Antigenic peptide-loaded cationic liposomes have shown promise as cancer vaccines. Quantification of both peptides and lipids is critical for quality control of such vaccines for clinical... Show moreAntigenic peptide-loaded cationic liposomes have shown promise as cancer vaccines. Quantification of both peptides and lipids is critical for quality control of such vaccines for clinical translation. In this work we describe a reversed phase ultra-performance liquid chromatography (RP-UPLC) method that separates lipids (DOTAP, DOPC and their degradation products) and two physicochemically different peptides within 12 min. Samples were prepared by dilution in a 1:1 (v/v) mixture of methanol and water. Peptide quantifi-cation was done via UV detection and lipids were quantified by an evaporative light scattering detector (ELSD), both coupled to the RP-UPLC system, with high precision (RSD < 3.5%). We showed that the presence of lipids and peptides did not mutually influence their quantification. Limit of detection (LOD) and limit of quantification (LOQ), as determined in the ICH guidelines, were 6 and 20 ng for DOTAP, 12 ng and 40 ng for DOPC, 3.0 ng and 8.0 ng for peptide A and 2.4 ng and 7.2 ng for the more hydrophobic peptide B. Finally, lipid degradation of DOTAP and DOPC was monitored in peptide loaded DOTAP:DOPC liposomes upon storage at 4 degrees C and 40 degrees C.(c) 2022 The Authors. Published by Elsevier Inc. on behalf of American Pharmacists Association. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) Show less
The measurement of polydisperse protein aggregates and particles in biotherapeutics remains a challenge, especially for particles with diameters of ≈ 1 µm and below (sub-micrometer). This paper... Show moreThe measurement of polydisperse protein aggregates and particles in biotherapeutics remains a challenge, especially for particles with diameters of ≈ 1 µm and below (sub-micrometer). This paper describes an interlaboratory comparison with the goal of assessing the measurement variability for the characterization of a sub-micrometer polydisperse particle dispersion composed of five sub-populations of poly(methyl methacrylate) (PMMA) and silica beads. The study included 20 participating laboratories from industry, academia, and government, and a variety of state-of-the-art particle-counting instruments. The received datasets were organized by instrument class to enable comparison of intralaboratory and interlaboratory performance. The main findings included high variability between datasets from different laboratories, with coefficients of variation from 13 % to 189 %. Intralaboratory variability was, on average, 37 % of the interlaboratory variability for an instrument class and particle sub-population. Drop-offs at either end of the size range and poor agreement on maximum counts of particle sub-populations were noted. The mean distributions from an instrument class, however, showed the size-coverage range for that class. The study shows that a poly-disperse sample can be used to assess performance capabilities of an instrument set-up (including hardware, software, and user settings) and provides guidance for the development of polydisperse reference materials. Show less
Jacobse, J.; Voorde, W. ten; Tandon, A.; Romeijn, S.G.; Grievink, H.W.; Maaden, K. van der; ... ; Cate, R. ten 2021
Aims To evaluate feasibility of intradermal (i.d.) adalimumab administration using hollow microneedles, and to compare a single i.d. dose of adalimumab using a hollow microneedle with a single... Show moreAims To evaluate feasibility of intradermal (i.d.) adalimumab administration using hollow microneedles, and to compare a single i.d. dose of adalimumab using a hollow microneedle with a single subcutaneous (s.c.) dose using a conventional needle.Methods In this single-centre double-blind, placebo-controlled, double-dummy clinical trial in 24 healthy adults we compared 40 mg adalimumab (0.4 mL) administered i.d. using a hollow microneedle with a s.c. dose using a conventional needle. Primary parameters were pain, acceptability and local tolerability; secondary parameters safety, pharmacokinetics and immunogenicity. We explored usability of optical coherence tomography, clinical photography, thermal imaging, and laser speckle contrast