Faricimab maintains substance integrity and sterility after compounding and storage in two different polypropylene syringe types

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However, preservation of compound integrity and microbiological safety must be guaranteed. The aim of this study was to compare the chemical and physical stability, sterility, and binding affinity to vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) of faricimab, a novel bispecific anti-VEGF/Ang-2 biologic, after compounding and storage in two different polypropylene syringe types for up to 28 days. Methods: Faricimab was compounded into silicone oil-free and silicone oil-containing polypropylene syringes under controlled aseptic clean room conditions and stored under light protection at 2°C to 8°C for up to 28 days. Compound integrity was analyzed by size exclusion chromatography, nano differential scanning fluorimetry, UV-Vis and dynamic light scattering. The analysis of the simultaneous binding of VEGF and Ang-2 was performed by grating-coupled interferometry. Additionally, samples were tested for sterility and presence of bacterial endotoxins. One-way ANOVA test was used to analyze statistical significance (p ≤0.05). Results: No significant differences in VEGF and ANG-2 binding affinity were found in faricimab samples stored in either syringe type after 28 days compared to control. Chemical and physical stability testing revealed no statistically significant variation. Furthermore, sterility and the absence of bacterial endotoxins could be maintained. Conclusion: Our findings confirm the pharmaceutical safety of compounded faricimab after storage for up to 28 days. This may facilitate a cost effective off-label use of faricimab in clinical practice while maintaining safety in the treatment of patients. Health sciences/Health care/Therapeutics/Drug therapy Health sciences/Diseases/Eye diseases Angiogenesis Anti-VEGF Compounding Faricimab Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Intravitreal anti-vascular endothelial growth factor (anti-VEGF) therapy is the standard of care for retinal disorders such as neovascular age-related macular degeneration, diabetic macular edema and macular edema secondary to retinal vein occlusion. The recently approved IgG1 antibody faricimab (Roche, Switzerland) is the first anti-VEGF agent with a bispecific mode of action.[ 1 ] Additionally to VEGF, faricimab binds and neutralizes angiopoietin-2 (Ang-2), which plays an important role in angiogenesis, vessel homeostasis and vascular permeability through activation of the receptor tyrosine kinase Tie-2.[ 1 , 2 ] This may offer the possibility of prolonging injection intervals for up to 16 weeks using a treat-and-extend regimen, while simultaneously providing non-inferiority compared to aflibercept administered every 8 weeks.[ 3 – 5 ] Anti-VEGF drugs are typically provided in single-use glass vials. Splitting of these vials into individual doses and storage in syringes has commonly been performed e.g. in Europe and particularly within publicly funded health care systems to reduce waste, cost and time per injection.[ 6 ] Faricimab is also provided in a single-use glass vial containing 28.8mg of faricimab dissolved in 0.24ml. While only being administered at a dose of 6mg (0.05ml), this would theoretically allow a total of four doses to be extracted out of one vial. Other anti-VEGF drugs such as aflibercept [ 7 ], bevacizumab [ 8 ] and ranibizumab [ 9 ] have been shown to maintain their stability, sterility and drug binding properties after compounding into syringes for at least 30 days and in case of bevacizumab even up to 6 months. However, concerns have been raised regarding a higher incidence of elevated intraocular pressure and endophthalmitis for compounded bevacizumab and ranibizumab possibly due to the formation of protein aggregates.[ 10 – 12 ] These aggregates can be caused by poor storage conditions i.e. light exposure, but also by inadequate drug handling during compounding as well as the type and silicone oil content of the syringes used for storage.[ 13 ] Accordingly, a safe and consistent compounding procedure to transfer the drug from the vial into ready-to-use syringes is imperative to maintain drug properties and sterility. The present study focuses on the evaluation of the long-term stability and sterility of faricimab compounded in both silicone oil-coated and silicone oil-free polypropylene syringes, which are commonly used for intravitreal administration of anti-VEGF therapeutics. The establishment of the stability assays was done following the guidelines of Ph.Eur. and ICH. Materials and Methods General informations Every analysis was conducted at least in triplicates. Additional informations regarding reagents, sample preparation and the analytical setup are described in the supplementary methods. Sample preparation 50µL (6mg) of faricimab were compounded under aseptic conditions in a laminar air flow system (clean room level A) into silicone oil-containing syringes (BD Microfine®, Becton Dickinson, USA) and silicone oil-free syringes (ZeroResidual®, SJJ Solutions, The Netherlands). All samples were stored at 2°C to 8°C under light protection in sterile bags for up to 28 days. Faricimab taken directly from a freshly punctured vial was used for day 0 samples. Before analysis, samples were equilibrated to room temperature and the whole content was ejected into particle free centrifugation tubes (Eppendorf) and mixed by slow pipetting before any further procedure. Where not stated otherwise, sample stability was tested weekly for four weeks. Tertiary structure analysis Structural changes leading to alterations in the tertiary structure of faricimab were analyzed by second derivative UV-Vis spectrophotometry on a UV-1800 cuvette system (Shimadzu) in the spectral range between 250nm and 300nm. Variations in the microenvironment of phenylalanine, tyrosine and tryptophan in the antibody molecule are indicated by a red or blue shift of the UV-Vis absorption spectrum.[ 14 ] Additionally, changes in the tyrosine exposure were calculated based on the a/b ratio (a = distance of the absorbance at ~ λ 288nm and ~ λ 283nm ; b = distance of the absorbance at ~ λ 294nm and ~ λ 291nm ) as published by Katayama et al.[ 15 ] Size exclusion chromatography (SEC) The structural degradation and the formation of oligomers, which directly correlate to a loss of functionality and may also contribute to sterile intraocular inflammation after administration [ 16 ], were evaluated by SEC. Molecular weight determination was carried out on an OmniSEC system (Malvern) equipped with a Superdex 200 10/300 column. Phosphate buffered saline (pH 7.3) was used as mobile phase with a flow rate of 0.5mL/min and a run time of 55 minutes. Samples were analyzed for the relative quantity of the signals derived by monomer, oligomers and low molecular weight species (e.g. degradation products) in the elution profile of the antibody formulation. Additionally, the average molecular weight (Mw) and the dispersity (Mw/Mn) of each signal were evaluated. Nano Differential Scanning Fluorimetry (nanoDSF) Impaired structural properties of proteins are often connected to changes in the denaturation profile. We therefore evaluated the thermal stability of faricimab with a NanoTemper Prometheus® NT.48 system, which detects changes in the intrinsic protein fluorescence caused by tryptophan and tyrosine as well as in light scattering with increasing temperature in the environment. Excitation occured at 280nm and emission was measured at 330nm and 350nm. The ratio between the signals at 350nm and 330nm was plotted as a function of temperature. Back reflection optics were employed for the detection of light scattering created through protein aggregation. Turbidity analysis The formation of undissolved particles was analyzed by UV-Vis on a UV-1800 cuvette system (Shimadzu) based on the calculation of the aggregation index in accordance to the instructions of Jaccoulet et al [ 17 ] with the following equation: $$\text{Aggregation index }\left(\text{%}\right)\text{=}\frac{\text{OD350nm}}{\text{OD280nm-OD350nm}}\text{*100}$$ An increase of the aggregation index indicates the formation of light scattering particles. Freshly prepared reference suspension I (Ph. Eur.) was used as control compound. Hydrodynamic diameter of the antibody To strengthen the data created by SEC, the hydrodynamic size distribution of faricimab was analyzed by dynamic light scattering (DLS) on a Zetasizer Nano ZS system equipped with a 632.8nm laser (Malvern). The device monitors the presence of particles with a size ranging from 0.6nm to 6µm. Measurements were conducted with plastic cuvettes (light path 1cm; Brand). Before analysis, both cuvettes and pipette tips were washed with diluent to avoid contaminations. Data analysis was done based on the calculated z-average hydrodynamic diameter of the intensity-based particle size distribution. pH measurement Faricimab shows its highest stability at a pH of 5.5 based on the product information of the manufacturer. We