Jet Injection of a Monoclonal Antibody: A Preliminary Study* N. Catherine Hogan, A.M. Cloutier, and I.W. Hunter, Member, IEEE 

Abstract— Monoclonal antibodies (mAbs) represent a major group of biotherapeutics. The high concentration and volume of drug administered together with a shift to administration via the subcutaneous route have generated interest in alternative delivery technologies. The feasibility of using a novel, highly controllable jet injection technology to deliver a mAb is presented. The effect of delivery parameters on protein structure were evaluated and compared with delivery using a conventional needle and syringe. Injection of mAb into a rat model showed that jet injection using the device resulted in more rapid absorption and longer duration of exposure.

I. INTRODUCTION Monoclonal antibodies (mAbs) are a well-established class of biotherapeutics for treatment of a wide range of diseases including cancer, allergies, cardiovascular disease, and autoimmune disorders (e.g. arthritis, psoriasis, Crohn’s disease) [1-3]. With their development and use comes the need for more innovative and controllable delivery devices to deal with the issues associated with dispensing high volumes (up to 2 mL) of drug with relatively high viscosities (0.01 to 0.047 Pa·s) [4-6]. Jet injectors, and in particular electronically controllable platform technologies such as the linear Lorentz-force actuated jet injector (JI) developed in the BioInstrumentation Lab at MIT, may fill this void [7][8]. The voice coil motor together with a real-time controller, power supply, and user interface permits the generation of variable injection trajectories (or waveforms) that define a set of parameters optimal for delivery of a specific drug to a given target. Generally, the voice coil is accelerated to the initial velocity required for drug to penetrate the tissue to a desired depth (vjet). This is maintained for a user-defined time (tjet) after which the velocity is transitioned to a lower velocity (vft) to permit delivery of the remaining volume of drug at a reduced fluid pressure. While these parameters are currently determined empirically, [9] has shown that the viscosity of a drug can be estimated using a brief voltage pulse and this information used to refine subsequent delivery parameters. In this paper, we evaluate the feasibility of using the JI to deliver mAb CXCR5. Anti-CXCR5 antibody can be used in the treatment or prevention of CXCR5 related diseases or disorders resulting from altered CXCR5 activity or metabolism [10-12]. * Research supported by Sanofi S.A., Paris, France. N. Catherine Hogan is a Research Scientist with the BioInstrumentation Lab in the Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (phone: 617-324-6051; e-mail: hog@ mit.edu). A.M. Cloutier is a graduate of the BioInstrumentation Lab. She is now completing a second Masters’ degree in Prosthetics and Orthopedics at Georgia Institute of Technology, Atlanta, GA 30332 (phone: 860-550-4250; e-mail: [email protected]). I.W. Hunter is a Professor and Director of the BioInstrumentation Laboratory in the Mechanical Engineering Department at the Massachusetts Institute of Technology, Cambridge, MA 02130 (e-mail:[email protected]).

