channels meeting the criterion of containing a sufficient percentage of microspheres within a predetermined size range and discarding flow from channels that do not meet this criterion; and

d) recovering the resulting sterile-excipient composition comprising said hydrogel microspheres;

wherein pressure, flow rates and the retaining and discarding of step a)-c) are monitored and controlled by a computer operated supervisory control and data acquisition (SCADA) system.

15. The aseptic method of claim 14 which further includes introducing said retained microemulsion flows into one or more collection vessels and removing solvent and antisolvent and substituting sterile excipient.

16. The aseptic method of claim 15 wherein the hydrogel microspheres are to be linked to a drug wherein said method further includes, prior to step d) the step of introducing into said system any reagents needed to link the drug to the microspheres, whereby said drug-linked hydrogel microspheres are formed; and washing the drug-linked hydrogel microspheres and exchanging microspheres into and excipient which is a suitable dosing buffer.

17. The aseptic method of claim 14 wherein the pressure in a) is exerted by an inert gas.

18. The aseptic method of claim 17 wherein said inert gas is nitrogen.

19. The aseptic method of claim 14 wherein said multiplicity of channels is in the range of 2- 100.

20. The aseptic method of claim 14 wherein said visualizing of step b) is conducted with microphotographic equipment.

21. The aseptic method of claim 14 wherein said sufficient percentage in step c) is at least 50% of the microspheres in said channel.

22. The aseptic method of claim 14 wherein said predetermined size range in step c) is between 10 and 100 mih.

23. The aseptic method of claim 22 wherein said predetermined size range in step c) is between 30 and 70 mih.

24. The aseptic method of claim 15 wherein additional steps prior to step f) are conducted in two vessels in series wherein a first vessel is configured to remove anti-solvent and comprises a sieve to discard waste particles larger than the predetermined size range and an outlet to pass fluid comprising said hydrogel microspheres into a second vessel, and

wherein when the components are prepolymers first vessel contains reagents to effect completion of polymerization, and

wherein said second vessel is configured for washing said microspheres and to exchange said microspheres into excipient and comprises a sieve to discard particles smaller than the predetermined size range and an outlet for recovery of the hydrogel microspheres.

25. The aseptic method of claim 24 wherein the microspheres are linked to a drug and the recovering is by directly introducing the drug-linked hydrogel microspheres into a syringe.

26. The aseptic method of claim 14 wherein said sterilized system comprises 0.2 micron filters for removing contaminants from introduced fluids.

27. The aseptic method of claim 14 wherein the tanks in said system are made of stainless steel and USP VI polymers.

28. The method of claim 16 wherein the reagents to link the drug to the hydrogel microspheres comprise a linker with a first functional group reactive with an amine and a second functional group reactive with an azide.

29. The aseptic method of claim 28 wherein the second functional group comprises cyclooctyne.

30. A sterile composition of microspheres prepared by the method of any of claims 14-28.

31. A sterile composition of microspheres wherein the diameter of said microspheres varies by no more than ±15%.

32. The sterile composition of claim 31 wherein the average diameter of said

microspheres is 20-200 nm.

33. The sterile composition of claim 32 wherein the average diameter of said

microspheres is 20-80 nm and varies by no more than ±10%.

34. The sterile composition of any of claims 30-33 formed from prepolymers comprising biodegradable linkers.

35. The sterile composition of claim 34 wherein the biodegradable linkers are cleavable by hydrolysis or beta elimination.