therefore analyzed the pH of the undiluted samples with MQuant® pH strips (Merck) with a detection range of 4.0 to 7.0. Specific binding of VEGF and Ang-2 Binding experiments were conducted by grating-coupled interferometry (GCI) on a Creoptix WAVEdelta system (Malvern). The surface of the 4PCP Creoptix chip was covalently modified with an anti-human IgG (Fc) antibody to bind faricimab via its Fc domain. Channel 1 was used as reference channel, whereas differently stored faricimab (e.g. faricimab stored in BD syringes for 14 days and 28 days and the day 0 sample) was injected in channels 2 to 4 aiming a final immobilization level of 100pg/mm 2 before antigen injection. VEGF and Ang-2 were injected consecutively to measure their respective binding to faricimab. After chip regeneration, the experiment was repeated with a switched order of the VEGF- and Ang-2-injection. The generated binding signals of VEGF and Ang-2 were taken at the end of each injection/dissociation-cycle considering the total capturing level of faricimab on the chip surface. Samples were tested on day 0, 14 and 28. Bacterial endotoxins As contaminations with bacterial endotoxins may play a role in the development of intraocular inflammation after intravitreal injections [ 18 ], diluted samples were analyzed with a limulus amebocyte lysate based assay with an Endosafe® PTS system (Charles River). Measurements were conducted with 1/50 dilutions of faricimab samples in water for injection resulting in a minimum detection threshold of 2.5EU/mL. Samples were tested directly after compounding and after 28 days of storage. Sterility Sterility was determined as outlined in Ph.Eur. using direct inoculation of fluid thioglycolate medium (FTM) and tryptic soy broth with the whole syringe content in clean room level A environment. Tryptic soy broth primarily detects growth of yeasts and moulds as well as aerobic bacteria, whereas fluid thioglycolate medium detects both anaerobic and aerobic bacteria. In total 4 samples (2 samples per media) were tested for sterility directly after compounding and after 28 days of storage. Statistics Where mentioned in the results section, stastistical differences between the means of groups were calculated by one-way ANOVA test with α = 0.05 using Microsoft Excel and GraphPad Prism. Results Tertiary Structure Analysis As shown in Fig. 1 a, no spectral shift indicating changes of the tertiary structure was detected until 28 days of storage. A maximum deviation of the absorption minima positions of ± 0.3nm was present in all samples until day 28, independently of the syringe type, which lies in the accepted uncertainty range of the UV-Vis device as per Ph.Eur. standards (± 1nm at a wavelength area < 400nm). Values of the a/b ratio (Fig. 1 b) of 1.49 to 1.96, 1.54 to 1.98 and 1.55 to 1.83 were created by faricimab in BD Microfine® and ZeroResidual® syringes and the day 0 samples, respectively, without statistically relevant changes after 28 days (Fig. 1 c and 1 d). Molecular weight distribution A representative SEC chromatogram is depicted in Fig. 2 a. In total 3 areas were analyzed both for the calculated molecular weight and the relative quantity in the sample. Peak 1 with an elution time of around 11 minutes corresponds to the faricimab-dimer, peak 2 with an elution time of around 13 minutes to the faricimab-monomer and peak (or area) 3 with an elution time of > 13.5 minutes to traces, excipients of the formulation or possible degradation products. Compounds that contributed to peak 3 were not identified in this study. The calculated molecular weight of the monomer was around 150kDa with a maximum dispersity (Mw/Mn) of 1.001 in all samples, which is in accordance to the manufacturer´s information.[ 19 ] A dimerization grade of approximately 2% was already present in the day 0 samples and did not show any relevant changes after 28 days of storage in both syringe types (Figs. 2 b and 2 c). Other high molecular weight species were not present. Also peak 3 did not show a considerable trend, meaning that there is no quantifiable formation of degradation products with a lower molecular weight. Thermal Stability Thermal stability data of compounded faricimab was acquired by nanoDSF. The unfolding process was visualized with the ratio between the emission signal at 350nm and the signal at 330nm. This, combined with the graph´s first derivative, was used to extract the information about the unfolding process (Supplementary Fig. 1). Additionally, the formation of aggregates was quantified based on light scattering. In total two unfolding transitions could be detected with a peak maximum at around 73°C in the first derivative of the 350nm/330nm graph. The unfolding onset lied at approximately 57°C. The scattering inflection point was detected between 74.5°C and 75°C with an onset temperature at around 73°C. In case of the 350nm/330nm-graph and the light scattering-graph the inflection points of all samples are identical within the uncertainty of the measurement technique (Figs. 3 a and 3 b). Additionally, the onset temperatures showed no relevant changes over time (Figs. 3 c and 3 d). Particle distribution and pH based stability The turbidity of the unfiltered diluted samples was evaluated based on the aggregation index derived by the absorbance at 280nm and 350nm. At 350nm no absorption was caused by the protein content in solution. Therefore, any increase of the absorption at 350nm could be attributed to the light scattering effect of undissolved particles. All samples showed an absorbance value at 350nm between 0.004 to 0.006 and an aggregation index between 0.2–0.4%. In contrast, the reference suspension-I showed an absorption at 350nm of 0.014 to 0.018 and an aggregation index of 166.6–217.7%. This data suggests, that there is no formation of undissolved particles in the samples after 4 weeks of storage. The distribution of the hydrodynamic diameter of faricimab in the formulation was evaluated by DLS to further validate absence of aggregation. The calculated z-average hydrodynamic diameter of the intensity-based particle size distribution showed values between 11.6nm to 14.6nm in case of faricimab stored in BD Microfine® (Fig. 4 a) and 11.7nm to 15.3nm of faricimab stored in ZeroResidual® syringes (Fig. 4 b). The day 0 sample showed a hydrodynamic diameter of 12.4nm. There were no significant differences of the z-average hydrodynamic diameter of nanoparticles after a storage duration of 28 days in both syringe types compared to day 0. The average polydispersity index (PDI) after 4 weeks of storage was 0.096 in case of faricimab in BD Microfine® and 0.129 in ZeroResidual® syringes. Also in case of the pH of the undiluted faricimab solution no changes in comparison to the day 0 samples were detected in both syringe types. Specific Binding of VEGF and Ang-2 A representative sensorgram of one measurement cycle is shown in Supplementary Fig. 2 and the binding signals of VEGF and Ang-2 relative to the capture levels of faricimab are depicted in Fig. 5 . The average values of the relative VEGF binding signals lied at approximately 0.25, when VEGF was injected after Ang-2, and 0.31, when VEGF was injected before Ang-2. In both injection sequences, no statistically significant difference of the VEGF-binding efficacy between faricimab stored for up to 28 days in syringes and the day 0 samples was found. However, in case of day 0 samples and samples stored for 28 days we detected a statistically significant difference in dependence of the injection sequence of both antigens. In case of Ang-2 average relative binding signals between 0.92 and 1.16 were detected, also with no statistically significant difference between compounded faricimab and the day 0 samples, meaning there was no negative trend in the binding efficiency of faricimab against both VEGF and Ang-2 when compounded in syringes. Microbiological stability All samples were free of aerobic and anaerobic bacteria and showed an endotoxin concentration below the detection limit of 2.5EU/mL directly after compounding and afer 28 days of storage. Discussion The bispecific mode of action of faricimab comprises a new concept in the treatment of retinal diseases related to pathological angiogenesis and vascular leakage. Given this feature and the increasing demand of anti-VEGF drugs that demographic changes will bring, data regarding the long-term stability of faricimab in ready-to-use syringes is of clinical relevance for providers of anti-VEGF therapy. While being off-label, compounding of anti-VEGF drugs in syringes by qualified pharmacies provides some advantages in comparison to drug preparation by the clinician. It can increase patient safety by introducing higher standards of hygiene, save time by executing the same task more efficiently and by a larger quantity and reduce cost by splitting vials in a safe manner. While compounding and storage has been shown to be safe for other anti-VEGF agents, those findings cannot be extrapolated to faricimab due to its different antibody properties. Jørstad et al. recently published stability data for faricimab compounded in silicone oil-free syringes and did not find changes in binding affinity or stability after 37 days.