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II. MATERIALS AND METHODS A. Viscosity measurements The viscosity of a series of known concentrations of glycerol, mAb buffer (25 mM histidine, 50 mM arginine, 5% sucrose, and 0.2% Tween 20), and mAb CXCR5 were determined directly using a TA Instruments ARES-G2 rheometer or indirectly by interpolation from the measured viscosities of dilutions of a comparable mAb of known concentration. Viscosity was determined at shear rates ranging from 10-3 s-1 to 103 s-1 using a 60 mm diameter 2° cone and plate measuring system at 25°C. B. Gel electrophoresis and immunoblotting Comparable concentrations of samples were separated on native 4-20% polyacrylamide gels or native isoelectric focusing (IEF) gels containing amphoteric molecules generating a pH gradient of 3 to 10 across the gels. Native gels were run for 1 h at 120 V in buffer containing 25 mM Tris-HCl, 192 mM glycine, pH8.3 after which the gels were washed 3x for 5 minutes, stained for 1 h with Coomassie Blue (CBB) G-250, destained in water for 30 minutes and photographed. IEF gels were run for 1 h at 100 V, 1 h at 250 V, and 30 minutes at 500 V. Prior to staining with CBB G-250, carrier ampholytes were removed using a procedure modified from [13-15]. Molecular weight standards appropriate to each gel type were loaded into separate wells and co-run with samples to permit determination of relative molecular masses or isoelectric points. Gels to be blotted were rinsed 3x for 5 minutes with water, equilibrated in 25 mM Tris-HCl, 192 mM glycine, 0.01% sodium dodecyl sulfate (SDS), 10% methanol for 30 minutes with agitation, and then sandwiched in a cassette containing Hybond C-extra membrane onto which the proteins were electrophoretically transferred (4 h at 100 V on ice). The membrane was blocked, and probed with antibody as described by [16] with some modifications. Photographs were obtained while the membranes were still wet. C. Enzyme linked immunoabsorbant assay (ELISA) Checkerboard titration [17] was carried out to optimize the coating, secondary, and tertiary reactant concentrations. Peptide 1 (P1) was diluted to 0.5 µg/mL using 0.2 M sodium carbonate-bicarbonate buffer, pH9.4 and 50 µL aliquots were transferred to the appropriate wells of 96 well flat-bottom Nunc-Immuno™ plates. A comparable volume of buffer or non-specific antigen was transferred to duplicate wells to serve as a no antigen control or a negative and/or positive control for the assay respectively. Plates were sealed, incubated at 25°C for ~4 h, and then placed at 4°C overnight. Unbound P1 was removed by washing with 1x phosphate buffered saline (PBS), unoccupied sites blocked using Starting BlockTM T20 (SB), and wells washed again with PBS. Excess buffer was removed and 50 µL of an appropriate

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dilution of 1) mAb CXCR5 ejected into tubes, 2) extracellular fluid from post mortem tissue injected with mAb, or 3) serum from animals injected with mAb was added to appropriate wells; mAbs serving as controls were also included. Plates were sealed, incubated, washed, and blocked as above, after which 50 µL of appropriate HRPconjugated secondary antibody in SB was added to each well. Plates were sealed and incubated for ~2 h at 25°C. Unbound antibody was removed by washing, the plates were blocked again, washed, and 50 µL of 3,3’,5,5’-tetramethylbenzidine substrate was added to each well. Plates were incubated for 15 minutes to maximize color detection, the reaction stopped by the addition of 50 µL of 2 M sulfuric acid to each well, and absorbance read using a Molecular Devices SpectraMax Plus384 spectrophotometer at 450 nm. Data sets were compared using one way analysis of variance. D. Dynamic light scattering (DLS) DLS analysis used a DynaPro®NanoStar™ (Wyatt Technology Corp) cuvette-based DLS system. The refractive index of the mAb buffer and choice of the shape of the molecule, in this case a uniform sphere, were input into the software. A cuvette holding 50 µL of mAb buffer or ejected sample was placed into the sample cell, the sample illuminated by a laser at 658 nm and the scattered light correlated. Analysis of the data was done using DYNAMICS® software (Wyatt Technology Corp). E. Animals, injections, and blood collection Experimental protocols were approved by the IUCAC at MIT (Protocol no. 0315-024-18) and were conducted in accordance with the NIH Guide for the Use and Care of Laboratory Animals. Male Wistar rats were acclimated to the facility and then acclimated over a 3 week period to the investigators as injections and blood draws were to be done in the absence of anaesthesia. During this time, the hair between the scapulae shaved in order to inject mAb into the pigmented fat in this region. On the day of the experiment, animals were weighed and separated into three groups; the first group were administered a single bolus of mAb CXCR5 (2 mg/kg) between the scapulae using the JI, the second group were administered the same dose of mAb to the same site using a 27G needle and syringe (27G NS), and the animal in the third group received a single injection of mAb buffer of comparable volume using the JI (no mAb control). Prior to each injection, the area between the scapulae was cleansed with water and alcohol and 200 µL of blood was collected from the lateral tail vein using aseptic technique. Additional blood collections (400 µL each) from the lateral tail veins of each animal were made at 2, 4, 6, 12, 24, 48, 96, 192, and 336 h post injection. Injection sites were monitored throughout the course of the experiment. Serum was collected from whole blood for analysis. III. RESULTS AND DISCUSSION The feasibility of using the current jet injector to deliver monoclonal antibodies in general first required that we determine the range of viscosities that the device was capable of delivering. Variable concentrations of glycerol were used to evaluate the ability of the jet injector to repeatably deliver a consistent volume at velocities of 50, 100, 150, and 200 m/s