imaging to evaluate skin reaction after i.d. injections. In vitro protein analysis was performed to assess compatibility of adalimumab with the hollow microneedle device.Results While feasible and safe, injection pain of i.d. adalimumab was higher compared to s.c. adalimumab (35.4 vs. 7.9 on a 100-point visual analogue scale). Initial absorption rate and relative bioavailability were higher after i.d. adalimumab (time to maximum plasma concentration = 95 h [47-120]; F-rel = 129% [6.46%]) compared to s.c. adalimumab (time to maximum plasma concentration = 120 h [96-221]). Anti-adalimumab antibodies were detected in 50% and 83% of the subjects after i.d. and s.c. adalimumab, respectively. We observed statistically significantly more erythema and skin perfusion after i.d. adalimumab, compared to s.c. adalimumab and placebo injections (P < .0001). Cytokine secretion after whole blood lipopolysaccharide challenge was comparable between administration routes.Conclusions Intradermal injection of adalimumab using hollowing microneedles was perceived as more painful and less accepted than s.c. administration, but yields a higher relative bioavailability with similar safety and pharmacodynamic effects. Show less
Jacobse, J.; Voorde, W. ten; Tandon, A.; Romeijn, S.G.; Grievink, H.W.; Maaden, K. van der; ... ; Cate, R. ten 2021
Aims To evaluate feasibility of intradermal (i.d.) adalimumab administration using hollow microneedles, and to compare a single i.d. dose of adalimumab using a hollow microneedle with a single... Show moreAims To evaluate feasibility of intradermal (i.d.) adalimumab administration using hollow microneedles, and to compare a single i.d. dose of adalimumab using a hollow microneedle with a single subcutaneous (s.c.) dose using a conventional needle.Methods In this single-centre double-blind, placebo-controlled, double-dummy clinical trial in 24 healthy adults we compared 40 mg adalimumab (0.4 mL) administered i.d. using a hollow microneedle with a s.c. dose using a conventional needle. Primary parameters were pain, acceptability and local tolerability; secondary parameters safety, pharmacokinetics and immunogenicity. We explored usability of optical coherence tomography, clinical photography, thermal imaging, and laser speckle contrast imaging to evaluate skin reaction after i.d. injections. In vitro protein analysis was performed to assess compatibility of adalimumab with the hollow microneedle device.Results While feasible and safe, injection pain of i.d. adalimumab was higher compared to s.c. adalimumab (35.4 vs. 7.9 on a 100-point visual analogue scale). Initial absorption rate and relative bioavailability were higher after i.d. adalimumab (time to maximum plasma concentration = 95 h [47-120]; F-rel = 129% [6.46%]) compared to s.c. adalimumab (time to maximum plasma concentration = 120 h [96-221]). Anti-adalimumab antibodies were detected in 50% and 83% of the subjects after i.d. and s.c. adalimumab, respectively. We observed statistically significantly more erythema and skin perfusion after i.d. adalimumab, compared to s.c. adalimumab and placebo injections (P < .0001). Cytokine secretion after whole blood lipopolysaccharide challenge was comparable between administration routes.Conclusions Intradermal injection of adalimumab using hollowing microneedles was perceived as more painful and less accepted than s.c. administration, but yields a higher relative bioavailability with similar safety and pharmacodynamic effects. Show less
in a 15 times higher dose of DT, as well as subcutaneous injected DT-poly(I:C) in a similar DT dose. Summarizing, adjuvant-free dMNs showed to be a promising delivery tool for vaccination performed... Show morein a 15 times higher dose of DT, as well as subcutaneous injected DT-poly(I:C) in a similar DT dose. Summarizing, adjuvant-free dMNs showed to be a promising delivery tool for vaccination performed in SFD administration. Show less
Jacobse, J.; Romeijn, S.G.; Tandon, A.; Voorde, W. ten; Grievink, W.; Rissmann, R.; ... ; Jiskoot, W. 2019