[ 20 ] However, in contrast to our study, evaluation of sterility was not included in their analysis and only silicone oil-free syringes were used, without a side-by-side comparison with other syringe types. Moreover, a different assay was employed to test the binding affinity to faricimab’s targets. In this study, compounding and storage in silicone oil-free as well as silicone oil-containing syringes were evaluated. While the silicone oil content of syringes has been associated with an increased presence of protein aggregates and particle formation in the past [ 13 ], the protective silicone layer could also serve as a barrier for interference between certain components of the syringe and the structural integrity of the biologic.[ 21 ] Thus, both syringe types were included in this study to investigate possible differences in drug integrity due to silicone-oil coating. As previously highlighted, a significant concern with compounded antibodies is the potential formation of aggregates and structural degradation, which can lead to reduced compound activity and intraocular inflammation.[ 12 , 22 ] In the present study, neither signs of oligomerization, nor changes in the size distribution, nor the formation of undissolved particles or aggregates could be found after 28 days of storage by SEC, UV-VIS and DLS. Additionally, SEC did not show structural degradation of the andibody. The presence of a second nanoparticle population in the range between 300 to 400nm derived by oligomerization processes over time, which was reported by Paul et al. for bevacizumab in syringes [ 14 ], was not observed with faricimab. Furthermore, the detected z-average hydrodynamic diameter is in accordance with other publications about the hydrodynamic diameter of particles formed by IgG1 antibodies with a molecular weight of 150kDa.[ 23 ] Also, the tertiary protein structure of faricimab remained stable. The stability of protein based compounds is also strictly dependent on the pH of the solution. Regarding monoclonal antibodies of the IgG1 type, Zheng et al. could show that a decreased pH provokes conformational changes and an increased degradation.[ 24 ] However, the buffer capacity of the faricimab formulation maintained a stable pH of 5.5 during the whole storage duration. The adequate binding of the biologic to its dedicated targets after compounding is of paramount importance for intravitreal antibody drugs. In contrast to Jørstad et al., who used ELISA to investigate the binding affinity to VEGF and Ang-2 separately, we established a GCI-based method similar to the surface plasmon resonance method used by Regula et al. in their evaluation of the binding properties of faricimab.[ 1 ] In our approach, faricimab was immobilized non-covalently on the measurement chip via an anti-human IgG (Fc) antibody. This gave the possibility of testing the simultaneous binding mechanism of both VEGF and Ang-2, closely mimicking the mode of action after intravitreal administration, which otherwise would require the establishment of a dual-binding ELISA method. In comparison to ELISA, GCI shows a significantly higher sensitivity and monitors the binding mechanisms in a real-time manner. Additionally, GCI works entirely label-free and therefore avoids possible negative interference with the antibody-antigen interaction.[ 25 , 26 ] For both antigens, the average signal intensity seemed to be dependent on the order in which the antigens were injected. In case of VEGF, a reduction of the average signal intensity by 0.06 was visible, when it was injected after Ang-2. With Ang-2, there was an increase by 0.13, when it was injected second. Such differences created by the injection order might occur because of steric hindrances. However, in the respective replicates there was no statistically relevant difference between faricimab samples compounded and stored in syringes and the day 0 samples. As microbiological contamination can result in endophthalmitis after intravitreal injection, we further analyzed both the safety of the compounding process and the reliability of the primary packaging system during storage based on the sterility of the product. Directly after transfering of faricimab into syringes as well as after 28 days of storage an endotoxine concentration below the detection limit and no signs of bacterial growth proof the quality of the compounding process. The limitations of this study are as follows. First, binding affinity was tested for VEGF as well as Ang-2 only, and not for Fcγ-receptors and the neonatal Fc-receptor as performed by Jørstad et al.[ 20 ] However, the acquired stability data in this study does not implicate any structural changes of faricimab after compounding and storage. Second, this study was not specifically designed to investigate the release of silicone oil-based coating into the formulation. Such analyses would require techniques such as flow cytometry in combination with specific labelling of protein particles and silicone oil droplets, which was not within the scope of this study.[ 27 ] Third, this was a laboratory study and the compounded faricimab was not tested clinically. In conclusion, the establishment of a safe compounding process for anti-VEGF drugs for intravitreal injections to avoid contamination as well as any negative interference with the integrity of the compound is imperative to ensure the highest possible patient safety and treatment efficacy. This study evaluated the effect of the compounding process of faricimab into silicone oil-containing and silicone oil-free polypropylene syringes and subsequent storage for up to 28 days at 2°C to 8°C under light protection. The applied methods did not show impaired chemical or physical stability nor changes in binding affinity to VEGF and Ang-2 while maintaining sterility. These findings suggest a safe use of pharmaceutically compounded faricimab in syringes after storage for at least 28 days and provide a way to increase patient safety and cost-effectiveness for intravitreal injections in real-world settings. Declarations Acknowledgements We would like to thank all colleagues of the pharmacy of the University Hospital in St. Pölten, who participated in the sample preparation for the stability study. Sterility tests were conducted by the Institute of Medical Microbiology and Hygiene of the Austrian Agency for Health and Food Safety (Graz). The equipment for compounding faricimab into silicone oil-free syringes was kindly supplied by SJJ Solutions. Conflict of interest Sebastian Waldstein: Consultancy for Apellis, Bayer, Böhringer Ingelheim, Roche, Santen; Research support by Böhringer Ingelheim and Roche. Funding We acknowledge the support by the Open Access Publishing Fund of Karl Landsteiner University of Health Sciences, Krems, Austria. References Regula JT, Lundh von Leithner P, Foxton R, Barathi VA, Cheung CM, Bo Tun SB, et al. Targeting key angiogenic pathways with a bispecific CrossMAb optimized for neovascular eye diseases. EMBO Mol Med. 2016;8(11):1265-88. 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Additional Declarations There is conflict of interest Supplementary Files 20240304SupplementaryInformation.docx SuppFigure1.tif SuppFigure2.tif Cite Share Download PDF Status: Published Journal Publication published 04 Dec, 2024 Read the published version in Eye → Version 1 posted Editorial decision: revise 19 Aug, 2024 Review # 2 received at journal 16 Aug, 2024 Reviewer # 2 agreed at journal 19 Jul, 2024 Review # 1 received at journal 23 Jun, 2024 Reviewer # 1 agreed at journal 25 May, 2024 Reviewers invited by journal 08 May, 2024 Editor assigned by journal 29 Apr, 2024 Submission checks completed at journal 12 Mar, 2024 First submitted to journal 11 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4077362","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":278666486,"identity":"f99b2c26-6f6c-4ae4-bfe2-37fe5bf855b7","order_by":0,"name":"Alexander Taschauer","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Alexander","middleName":"","lastName":"Taschauer","suffix":""},{"id":278666487,"identity":"8e9646ce-f3d6-4585-a4df-fe85b8d35bb8","order_by":1,"name":"Arthur Sedivy","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Arthur","middleName":"","lastName":"Sedivy","suffix":""},{"id":278666488,"identity":"c1369a62-a1cf-484b-ae03-086531ea1ffd","order_by":2,"name":"Daniel Egger","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Egger","suffix":""},{"id":278666489,"identity":"6db8f8b5-0fd4-4d8f-80ed-18577482fa38","order_by":3,"name":"Reinhard Angermann","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Reinhard","middleName":"","lastName":"Angermann","suffix":""},{"id":278666490,"identity":"a72ea0a5-59fa-4c8a-94ae-b3e34136c5af","order_by":4,"name":"Haider Sami","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Haider","middleName":"","lastName":"Sami","suffix":""},{"id":278666491,"identity":"f3626bd9-71d6-485c-a125-a5352e39a91f","order_by":5,"name":"Claudia