(~20 MPa given an ampoule and nozzle orifice diameter of approximately 3.57 mm and 193 µm respectively) (Fig. 1). Good repeatability was observed for each volume tested at each velocity up to 70% glycerol (~0.02 Pa·s). The viscosity of the mAb buffer was shown to be 9.3×10 -4 Pa·s while neat mAb CXCR5 had a viscosity of 9.5×10-4 Pa·s; the viscosities of both solutions are on the order of water at 1.0×10-3 Pa·s.

Figure 1: Delivery of variable glycerol concentrations at variable jet velocities (vjet), constant tjet (10 m/s) and vft (50 m/s) using the current jet injector. (a) Plot showing the relationship between glycerol concentration and viscosity at 25°C. Cited viscosity of glycerol at 25°C is 1.2 Pa·s [18]. (b) Repeatability of delivery of variable glycerol volumes (50, 100, 150, and 200 µL) and concentrations (0, 1, 10, 30, 50, and 70%) with increasing jet velocities. Each point with error bars represents the mean and standard deviation respectively of 10 ejections.

A. Analysis of the Integrity of mAb CXCR5 Post Ejection The effect of ejecting a mAb having a viscosity comparable to that of water at 25°C through a narrow orifice under high pressure was evaluated using three different approaches; the first looked for changes in migration patterns using gel electrophoresis, the second assessed disruption of binding using ELISA’s, and the third measured changes in particle size using DLS. A1. Gel electrophoresis and blotting. Native gel electrophoresis permits detection of aggregates. In the absence of sodium dodecyl sulfate, proteins retain native conformation and as such migration is a function of both charge and size (i.e. hydrodynamic size) [19]. Changes in stability due to degradation or aggregation will alter the migration pattern of the molecule in the gel. mAb ejected using the JI at variable velocities and constant tjet and vft showed no difference in migration on native gels when compared to untreated mAb (i.e. pre-ejectate) and mAb ejected using either a 27G NS or two commercially available jet injectors, the Injex30 and the Medi-Jector VISION (Fig. 2a). In addition, there was no evidence of protein in the gel wells which can be indicative of aggregation. Heating the mAb to cause aggregation was not run as a control. Migration of untreated mAb and mAb ejected at variable velocities using the JI was also analyzed using native IEF slab gels. All ejected mAb CXCR5 samples exhibited an isoelectric point (pI) of approximately 6.6 regardless of the delivery mode and consistent with that observed for untreated sample on gels stained with CBB G-250 (Fig. 2b). The mAb appears to be relatively pure as is suggested by the absence of

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charge heterogeneity which is observed in a commercial mAb (C86322M) that served as a control. In some cases, mAb separated by native IEF gel electrophoresis following ejection using a 27G NS, an Injex 30 jet injector, or the JI was transferred to a solid-phase membrane support for immunological analysis in order to evaluate the presence of additional bands that might not be detected by staining using CBB G-250. Bio-Safe CBB G-250 has a detection range of 8 to 29 ng of protein per band. Immunoblotting can detect as little as 0.1 ng of protein per band and is selective for the protein of interest. Blots probed with affinity purified Rat Anti-Mouse IgG resolved mAb CXCR5 into two charged isoforms with both isoforms being present in all samples analyzed (Fig. 2c).