PurposePersonalized peptide-based cancer vaccines will be composed of multiple patient specific synthetic long peptides (SLPs) which may have various physicochemical properties. To formulate such... Show morePurposePersonalized peptide-based cancer vaccines will be composed of multiple patient specific synthetic long peptides (SLPs) which may have various physicochemical properties. To formulate such SLPs, a flexible vaccine delivery system is required. We studied whether cationic liposomes are suitable for this purpose.MethodsFifteen SIINFEKL T cell epitope-containing SLPs, widely differing in hydrophobicity and isoelectric point, were separately loaded in cationic liposomes via the dehydration-rehydration method. Particle size and polydispersity index (PDI) were measured via dynamic light scattering (DLS), and zeta potential with laser Doppler electrophoresis. Peptide loading was fluorescently determined and the immunogenicity of the formulated peptides was assessed in co-cultures of dendritic cells (DCs) and CD8(+) T-cells in vitro.ResultsAll SLPs were loaded in cationic liposomes by using three different loading method variants, depending on the SLP characteristics. The fifteen liposomal formulations had a comparable size (< 200nm), PDI (< 0.3) and zeta potential (22-30mV). Cationic liposomes efficiently delivered the SLPs to DCs that subsequently activated SIINFEKL-specific CD8(+) T-cells, indicating improved immunological activity of the SLPs.ConclusionCationic liposomes can accommodate a wide range of different SLPs and are therefore a potential delivery platform for personalized cancer vaccines. Show less
Mönkare, J.; Pontier, M.J.; Kampen, E.E.M. van; Du, G.; Leone, M.; Romeijn, S.G.; ... ; Bouwstra, J.A. 2018
Skin is an attractive but also very challenging immunisation site for particulate subunit vaccines. The aim of this study was to develop hyaluronan (HA)-based dissolving microneedles (MNs) loaded... Show moreSkin is an attractive but also very challenging immunisation site for particulate subunit vaccines. The aim of this study was to develop hyaluronan (HA)-based dissolving microneedles (MNs) loaded with PLGA nanoparticles (NPs) co-encapsulating ovalbumin (OVA) and poly(I:C) for intradermal immunisation. The NP:HA ratio used for the preparation of dissolving MNs appeared to be critical for the quality of MNs and their dissolution in ex vivo human skin. Asymmetrical flow field-flow fractionation and dynamic light scattering were used to analyse the NPs released from the MNs in vitro. Successful release of the NPs depended on the drying conditions during MN preparation. The delivered antigen dose from dissolving MNs in mice was determined to be 1 µg OVA, in NPs or as free antigen, by using near-infrared fluorescence imaging. Finally, the immunogenicity of the NPs after administration of dissolving MNs (NP:HA weight ratio 1:4) was compared with that of hollow MN-delivered NPs in mice. Immunization with free antigen in dissolving MNs resulted in equally strong immune responses compared to delivery by hollow MNs. However, humoral and cellular immune responses evoked by NP-loaded dissolving MNs were inferior to those elicited by NPs delivered through a hollow MN. In conclusion, we identified several critical formulation parameters for the further development of NP-loaded dissolving MNs. Show less
Du, G.; Woythe, L.; Maaden, K. van der; Leone, M.; Romeijn, S.G.; Kros, A.; ... ; Bouwstra, J.A. 2018
To examine the immunogenicity of diphtheria toxoid (DT) loaded mesoporous silica nanoparticles (MSNs) after coated and hollow microneedle-mediated intradermal immunization in mice. DT was loaded... Show moreTo examine the immunogenicity of diphtheria toxoid (DT) loaded mesoporous silica nanoparticles (MSNs) after coated and hollow microneedle-mediated intradermal immunization in mice. DT was loaded into MSNs and the nanoparticle surface was coated with a lipid bilayer (LB-MSN-DT). To prepare coated microneedles, alternating layers of negatively charged LB-MSN-DT and positively charged N-trimethyl chitosan (TMC) were coated onto pH-sensitive microneedle arrays via a layer-by-layer approach. Microneedle arrays coated with 5 or 3 layers of LB-MSN-DT were used to immunize mice and the elicited antibody responses were compared with those induced by hollow microneedle-injected liquid formulation of LB-MSN-DT. Liquid DT formulation with and without TMC (DT/TMC) injected by a hollow microneedle were used as controls. LB-MSN-DT had an average size of about 670 nm and a zeta potential of -35 mV. The encapsulation efficiency of DT in the nanoparticles was 77%. The amount of nano-encapsulated DT coated onto the microneedle array increased linearly with increasing number of the coating layers. Nano-encapsulated DT induced stronger immune responses than DT solution when delivered intradermally via hollow microneedles, but not when delivered via coated microneedles. Both the nano-encapsulation of DT and the type of microneedles affect the immunogenicity of the antigen. PURPOSE METHODS RESULTS CONCLUSION Show less