Wunder","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"","lastName":"Wunder","suffix":""},{"id":278666485,"identity":"ab56e729-20f1-46ca-a3a4-c4d2b742ca68","order_by":6,"name":"Sebastian Waldstein","email":"data:image/png;base64,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","orcid":"","institution":"Karl Landsteiner University of Health Sciences","correspondingAuthor":true,"prefix":"","firstName":"Sebastian","middleName":"","lastName":"Waldstein","suffix":""}],"badges":[],"createdAt":"2024-03-11 18:20:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4077362/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4077362/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41433-024-03511-5","type":"published","date":"2024-12-04T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":55508107,"identity":"4128a686-eb42-4e69-9300-127f4816847a","added_by":"auto","created_at":"2024-04-29 12:11:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":33810,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of the tertiary structure of faricimab. \u003c/strong\u003e(a) Representative second derivative absorbance spectrum of faricimab diluted to 1mg/mL at pH 5.5 after 0 and 28 days of storage in BD Microfine® and ZeroResidual® syringes. (b) Detection of a and b in the second derivative absorbance spectrum for analyzing tyrosine exposure in the tertiary protein structure. (c) a/b ratio of faricimab diluted to 1mg/mL at pH 5.5 stored in BD Microfine® syringes and (d) in ZeroResidual® syringes (n=3; error bar ranges from max. value to min. value).\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4077362/v1/64ca04a7c470f03d31b8a26c.png"},{"id":55508106,"identity":"0f8b4c7d-8e03-45a8-a04e-cbcf9bf4cf1d","added_by":"auto","created_at":"2024-04-29 12:11:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":26229,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSEC analysis of faricimab. \u003c/strong\u003e(a) Representative chromatogram of faricimab diluted to 1mg/mL at pH 5.5 at day 0. The absorbance at 280nm is depicted as a violet line, the refractive index as a red line and the right-angle light scattering as a green line. The borders of the 3 areas which were analyzed for the existence of peaks/signals are shown as vertical blue lines. (b) Percentage of peak/area 1 (dimer), 2 (monomer) and 3 (e.g. degradation products or excipients) after storage for up to 28 days of faricimab in BD-Microfine® syringes and (c) in ZeroResidual® syringes (n=3; ± stddev.).\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4077362/v1/bfe35ea39f645d226662c331.png"},{"id":55509075,"identity":"a5504f85-22ce-4dd4-8282-192782a3d75f","added_by":"auto","created_at":"2024-04-29 12:19:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":34705,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003enanoDSF analysis of faricimab for thermal stability. \u003c/strong\u003e(a) Inflection point values of the 350nm/330nm graph and the light scattering graph after storage for up to 28 days in BD-Microfine® syringes and (b) in ZeroResidual® syringes. (c) Onset temperature values of the 350nm/330nm graph and the light scattering graph after storage for up to 28 days in BD-Microfine® syringes and (d) in ZeroResidual® syringes (n=3; ± stddev.).\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4077362/v1/a4bdf0b69207f68eb49cbda0.png"},{"id":55508104,"identity":"8e19adf1-2e76-4de6-94c7-8b7f9a65f9a1","added_by":"auto","created_at":"2024-04-29 12:11:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":14896,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDLS analysis of faricimab. \u003c/strong\u003e(a) z-average hydrodynamic diameter of particles at a concentration of 3mg/mL and pH 5.5 after storage for up to 28 days in BD Microfine® syringes and (b) ZeroResidual® syringes.\u003c/p\u003e","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4077362/v1/b8374a8682830fd1a5befa1c.png"},{"id":55508102,"identity":"4b384b11-449f-45ef-b8ed-7ee227d8a740","added_by":"auto","created_at":"2024-04-29 12:11:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":41200,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBinding efficiency of faricimab to VEGF and Ang-2 by GCI. \u003c/strong\u003e(a) Relative VEGF-binding signal when injected before Ang-2 (orange bar) and when injected after Ang-2 (grey bar) after storage for up to 28 days in BD Microfine® syringes and (b) ZeroResidual® syringes. (c) Relative Ang-2-binding signal when injected before VEGF (green bar) and when injected after VEGF (blue bar) after storage for up to 28 days in BD Microfine® syringes and (d) ZeroResidual® syringes (n=6 (day 0); n=3 (Week 2 and 4); error bar ranges from max. value to min. value). The difference between 0, 2 and 4 weeks of storage per injection sequence (e.g. VEGF before Ang-2) as well as the difference between the relative binding signals, when one antigen was injected before and after the other antigen, were analyzed for their statistical relevance (* p ≤0.05; One-way Anova)\u003c/p\u003e","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4077362/v1/65e58e265198e01c14193a2a.png"},{"id":70645648,"identity":"af46ff5f-1493-46b4-8a4e-65b8d2257c88","added_by":"auto","created_at":"2024-12-05 08:13:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":773181,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4077362/v1/f2b5211a-e956-4038-93f1-67700b92167c.pdf"},{"id":55508094,"identity":"5d03332d-396b-4648-8390-4d3d348d2aba","added_by":"auto","created_at":"2024-04-29 12:11:56","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4743364,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"20240304SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4077362/v1/5600beababecfa5b2d99ba2b.docx"},{"id":55508101,"identity":"13e663fa-baf1-4e3b-816d-986609dd0b53","added_by":"auto","created_at":"2024-04-29 12:11:56","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2315238,"visible":true,"origin":"","legend":"","description":"","filename":"SuppFigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4077362/v1/02e3a642f5f125c3b290627c.tif"},{"id":55509076,"identity":"023c2699-202b-4a88-a670-00ca84a50da3","added_by":"auto","created_at":"2024-04-29 12:19:57","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2407050,"visible":true,"origin":"","legend":"","description":"","filename":"SuppFigure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-4077362/v1/1790033882df7945f9fbf09d.tif"}],"financialInterests":"There is conflict of interest","formattedTitle":"Faricimab maintains substance integrity and sterility after compounding and storage in two different polypropylene syringe types","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIntravitreal anti-vascular endothelial growth factor (anti-VEGF) therapy is the standard of care for retinal disorders such as neovascular age-related macular degeneration, diabetic macular edema and macular edema secondary to retinal vein occlusion.\u003c/p\u003e \u003cp\u003eThe recently approved IgG1 antibody faricimab (Roche, Switzerland) is the first anti-VEGF agent with a bispecific mode of action.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] Additionally to VEGF, faricimab binds and neutralizes angiopoietin-2 (Ang-2), which plays an important role in angiogenesis, vessel homeostasis and vascular permeability through activation of the receptor tyrosine kinase Tie-2.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] This may offer the possibility of prolonging injection intervals for up to 16 weeks using a treat-and-extend regimen, while simultaneously providing non-inferiority compared to aflibercept administered every 8 weeks.[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eAnti-VEGF drugs are typically provided in single-use glass vials. Splitting of these vials into individual doses and storage in syringes has commonly been performed e.g. in Europe and particularly within publicly funded health care systems to reduce waste, cost and time per injection.[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] Faricimab is also provided in a single-use glass vial containing 28.8mg of faricimab dissolved in 0.24ml. While only being administered at a dose of 6mg (0.05ml), this would theoretically allow a total of four doses to be extracted out of one vial.\u003c/p\u003e \u003cp\u003eOther anti-VEGF drugs such as aflibercept [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], bevacizumab [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and ranibizumab [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] have been shown to maintain their stability, sterility and drug binding properties after compounding into syringes for at least 30 days and in case of bevacizumab even up to 6 months. However, concerns have been raised regarding a higher incidence of elevated intraocular pressure and endophthalmitis for compounded bevacizumab and ranibizumab possibly due to the formation of protein aggregates.[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] These aggregates can be caused by poor storage conditions i.e. light exposure, but also by inadequate drug handling during compounding as well as the type and silicone oil content of the syringes used for storage.