Figure 2. Migration of mAb CXCR5 on (a) native and (b) native IEF polyacrylamide gels after ejection of antibody using a 27G NS, the JI (variable vjet, tjet = 10 ms, and vft = 50 m/s), the Injex30, or Medi-Jector VISION (M-J). Untreated mAb served as a control. Appropriate protein standards were co-electrophoresed on each gel. Gels were stained with CBB G-250, destained, and photographed. (c) mAb separated on native IEF gels was transferred by means of electrotransfer to membrane, probed with 1:20,000 dilution of Biotin-SP-conjugated AffiniPure Rat Anti-Mouse IgG, and visualized using a 1:1,000 dilution of peroxidase-conjugated streptavidin.

A2. mAb CXCR5 binding pre- and post-ejection using ELISAs. Changes in the ability of the antibody to bind specific antigen following delivery through a narrow orifice under high pressure (velocity) were evaluated using indirect ELISA’s to determine the effect of high shear forces on the mAb (Fig. 3). Ejection of a consistent volume and concentration of mAb CXCR5 at 100, 125, and 150 m/s using the JI did not result in a significant change in binding of the mAb to P1 when compared to the binding detected for a comparable volume and concentration of untreated mAb CXCR5 and P1 (p = 0.415) (Fig. 3a). The ELISA in Fig. 3a also shows that mAb CXCR5 binds specifically to P1 and does not bind non-specific antigen (i.e. commercial sources of collagen III protein or CXCR5 peptide). Nor does P1 bind non-specific mAbs (data not shown). In order to obtain a more resolute measure of the effect of jet ejection on antibody binding, serial dilutions of pre-ejected mAb and mAb ejected using the JI, a NS, and two commercial jet

injectors were compared. Figure 3b shows the titration data. As expected, dilution of the antibody resulted in a decline in binding activity as determined indirectly by ELISA. Integration of a linear interpolation to a mAb concentration of 412 ng/mL (chosen as the curves plateau at higher concentrations) indicated that the relative amount of antibody bound varied dependent on the mode of ejection. For JI ejected mAb, calculated values ranged from 3% lower to 1.7% higher than those calculated for untreated mAb (preejectate) while values for mAb ejected using a NS, the Injex 30, or the Medi-Jector VISION were all lower with values of -6.0 %, -11.6%, and -13.5% respectively.

Figure 3. Binding of mAb CXCR5 post ejection as determined by indirect ELISAs. (a) Plate wells were coated with 0.5 µg/mL of peptide 1 (P1), 10 µg/mL collagen III protein (CIII), or 1.0 µg/mL CXCR5 peptide (cCXCR5). CIII and cCXCR5 served as non-specific controls while no antigen (Ag) provided a negative control. mAb CXCR5 was added to the appropriate wells and antibody detected using HRP-conjugated GAM secondary antibody. (b) Plot showing binding activity as a function of decreasing mAb concentration following ejection. Untreated mAb (preejectate) served as a control. 27G refers to the NS.

A3. Size distribution of mAb CXCR5 post-ejection using DLS. To determine if the difference in binding observed in the titration experiments might be due to aggregation, the size distribution of untreated and ejected mAb CXCR5 was evaluated using DLS. DLS inspects fluctuations in the intensity of light scattered from a protein solution over time scales ranging from ~0.1 µs to ~0.1 ms due to Brownian motion of the molecules. Because larger molecules move more slowly than smaller ones, the time scale of the scattering fluctuations provides a measure of molecular size [20]. As shown in Fig. 4, all samples analyzed including the untreated sample contained aggregates. Untreated mAb, mAb ejected using a NS, and mAb ejected at 100 and 135 m/s using the JI displayed two types of particles (multimodal distribution), the monomer with an average radius of 5.569 nm, consistent with that observed in [21] and a large aggregate that was at least 5x the radius of the monomer. The molecules comprising each group of particles were homogenous with a percent polydispersity of