Size distribution and conformation of ADA-drug complexes were characterised by size-exclusion chromatography and electron microscopy. Internalisation of and immune activation by complexes of... Show moreSize distribution and conformation of ADA-drug complexes were characterised by size-exclusion chromatography and electron microscopy. Internalisation of and immune activation by complexes of defined size was visualised with flow imaging, whole blood cell assay and C4b/c ELISA. Therapeutic antibodies can provoke an antidrug antibody (ADA) response, which can form soluble immune complexes with the drug in potentially high amounts. Nevertheless, ADA-associated adverse events are usually rare, although with notable exceptions including infliximab. The immune activating effects and the eventual fate of these 'anti-idiotype' complexes are poorly studied, hampering assessment of ADA-associated risk of adverse events. We investigated the in vitro formation and biological activities of ADA-drug anti-idiotype immune complexes using patient-derived monoclonal anti-infliximab antibodies. Anti-idiotype ADA-drug complexes generally have restricted immune activation capacity. Large, irregularly shaped complexes only form at high concentrations of both drug and ADA, as may be achieved during intravenous infusion of infliximab, explaining the rarity of serious ADA-associated adverse events. Size and conformation of immune complexes depended on the concentrations and ratio of drug and ADA; large complexes (>6 IgGs) formed only with high ADA titres. Macrophages efficiently internalised tetrameric and bigger complexes in vitro, but not dimers. Corroborating these results, ex vivo analysis of patient sera demonstrated only dimeric complexes in circulation.No activation of immune cells by anti-idiotype complexes was observed, and only very large complexes activated complement. Unlike Fc-linked hexamers, anti-idiotype hexamers did not activate complement, demonstrating that besides size, conformation governs immune complex potential for triggering effector functions. METHODS OBJECTIVES CONCLUSIONS RESULTS Show less
Du, G.; Leone, M.; Romeijn, S.G.; Kersten, G.F.A.; Jiskoot, W.; Bouwstra, J.A. 2018
In this study, we aimed to investigate the immunogenicity of cationic liposomes loaded with diphtheria toxoid (DT) and poly(I:C) after hollow microneedle-mediated intradermal vaccination in mice.... Show moreIn this study, we aimed to investigate the immunogenicity of cationic liposomes loaded with diphtheria toxoid (DT) and poly(I:C) after hollow microneedle-mediated intradermal vaccination in mice. The following liposomal formulations were studied: DT loaded liposomes, a mixture of free DT and poly(I:C)-loaded liposomes, a mixture of DT-loaded liposomes and free poly(I:C), and liposomal formulations with DT and poly(I:C) either individually or co-encapsulated in the liposomes. Reference groups were DT solution adjuvanted with or without poly(I:C) (DT/poly(I:C)). The liposomal formulations were characterized in terms of particle size, zeta potential, loading and release of DT and poly(I:C). After intradermal injection of BALB/c mice with the formulations through a hollow microneedle, the immunogenicity was assessed by DT-specific ELISAs. All formulations induced similar total IgG and IgG1 titers. However, all the liposomal groups containing both DT and poly(I:C) showed enhanced IgG2a titers compared to DT/poly(I:C) solution, indicating that the immune response was skewed towards a Th1 direction. This enhancement was similar for all liposomal groups that contain both DT and poly(I:C) in the formulations. Our results reveal that a mixture of DT encapsulated liposomes and poly(I:C) encapsulated liposomes have a similar effect on the antibody responses as DT and poly(I:C) co-encapsulated liposomes. These findings may have implications for future design of liposomal vaccine delivery systems. Show less