[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] Accordingly, a safe and consistent compounding procedure to transfer the drug from the vial into ready-to-use syringes is imperative to maintain drug properties and sterility.\u003c/p\u003e \u003cp\u003eThe present study focuses on the evaluation of the long-term stability and sterility of faricimab compounded in both silicone oil-coated and silicone oil-free polypropylene syringes, which are commonly used for intravitreal administration of anti-VEGF therapeutics. The establishment of the stability assays was done following the guidelines of Ph.Eur. and ICH.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eGeneral informations\u003c/h2\u003e\n \u003cp\u003eEvery analysis was conducted at least in triplicates. Additional informations regarding reagents, sample preparation and the analytical setup are described in the supplementary methods.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eSample preparation\u003c/h2\u003e\n \u003cp\u003e50\u0026micro;L (6mg) of faricimab were compounded under aseptic conditions in a laminar air flow system (clean room level A) into silicone oil-containing syringes (BD Microfine\u0026reg;, Becton Dickinson, USA) and silicone oil-free syringes (ZeroResidual\u0026reg;, SJJ Solutions, The Netherlands). All samples were stored at 2\u0026deg;C to 8\u0026deg;C under light protection in sterile bags for up to 28 days. Faricimab taken directly from a freshly punctured vial was used for day 0 samples. Before analysis, samples were equilibrated to room temperature and the whole content was ejected into particle free centrifugation tubes (Eppendorf) and mixed by slow pipetting before any further procedure. Where not stated otherwise, sample stability was tested weekly for four weeks.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eTertiary structure analysis\u003c/h2\u003e\n \u003cp\u003eStructural changes leading to alterations in the tertiary structure of faricimab were analyzed by second derivative UV-Vis spectrophotometry on a UV-1800 cuvette system (Shimadzu) in the spectral range between 250nm and 300nm. Variations in the microenvironment of phenylalanine, tyrosine and tryptophan in the antibody molecule are indicated by a red or blue shift of the UV-Vis absorption spectrum.[\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e] Additionally, changes in the tyrosine exposure were calculated based on the a/b ratio (a\u0026thinsp;=\u0026thinsp;distance of the absorbance at\u0026thinsp;~\u0026thinsp;\u0026lambda;\u003csub\u003e288nm\u003c/sub\u003e and\u0026thinsp;~\u0026thinsp;\u0026lambda;\u003csub\u003e283nm\u003c/sub\u003e; b\u0026thinsp;=\u0026thinsp;distance of the absorbance at\u0026thinsp;~\u0026thinsp;\u0026lambda;\u003csub\u003e294nm\u003c/sub\u003e and\u0026thinsp;~\u0026thinsp;\u0026lambda;\u003csub\u003e291nm\u003c/sub\u003e) as published by Katayama et al.[\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003eSize exclusion chromatography (SEC)\u003c/h2\u003e\n \u003cp\u003eThe structural degradation and the formation of oligomers, which directly correlate to a loss of functionality and may also contribute to sterile intraocular inflammation after administration [\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e], were evaluated by SEC. Molecular weight determination was carried out on an OmniSEC system (Malvern) equipped with a Superdex 200 10/300 column. Phosphate buffered saline (pH 7.3) was used as mobile phase with a flow rate of 0.5mL/min and a run time of 55 minutes.\u003c/p\u003e\n \u003cp\u003eSamples were analyzed for the relative quantity of the signals derived by monomer, oligomers and low molecular weight species (e.g. degradation products) in the elution profile of the antibody formulation. Additionally, the average molecular weight (Mw) and the dispersity (Mw/Mn) of each signal were evaluated.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eNano Differential Scanning Fluorimetry (nanoDSF)\u003c/h2\u003e\n \u003cp\u003eImpaired structural properties of proteins are often connected to changes in the denaturation profile. We therefore evaluated the thermal stability of faricimab with a NanoTemper Prometheus\u0026reg; NT.48 system, which detects changes in the intrinsic protein fluorescence caused by tryptophan and tyrosine as well as in light scattering with increasing temperature in the environment. Excitation occured at 280nm and emission was measured at 330nm and 350nm. The ratio between the signals at 350nm and 330nm was plotted as a function of temperature. Back reflection optics were employed for the detection of light scattering created through protein aggregation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eTurbidity analysis\u003c/h2\u003e\n \u003cp\u003eThe formation of undissolved particles was analyzed by UV-Vis on a UV-1800 cuvette system (Shimadzu) based on the calculation of the aggregation index in accordance to the instructions of Jaccoulet et al [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e] with the following equation:\u003c/p\u003e\n \u003cdiv id=\"Equa\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e$$\\text{Aggregation index }\\left(\\text{%}\\right)\\text{=}\\frac{\\text{OD350nm}}{\\text{OD280nm-OD350nm}}\\text{*100}$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003eAn increase of the aggregation index indicates the formation of light scattering particles. Freshly prepared reference suspension I (Ph. Eur.) was used as control compound.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eHydrodynamic diameter of the antibody\u003c/h2\u003e\u003cp\u003eTo strengthen the data created by SEC, the hydrodynamic size distribution of faricimab was analyzed by dynamic light scattering (DLS) on a Zetasizer Nano ZS system equipped with a 632.8nm laser (Malvern). The device monitors the presence of particles with a size ranging from 0.6nm to 6\u0026micro;m. Measurements were conducted with plastic cuvettes (light path 1cm; Brand). Before analysis, both cuvettes and pipette tips were washed with diluent to avoid contaminations. Data analysis was done based on the calculated z-average hydrodynamic diameter of the intensity-based particle size distribution.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003epH measurement\u003c/h2\u003e\u003cp\u003eFaricimab shows its highest stability at a pH of 5.5 based on the product information of the manufacturer. We therefore analyzed the pH of the undiluted samples with MQuant\u0026reg; pH strips (Merck) with a detection range of 4.0 to 7.0.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eSpecific binding of VEGF and Ang-2\u003c/h2\u003e\u003cp\u003eBinding experiments were conducted by grating-coupled interferometry (GCI) on a Creoptix WAVEdelta system (Malvern). The surface of the 4PCP Creoptix chip was covalently modified with an anti-human IgG (Fc) antibody to bind faricimab via its Fc domain. Channel 1 was used as reference channel, whereas differently stored faricimab (e.g. faricimab stored in BD syringes for 14 days and 28 days and the day 0 sample) was injected in channels 2 to 4 aiming a final immobilization level of 100pg/mm\u003csup\u003e2\u003c/sup\u003e before antigen injection. VEGF and Ang-2 were injected consecutively to measure their respective binding to faricimab. After chip regeneration, the experiment was repeated with a switched order of the VEGF- and Ang-2-injection. The generated binding signals of VEGF and Ang-2 were taken at the end of each injection/dissociation-cycle considering the total capturing level of faricimab on the chip surface. Samples were tested on day 0, 14 and 28.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eBacterial endotoxins\u003c/h2\u003e\u003cp\u003eAs contaminations with bacterial endotoxins may play a role in the development of intraocular inflammation after intravitreal injections [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e], diluted samples were analyzed with a limulus amebocyte lysate based assay with an Endosafe\u0026reg; PTS system (Charles River). Measurements were conducted with 1/50 dilutions of faricimab samples in water for injection resulting in a minimum detection threshold of 2.5EU/mL. Samples were tested directly after compounding and after 28 days of storage.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eSterility\u003c/h2\u003e\u003cp\u003eSterility was determined as outlined in Ph.Eur. using direct inoculation of fluid thioglycolate medium (FTM) and tryptic soy broth with the whole syringe content in clean room level A environment. Tryptic soy broth primarily detects growth of yeasts and moulds as well as aerobic bacteria, whereas fluid thioglycolate medium detects both anaerobic and aerobic bacteria. In total 4 samples (2 samples per media) were tested for sterility directly after compounding and after 28 days of storage.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eStatistics\u003c/h2\u003e\u003cp\u003eWhere mentioned in the \u003cspan class=\"InternalRef\"\u003eresults\u003c/span\u003e section, stastistical differences between the means of groups were calculated by one-way ANOVA test with \u0026alpha;\u0026thinsp;=\u0026thinsp;0.05 using Microsoft Excel and GraphPad Prism.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eTertiary Structure Analysis\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, no spectral shift indicating changes of the tertiary structure was detected until 28 days of storage. A maximum deviation of the absorption minima positions of \u0026plusmn;\u0026thinsp;0.3nm was present in all samples until day 28, independently of the syringe type, which lies in the accepted uncertainty range of the UV-Vis device as per Ph.Eur. standards (\u0026plusmn;\u0026thinsp;1nm at a wavelength area\u0026thinsp;\u0026lt;\u0026thinsp;400nm).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eValues of the a/b ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) of 1.49 to 1.96, 1.54 to 1.98 and 1.55 to 1.83 were created by faricimab in BD Microfine\u0026reg; and ZeroResidual\u0026reg; syringes and the day 0 samples, respectively, without statistically relevant changes after 28 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMolecular weight distribution\u003c/h2\u003e \u003cp\u003eA representative SEC chromatogram is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea. In total 3 areas were analyzed both for the calculated molecular weight and the relative quantity in the sample. Peak 1 with an elution time of around 11 minutes corresponds to the faricimab-dimer, peak 2 with an elution time of around 13 minutes to the faricimab-monomer and peak (or area) 3 with an elution time of \u0026gt;\u0026thinsp;13.5 minutes to traces, excipients of the formulation or possible degradation products. Compounds that contributed to peak 3 were not identified in this study. The calculated molecular weight of the monomer was around 150kDa with a maximum dispersity (Mw/Mn) of 1.001 in all samples, which is in accordance to the manufacturer\u0026acute;s information.[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] A dimerization grade of approximately 2% was already present in the day 0 samples and did not show any relevant changes after 28 days of storage in both syringe types (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Other high molecular weight species were not present. Also peak 3 did not show a considerable trend, meaning that there is no quantifiable formation of degradation products with a lower molecular weight.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eThermal Stability\u003c/h2\u003e \u003cp\u003eThermal stability data of compounded faricimab was acquired by nanoDSF. The unfolding process was visualized with the ratio between the emission signal at 350nm and the signal at 330nm. This, combined with the graph\u0026acute;s first derivative, was used to extract the information about the unfolding process (Supplementary Fig.\u0026nbsp;1). Additionally, the formation of aggregates was quantified based on light scattering. In total two unfolding transitions could be detected with a peak maximum at around 73\u0026deg;C in the first derivative of the 350nm/330nm graph. The unfolding onset lied at approximately 57\u0026deg;C. The scattering inflection point was detected between 74.5\u0026deg;C and 75\u0026deg;C with an onset temperature at around 73\u0026deg;C. In case of the 350nm/330nm-graph and the light scattering-graph the inflection points of all samples are identical within the uncertainty of the measurement technique (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Additionally, the onset temperatures showed no relevant changes over time (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eParticle distribution and pH based stability\u003c/h2\u003e \u003cp\u003eThe turbidity of the unfiltered diluted samples was evaluated based on the aggregation index derived by the absorbance at 280nm and 350nm. At 350nm no absorption was caused by the protein content in solution. Therefore, any increase of the absorption at 350nm could be attributed to the light scattering effect of undissolved particles. All samples showed an absorbance value at 350nm between 0.004 to 0.006 and an aggregation index between 0.2\u0026ndash;0.4%. In contrast, the reference suspension-I showed an absorption at 350nm of 0.014 to 0.018 and an aggregation index of 166.6\u0026ndash;217.7%. This data suggests, that there is no formation of undissolved particles in the samples after 4 weeks of storage.\u003c/p\u003e \u003cp\u003eThe distribution of the hydrodynamic diameter of faricimab in the formulation was evaluated by DLS to further validate absence of aggregation. The calculated z-average hydrodynamic diameter of the intensity-based particle size distribution showed values between 11.6nm to 14.6nm in case of faricimab stored in BD Microfine\u0026reg; (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) and 11.7nm to 15.3nm of faricimab stored in ZeroResidual\u0026reg; syringes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The day 0 sample showed a hydrodynamic diameter of 12.4nm. There were no significant differences of the z-average hydrodynamic diameter of nanoparticles after a storage duration of 28 days in both syringe types compared to day 0. The average polydispersity index (PDI) after 4 weeks of storage was 0.096 in case of faricimab in BD Microfine\u0026reg; and 0.129 in ZeroResidual\u0026reg; syringes. Also in case of the pH of the undiluted faricimab solution no changes in comparison to the day 0 samples were detected in both syringe types.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eSpecific Binding of VEGF and Ang-2\u003c/h2\u003e \u003cp\u003eA representative sensorgram of one measurement cycle is shown in Supplementary Fig.\u0026nbsp;2 and the binding signals of VEGF and Ang-2 relative to the capture levels of faricimab are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The average values of the relative VEGF binding signals lied at approximately 0.25, when VEGF was injected after Ang-2, and 0.31, when VEGF was injected before Ang-2. In both injection sequences, no statistically significant difference of the VEGF-binding efficacy between faricimab stored for up to 28 days in syringes and the day 0 samples was found. However, in case of day 0 samples and samples stored for 28 days we detected a statistically significant difference in dependence of the injection sequence of both antigens. In case of Ang-2 average relative binding signals between 0.92 and 1.16 were detected, also with no statistically significant difference between compounded faricimab and the day 0 samples, meaning there was no negative trend in the binding efficiency of faricimab against both VEGF and Ang-2 when compounded in syringes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eMicrobiological stability\u003c/h2\u003e \u003cp\u003eAll samples were free of aerobic and anaerobic bacteria and showed an endotoxin concentration below the detection limit of 2.5EU/mL directly after compounding and afer 28 days of storage.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe bispecific mode of action of faricimab comprises a new concept in the treatment of retinal diseases related to pathological angiogenesis and vascular leakage. Given this feature and the increasing demand of anti-VEGF drugs that demographic changes will bring, data regarding the long-term stability of faricimab in ready-to-use syringes is of clinical relevance for providers of anti-VEGF therapy. While being off-label, compounding of anti-VEGF drugs in syringes by qualified pharmacies provides some advantages in comparison to drug preparation by the clinician. It can increase patient safety by introducing higher standards of hygiene, save time by executing the same task more efficiently and by a larger quantity and reduce cost by splitting vials in a safe manner. While compounding and storage has been shown to be safe for other anti-VEGF agents, those findings cannot be extrapolated to faricimab due to its different antibody properties. J\u0026oslash;rstad et al. recently published stability data for faricimab compounded in silicone oil-free syringes and did not find changes in binding affinity or stability after 37 days.[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] However, in contrast to our study, evaluation of sterility was not included in their analysis and only silicone oil-free syringes were used, without a side-by-side comparison with other syringe types. Moreover, a different assay was employed to test the binding affinity to faricimab\u0026rsquo;s targets.\u003c/p\u003e \u003cp\u003eIn this study, compounding and storage in silicone oil-free as well as silicone oil-containing syringes were evaluated. While the silicone oil content of syringes has been associated with an increased presence of protein aggregates and particle formation in the past [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], the protective silicone layer could also serve as a barrier for interference between certain components of the syringe and the structural integrity of the biologic.