PurposePersonalized peptide-based cancer vaccines will be composed of multiple patient specific synthetic long peptides (SLPs) which may have various physicochemical properties. To formulate such... Show morePurposePersonalized peptide-based cancer vaccines will be composed of multiple patient specific synthetic long peptides (SLPs) which may have various physicochemical properties. To formulate such SLPs, a flexible vaccine delivery system is required. We studied whether cationic liposomes are suitable for this purpose.MethodsFifteen SIINFEKL T cell epitope-containing SLPs, widely differing in hydrophobicity and isoelectric point, were separately loaded in cationic liposomes via the dehydration-rehydration method. Particle size and polydispersity index (PDI) were measured via dynamic light scattering (DLS), and zeta potential with laser Doppler electrophoresis. Peptide loading was fluorescently determined and the immunogenicity of the formulated peptides was assessed in co-cultures of dendritic cells (DCs) and CD8(+) T-cells in vitro.ResultsAll SLPs were loaded in cationic liposomes by using three different loading method variants, depending on the SLP characteristics. The fifteen liposomal formulations had a comparable size (< 200nm), PDI (< 0.3) and zeta potential (22-30mV). Cationic liposomes efficiently delivered the SLPs to DCs that subsequently activated SIINFEKL-specific CD8(+) T-cells, indicating improved immunological activity of the SLPs.ConclusionCationic liposomes can accommodate a wide range of different SLPs and are therefore a potential delivery platform for personalized cancer vaccines. Show less
Schipper, P.; Maaden, K. van der; Groeneveld, V.G.D.; Ruigrok, M.J.R.; Romeijn, S.G.; Uleman, S.; ... ; Bouwstra, J.A. 2017
Dermal immunization using antigen-coated microneedle arrays is a promising vaccination strategy. However, reduction of microneedle sharpness and the available surface area for antigen coating is a... Show moreDermal immunization using antigen-coated microneedle arrays is a promising vaccination strategy. However, reduction of microneedle sharpness and the available surface area for antigen coating is a limiting factor. To overcome these obstacles, a layer-by-layer coating approach can be applied onto pH-sensitive microneedles. Following this approach, pH-sensitive microneedle arrays (positively charged at coating pH 5.8 and nearly uncharged at pH 7.4) were alternatingly coated with negatively charged diphtheria toxoid (DT) and N-trimethyl chitosan (TMC), a cationic adjuvant. First, the optimal DT dose for intradermal immunization was determined in a dose-response study, which revealed that low-dose intradermal immunization was more efficient than subcutaneous immunization and that the EC50 dose of DT upon intradermal immunization is 3-fold lower, as compared to subcutaneous immunization. In a subsequent immunization study, microneedle arrays coated with an increasing number (2, 5, and 10) of DT/TMC bilayers resulted in step-wise increasing DT-specific immune responses. Dermal immunization with microneedle arrays coated with 10 bilayers of DT/TMC (corresponding with ± 0.6 μg DT delivered intradermally) resulted in similar DT-specific immune responses as subcutaneous immunization with 5 μg of DT adjuvanted with aluminum phosphate (8-fold dose reduction). Summarizing, the layer-by-layer coating approach onto pH-sensitive microneedles is a versatile method to precisely control the amount of coated and dermally-delivered antigen that is highly suitable for dermal immunization. Show less
Schipper, P.; Maaden, K. van der; Romeijn, S.G.; Oomens, C.; Kersten, G.F.A.; Jiskoot, W.; Bouwstra, J.A. 2016
PURPOSE\nThe aim of this study was to investigate the depth-dependent intradermal immunogenicity of inactivated polio vaccine (IPV) delivered by depth-controlled microinjections via hollow... Show morePURPOSE\nThe aim of this study was to investigate the depth-dependent intradermal immunogenicity of inactivated polio vaccine (IPV) delivered by depth-controlled microinjections via hollow microneedles (HMN) and to investigate antibody response enhancing effects of IPV immunization adjuvanted with CpG oligodeoxynucleotide 1826 (CpG) or cholera toxin (CT).\nMETHODS\nA novel applicator for HMN was designed to permit depth- and volume-controlled microinjections. The applicator was used to immunize rats intradermally with monovalent IPV serotype 1 (IPV1) at injection depths ranging from 50 to 550 μm, or at 400 μm for CpG and CT adjuvanted immunization, which were compared to intramuscular immunization.\nRESULTS\nThe applicator allowed accurate microinjections into rat skin at predetermined injection depths (50-900 μm), -volumes (1-100 μL) and -rates (up to 60 μL/min) with minimal volume loss (±1-2%). HMN-mediated intradermal immunization resulted in similar IgG and virus-neutralizing antibody titers as conventional intramuscular immunization. No differences in IgG titers were observed as function of injection depth, however IgG titers were significantly increased in the CpG and CT adjuvanted groups (7-fold).\nCONCLUSION\nIntradermal immunogenicity of IPV1 was not affected by injection depth. CpG and CT were potent adjuvants for both intradermal and intramuscular immunization, allowing effective vaccination upon a minimally-invasive single intradermal microinjection by HMN. Show less
Comprehensive physicochemical characterization and biological assays are essential parts in assessing quality attributes of biologicals. Here, we compared the quality of different marketed... Show moreComprehensive physicochemical characterization and biological assays are essential parts in assessing quality attributes of biologicals. Here, we compared the quality of different marketed recombinant human erythropoietin (epoetin) products: originators, Eprex and NeoRecormon as well as 2 biosimilars, Retacrit and Binocrit. In addition, assessment of batch-to-batch variability was included by collecting 2 or more batches of each product. Common assays which included sodium dodecyl sulfate-polyacrylamide gel electrophoresis, high-performance size-exclusion chromatography, asymmetrical flow field-flow fractionation, capillary zone electrophoresis, and potency testing were used. Of the tested products and among batches of single products, variations in epoetin content, isoform profiles, and potency were found. Ultimately, this study demonstrated the high quality of epoetin products with some degree of variation among products and batches, confirming the "similar but not identical" paradigm of biologicals. Show less
We investigated the influence of pH and sodium chloride concentration on aggregation kinetics of a monoclonal antibody. Aggregation was induced by sodium chloride addition at low pH. Protein... Show moreWe investigated the influence of pH and sodium chloride concentration on aggregation kinetics of a monoclonal antibody. Aggregation was induced by sodium chloride addition at low pH. Protein conformation before and after salt addition was determined as well as the reversibility of aggregation. Aggregation was monitored at pH values between 2 and 7 with NaCl up to 1.5M by turbidity measurement and size-exclusion chromatography. Particle size distribution was assessed by using size-exclusion chromatography as well as nanoparticle tracking analysis and flow imaging microscopy. Structural changes were monitored by circular dichroism, Fourier transform infrared and fluorescence spectroscopy. Thermal stability was measured by differential scanning fluorimetry. Aggregation propensity was maximal at low pH and high ionic strength. While thermal stability decreased with pH, the secondary structure remained unchanged down to pH 3.5 and up to 1.5M NaCl. Precipitated protein could be largely reverted to monomers by dilution into salt-free buffer. The re-solubilized antibody was indistinguishable in structure, solubility and monodispersity from the unstressed protein. Also, binding to Protein A was steady. Aggregation could be reduced in the presence of trehalose. The results suggest a reversible aggregation mechanism characterized by a limited change in tertiary structure at low pH and a subsequent loss of colloidal stability resulting from electrostatic repulsion once salt is added to the sample. The experimental setup is robust and allows high-throughput quantification of the effect of additives on aggregation kinetics. Show less