[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] Thus, both syringe types were included in this study to investigate possible differences in drug integrity due to silicone-oil coating.\u003c/p\u003e \u003cp\u003eAs previously highlighted, a significant concern with compounded antibodies is the potential formation of aggregates and structural degradation, which can lead to reduced compound activity and intraocular inflammation.[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] In the present study, neither signs of oligomerization, nor changes in the size distribution, nor the formation of undissolved particles or aggregates could be found after 28 days of storage by SEC, UV-VIS and DLS. Additionally, SEC did not show structural degradation of the andibody. The presence of a second nanoparticle population in the range between 300 to 400nm derived by oligomerization processes over time, which was reported by Paul et al. for bevacizumab in syringes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], was not observed with faricimab. Furthermore, the detected z-average hydrodynamic diameter is in accordance with other publications about the hydrodynamic diameter of particles formed by IgG1 antibodies with a molecular weight of 150kDa.[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] Also, the tertiary protein structure of faricimab remained stable.\u003c/p\u003e \u003cp\u003eThe stability of protein based compounds is also strictly dependent on the pH of the solution. Regarding monoclonal antibodies of the IgG1 type, Zheng et al. could show that a decreased pH provokes conformational changes and an increased degradation.[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] However, the buffer capacity of the faricimab formulation maintained a stable pH of 5.5 during the whole storage duration.\u003c/p\u003e \u003cp\u003eThe adequate binding of the biologic to its dedicated targets after compounding is of paramount importance for intravitreal antibody drugs. In contrast to J\u0026oslash;rstad et al., who used ELISA to investigate the binding affinity to VEGF and Ang-2 separately, we established a GCI-based method similar to the surface plasmon resonance method used by Regula et al. in their evaluation of the binding properties of faricimab.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] In our approach, faricimab was immobilized non-covalently on the measurement chip via an anti-human IgG (Fc) antibody. This gave the possibility of testing the simultaneous binding mechanism of both VEGF and Ang-2, closely mimicking the mode of action after intravitreal administration, which otherwise would require the establishment of a dual-binding ELISA method. In comparison to ELISA, GCI shows a significantly higher sensitivity and monitors the binding mechanisms in a real-time manner. Additionally, GCI works entirely label-free and therefore avoids possible negative interference with the antibody-antigen interaction.[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] For both antigens, the average signal intensity seemed to be dependent on the order in which the antigens were injected. In case of VEGF, a reduction of the average signal intensity by 0.06 was visible, when it was injected after Ang-2. With Ang-2, there was an increase by 0.13, when it was injected second. Such differences created by the injection order might occur because of steric hindrances. However, in the respective replicates there was no statistically relevant difference between faricimab samples compounded and stored in syringes and the day 0 samples.\u003c/p\u003e \u003cp\u003eAs microbiological contamination can result in endophthalmitis after intravitreal injection, we further analyzed both the safety of the compounding process and the reliability of the primary packaging system during storage based on the sterility of the product. Directly after transfering of faricimab into syringes as well as after 28 days of storage an endotoxine concentration below the detection limit and no signs of bacterial growth proof the quality of the compounding process.\u003c/p\u003e \u003cp\u003eThe limitations of this study are as follows. First, binding affinity was tested for VEGF as well as Ang-2 only, and not for Fcγ-receptors and the neonatal Fc-receptor as performed by J\u0026oslash;rstad et al.[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] However, the acquired stability data in this study does not implicate any structural changes of faricimab after compounding and storage. Second, this study was not specifically designed to investigate the release of silicone oil-based coating into the formulation. Such analyses would require techniques such as flow cytometry in combination with specific labelling of protein particles and silicone oil droplets, which was not within the scope of this study.[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] Third, this was a laboratory study and the compounded faricimab was not tested clinically.\u003c/p\u003e \u003cp\u003eIn conclusion, the establishment of a safe compounding process for anti-VEGF drugs for intravitreal injections to avoid contamination as well as any negative interference with the integrity of the compound is imperative to ensure the highest possible patient safety and treatment efficacy. This study evaluated the effect of the compounding process of faricimab into silicone oil-containing and silicone oil-free polypropylene syringes and subsequent storage for up to 28 days at 2\u0026deg;C to 8\u0026deg;C under light protection. The applied methods did not show impaired chemical or physical stability nor changes in binding affinity to VEGF and Ang-2 while maintaining sterility. These findings suggest a safe use of pharmaceutically compounded faricimab in syringes after storage for at least 28 days and provide a way to increase patient safety and cost-effectiveness for intravitreal injections in real-world settings.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank all colleagues of the pharmacy of the University Hospital in St. Pölten, who participated in the sample preparation for the stability study. Sterility tests were conducted by the Institute of Medical Microbiology and Hygiene of the Austrian Agency for Health and Food Safety (Graz). The equipment for compounding faricimab into silicone oil-free syringes was kindly supplied by SJJ Solutions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSebastian Waldstein: Consultancy for Apellis, Bayer, Böhringer Ingelheim, Roche, Santen; Research support by Böhringer Ingelheim and Roche.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge the support by the Open Access Publishing Fund of Karl Landsteiner University of Health Sciences, Krems, Austria.\u0026nbsp;\u003cbr\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eRegula JT, Lundh von Leithner P, Foxton R, Barathi VA, Cheung CM, Bo Tun SB, et al. Targeting key angiogenic pathways with a bispecific CrossMAb optimized for neovascular eye diseases. EMBO Mol Med. 2016;8(11):1265-88.\u003c/li\u003e\n \u003cli\u003eKim J, Park JR, Choi J, Park I, Hwang Y, Bae H, et al. Tie2 activation promotes choriocapillary regeneration for alleviating neovascular age-related macular degeneration. Sci Adv. 2019;5(2):eaau6732.\u003c/li\u003e\n \u003cli\u003eHeier JS, Khanani AM, Quezada Ruiz C, Basu K, Ferrone PJ, Brittain C, et al. Efficacy, durability, and safety of intravitreal faricimab up to every 16 weeks for neovascular age-related macular degeneration (TENAYA and LUCERNE): two randomised, double-masked, phase 3, non-inferiority trials. Lancet. 2022;399(10326):729-40.\u003c/li\u003e\n \u003cli\u003eWykoff CC, Abreu F, Adamis AP, Basu K, Eichenbaum DA, Haskova Z, et al. Efficacy, durability, and safety of intravitreal faricimab with extended dosing up to every 16 weeks in patients with diabetic macular oedema (YOSEMITE and RHINE): two randomised, double-masked, phase 3 trials. Lancet. 2022;399(10326):741-55.\u003c/li\u003e\n \u003cli\u003eHattenbach LO, Abreu F, Arrisi P, Basu K, Danzig CJ, Guymer R, et al. BALATON and COMINO: Phase III Randomized Clinical Trials of Faricimab for Retinal Vein Occlusion: Study Design and Rationale. Ophthalmol Sci. 2023;3(3):100302.\u003c/li\u003e\n \u003cli\u003eSubhi Y, Kjer B, Munch IC. Prefilled syringes for intravitreal injection reduce preparation time. Dan Med J. 2016;63(4).\u003c/li\u003e\n \u003cli\u003eSivertsen MS, J\u0026oslash;rstad \u0026Oslash; K, Grevys A, Foss S, Moe MC, Andersen JT. Pharmaceutical compounding of aflibercept in prefilled syringes does not affect structural integrity, stability or VEGF and Fc binding properties. Sci Rep. 2018;8(1):2101.\u003c/li\u003e\n \u003cli\u003eBakri SJ, Snyder MR, Pulido JS, McCannel CA, Weiss WT, Singh RJ. Six-month stability of bevacizumab (Avastin) binding to vascular endothelial growth factor after withdrawal into a syringe and refrigeration or freezing. Retina. 2006;26(5):519-22.\u003c/li\u003e\n \u003cli\u003eCao S, Cui J, Matsubara J, Forooghian F. Long-term in vitro functional stability of compounded ranibizumab and aflibercept. Can J Ophthalmol. 2017;52(3):273-6.\u003c/li\u003e\n \u003cli\u003eSniegowski M, Mandava N, Kahook MY. Sustained intraocular pressure elevation after intravitreal injection of bevacizumab and ranibizumab associated with trabeculitis. Open Ophthalmol J. 2010;4:28-9.\u003c/li\u003e\n \u003cli\u003eYamashiro K, Tsujikawa A, Miyamoto K, Oh H, Otani A, Tamuara H, et al. Sterile endophthalmitis after intravitreal injection of bevacizumab obtained from a single batch. Retina. 2010;30(3):485-90.\u003c/li\u003e\n \u003cli\u003eKahook MY, Liu L, Ruzycki P, Mandava N, Carpenter JF, Petrash JM, et al. High-molecular-weight aggregates in repackaged bevacizumab. Retina. 2010;30(6):887-92.\u003c/li\u003e\n \u003cli\u003eLiu L, Ammar DA, Ross LA, Mandava N, Kahook MY, Carpenter JF. Silicone oil microdroplets and protein aggregates in repackaged bevacizumab and ranibizumab: effects of long-term storage and product mishandling. Invest Ophthalmol Vis Sci. 2011;52(2):1023-34.\u003c/li\u003e\n \u003cli\u003ePaul M, Vieillard V, Roumi E, Cauvin A, Despiau MC, Laurent M, et al. Long-term stability of bevacizumab repackaged in 1mL polypropylene syringes for intravitreal administration. Ann Pharm Fr. 2012;70(3):139-54.\u003c/li\u003e\n \u003cli\u003eKatayama DS, Nayar R, Chou DK, Campos J, Cooper J, Vander Velde DG, et al. Solution behavior of a novel type 1 interferon, interferon-tau. J Pharm Sci. 2005;94(12):2703-15.\u003c/li\u003e\n \u003cli\u003eAnderson WJ, da Cruz NFS, Lima LH, Emerson GG, Rodrigues EB, Melo GB. Mechanisms of sterile inflammation after intravitreal injection of antiangiogenic drugs: a narrative review. Int J Retina Vitreous. 2021;7(1):37.\u003c/li\u003e\n \u003cli\u003eJaccoulet E, Daniel T, Dammak D, Prognon P, Caudron E. Interest of flow injection spectrophotometry as an orthogonal method for analyzing biomolecule aggregates: Application to stressed monoclonal antibody study. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2021;251:119436.\u003c/li\u003e\n \u003cli\u003eWang F, Yu S, Liu K, Chen FE, Song Z, Zhang X, et al. Acute intraocular inflammation caused by endotoxin after intravitreal injection of counterfeit bevacizumab in Shanghai, China. Ophthalmology. 2013;120(2):355-61.\u003c/li\u003e\n \u003cli\u003eNicol\u0026ograve; M, Ferro Desideri L, Vagge A, Traverso CE. Faricimab: an investigational agent targeting the Tie-2/angiopoietin pathway and VEGF-A for the treatment of retinal diseases. Expert Opin Investig Drugs. 2021;30(3):193-200.\u003c/li\u003e\n \u003cli\u003eJ\u0026oslash;rstad \u0026Oslash; K, Foss S, Gj\u0026oslash;lberg TT, Mester S, Nyquist-Andersen M, Sivertsen MS, et al. Pharmaceutical compounding and storage of faricimab in a syringe for intravitreal injection do not impair stability and bi-specific binding properties. Int J Retina Vitreous. 2023;9(1):65.\u003c/li\u003e\n \u003cli\u003eLode HE, Gj\u0026oslash;lberg TT, Foss S, Sivertsen MS, Brustugun J, Andersson Y, et al. A new method for pharmaceutical compounding and storage of anti-VEGF biologics for intravitreal use in silicone oil-free prefilled plastic syringes. Sci Rep. 2019;9(1):18021.\u003c/li\u003e\n \u003cli\u003eCrul M, Zandvliet A, Moes JR, Veenbaas T, Smeets O. Bevacizumab for Intravitreal Injection: Impact of Sub-Visible Particles on the Shelf-Life of Repackaged Bevacizumab. J Ocul Pharmacol Ther. 2019;35(6):372-5.\u003c/li\u003e\n \u003cli\u003eKhalili H, Sharma G, Froome A, Khaw PT, Brocchini S. Storage stability of bevacizumab in polycarbonate and polypropylene syringes. Eye. 2015;29(6):820-7.\u003c/li\u003e\n \u003cli\u003eZheng S, Qiu D, Adams M, Li J, Mantri RV, Gandhi R. Investigating the Degradation Behaviors of a Therapeutic Monoclonal Antibody Associated with pH and Buffer Species. AAPS PharmSciTech. 2017;18(1):42-8.\u003c/li\u003e\n \u003cli\u003eKozma P, H\u0026aacute;mori A, Kurunczi S, Cottier K, Horvath R. Grating coupled optical waveguide interferometer for label-free biosensing. Sensors and Actuators B: Chemical. 2011;155(2):446-50.\u003c/li\u003e\n \u003cli\u003eCooper MA. Optical biosensors in drug discovery. Nature Reviews Drug Discovery. 2002;1(7):515-28.\u003c/li\u003e\n \u003cli\u003eProbst C. Characterization of Protein Aggregates, Silicone Oil Droplets, and Protein-Silicone Interactions Using Imaging Flow Cytometry. Journal of Pharmaceutical Sciences. 2020;109(1):364-74.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"eye","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"eye","sideBox":"Learn more about [Eye](http://www.nature.com/eye/)","snPcode":"41433","submissionUrl":"https://mts-eye.nature.com/cgi-bin/main.plex","title":"Eye","twitterHandle":"@eye_journal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Angiogenesis, Anti-VEGF, Compounding, Faricimab","lastPublishedDoi":"10.21203/rs.3.rs-4077362/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4077362/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompounding and storage of intravitreal anti-vascular endothelial growth factor (anti-VEGF) agents in syringes is commonly performed in an off-label manner. However, preservation of compound integrity and microbiological safety must be guaranteed. The aim of this study was to compare the chemical and physical stability, sterility, and binding affinity to vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) of faricimab, a novel bispecific anti-VEGF/Ang-2 biologic, after compounding and storage in two different polypropylene syringe types for up to 28 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFaricimab was compounded into silicone oil-free and silicone oil-containing polypropylene syringes under controlled aseptic clean room conditions and stored under light protection at 2°C to 8°C for up to 28 days. Compound integrity was analyzed by size exclusion chromatography, nano differential scanning fluorimetry, UV-Vis and dynamic light scattering. The analysis of the simultaneous binding of VEGF and Ang-2 was performed by grating-coupled interferometry. Additionally, samples were tested for sterility and presence of bacterial endotoxins. One-way ANOVA test was used to analyze statistical significance (p ≤0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003cbr\u003e\n \u003c/strong\u003eNo significant differences in VEGF and ANG-2 binding affinity were found in faricimab samples stored in either syringe type after 28 days compared to control. Chemical and physical stability testing revealed no statistically significant variation. Furthermore, sterility and the absence of bacterial endotoxins could be maintained.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003cbr\u003e\n \u003c/strong\u003eOur findings confirm the pharmaceutical safety of compounded faricimab after storage for up to 28 days. This may facilitate a cost effective off-label use of faricimab in clinical practice while maintaining safety in the treatment of patients.\u003c/p\u003e","manuscriptTitle":"Faricimab maintains substance integrity and sterility after compounding and storage in two different polypropylene syringe types","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-29 12:11:52","doi":"10.21203/rs.3.rs-4077362/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-08-19T14:23:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-08-17T02:40:07+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-07-19T13:58:37+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-06-23T18:56:05+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-05-25T18:30:59+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-05-08T14:34:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-29T15:28:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-12T09:31:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Eye","date":"2024-03-11T18:17:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"eye","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"eye","sideBox":"Learn more about [Eye](http://www.nature.com/eye/)","snPcode":"41433","submissionUrl":"https://mts-eye.nature.com/cgi-bin/main.plex","title":"Eye","twitterHandle":"@eye_journal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2d7f01eb-9bc4-42a5-94d1-efe442cdefe1","owner":[],"postedDate":"April 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":29348824,"name":"Health sciences/Health care/Therapeutics/Drug therapy"},{"id":29348825,"name":"Health sciences/Diseases/Eye diseases"}],"tags":[],"updatedAt":"2024-12-05T08:12:58+00:00","versionOfRecord":{"articleIdentity":"rs-4077362","link":"https://doi.org/10.1038/s41433-024-03511-5","journal":{"identity":"eye","isVorOnly":false,"title":"Eye"},"publishedOn":"2024-12-04 05:00:00","publishedOnDateReadable":"December 4th, 2024"},"versionCreatedAt":"2024-04-29 12:11:52","video":"","vorDoi":"10.1038/s41433-024-03511-5","vorDoiUrl":"https://doi.org/10.1038/s41433-024-03511-5","workflowStages":[]},"version":"v1","identity":"rs-4077362","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4077362","identity":"rs-4077362","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}