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author:("wiskott, Wim")
2.  Immunogenicity of different stressed IgG monoclonal antibody formulations in immune tolerant transgenic mice 
mAbs  2012;4(6):740-752.
The presence of protein aggregates in biopharmaceutical formulations is of great concern for safety and efficacy reasons. The aim of this study was to correlate the type and amount of IgG monoclonal antibody aggregates with their immunogenic potential. IgG degradation was obtained by freeze-thawing cycles, pH-shift cycles, heating, shaking and metal-catalyzed oxidation. The size, amount, morphology and type of intermolecular bonds of aggregates, as well as structural changes and epitope integrity were characterized. These formulations were injected in mice transgenic (TG) for human genes for Ig heavy and light chains and their non-transgenic (NTG) counterparts. Anti-drug antibody (ADA) titers were determined by bridging ELISA. Both unstressed IgG and freeze-thawed formulation did not induce measurable ADA levels. A mild antibody response was obtained in a fairly small percentage of mice, when injected with shaken, pH-shifted and heated formulations. The metal-catalyzed oxidized IgG formulation was the most immunogenic one, in both ADA titers and number of responders. The overall titers of NTG responders were significantly higher than the ones produced by TG mice, whereas there was no significant difference between the overall number of TG and NTG responders. This study reinforces the important role of protein aggregates on immunogenicity of therapeutic proteins and provides new insight into the immunogenic potential of different types of IgG aggregates. The results indicate that the quality of the IgG aggregates has more impact on the development of an immune response than their quantity or size.
doi:10.4161/mabs.22066
PMCID: PMC3502241  PMID: 22951518
IgG; accelerated stress conditions; protein aggregates; immunogenicity; transgenic mice
3.  Photodynamic therapy with conventional and PEGylated liposomal formulations of mTHPC (temoporfin): comparison of treatment efficacy and distribution characteristics in vivo 
A major challenge in the application of a nanoparticle-based drug delivery system for anticancer agents is the knowledge of the critical properties that influence their in vivo behavior and the therapeutic performance of the drug. The effect of a liposomal formulation, as an example of a widely-used delivery system, on all aspects of the drug delivery process, including the drug’s behavior in blood and in the tumor, has to be considered when optimizing treatment with liposomal drugs, but that is rarely done. This article presents a comparison of conventional (Foslip®) and polyethylene glycosylated (Fospeg®) liposomal formulations of temoporfin (meta-tetra[hydroxyphenyl]chlorin) in tumor-grafted mice, with a set of comparison parameters not reported before in one model. Foslip® and Fospeg® pharmacokinetics, drug release, liposome stability, tumor uptake, and intratumoral distribution are evaluated, and their influence on the efficacy of the photodynamic treatment at different light–drug intervals is discussed. The use of whole-tumor multiphoton fluorescence macroscopy imaging is reported for visualization of the in vivo intratumoral distribution of the photosensitizer. The combination of enhanced permeability and retention-based tumor accumulation, stability in the circulation, and release properties leads to a higher efficacy of the treatment with Fospeg® compared to Foslip®. A significant advantage of Fospeg® lies in a major decrease in the light–drug interval, while preserving treatment efficacy.
doi:10.2147/IJN.S51002
PMCID: PMC3797282  PMID: 24143087
mTHPC; liposomes; drug release; liposomal pharmacokinetics; biodistribution; photodynamic therapy
4.  Adjuvant Effect of Cationic Liposomes for Subunit Influenza Vaccine: Influence of Antigen Loading Method, Cholesterol and Immune Modulators 
Pharmaceutics  2013;5(3):392-410.
Cationic liposomes are potential adjuvants for influenza vaccines. In a previous study we reported that among a panel of cationic liposomes loaded with influenza hemagglutinin (HA), DC-Chol:DPPC (1:1 molar ratio) liposomes induced the strongest immune response. However, it is not clear whether the cholesterol (Chol) backbone or the tertiary amine head group of DC-Chol was responsible for this. Therefore, in the present work we studied the influence of Chol in the lipid bilayer of cationic liposomes. Moreover, we investigated the effect of the HA loading method (adsorption versus encapsulation) and the encapsulation of immune modulators in DC-Chol liposomes on the immunogenicity of HA. Liposomes consisting of a neutral lipid (DPPC or Chol) and a cationic compound (DC-Chol, DDA, or eDPPC) were produced by film hydration-extrusion with/without an encapsulated immune modulator (CpG or imiquimod). The liposomes generally showed comparable size distribution, zeta potential and HA loading. In vitro studies with monocyte-derived human dendritic cells and immunization studies in C57Bl/6 mice showed that: (1) liposome-adsorbed HA is more immunogenic than encapsulated HA; (2) the incorporation of Chol in the bilayer of cationic liposomes enhances their adjuvant effect; and (3) CpG loaded liposomes are more efficient at enhancing HA-specific humoral responses than plain liposomes or Alhydrogel.
doi:10.3390/pharmaceutics5030392
PMCID: PMC3836624  PMID: 24300513
adjuvant; cationic liposomes; cholesterol; CpG; H3N2; hemagglutinin; imiquimod; immunogenicity; influenza
5.  Chemical Modifications in Aggregates of Recombinant Human Insulin Induced by Metal-Catalyzed Oxidation: Covalent Cross-Linking via Michael Addition to Tyrosine Oxidation Products 
Pharmaceutical Research  2012;29(8):2276-2293.
Purpose
To elucidate the chemical modifications in covalent aggregates of recombinant human insulin induced by metal catalyzed oxidation (MCO).
Methods
Insulin was exposed for 3 h at room temperature to the oxidative system copper(II)/ascorbate. Chemical derivatization with 4-(aminomethyl) benzenesulfonic acid (ABS) was performed to detect 3,4-dihydroxyphenylalanine (DOPA) formation. Electrospray ionization-mass spectrometry (ESI-MS) was employed to localize the amino acids targeted by oxidation and the cross-links involved in insulin aggregation. Oxidation at different pH and temperature was monitored with size exclusion chromatography (SEC) and ESI-MS analysis to further investigate the chemical mechanism(s), to estimate the aggregates content and to quantify DOPA in aggregated insulin.
Results
The results implicate the formation of DOPA and 2-amino-3-(3,4-dioxocyclohexa-1,5-dien-1-yl) propanoic acid (DOCH), followed by Michael addition, as responsible for new cross-links resulting in covalent aggregation of insulin during MCO. Michael addition products were detected between DOCH at positions B16, B26, A14, and A19, and free amino groups of the N-terminal amino acids Phe B1 and Gly A1, and side chains of Lys B29, His B5 and His B10. Fragments originating from peptide bond hydrolysis were also detected.
Conclusion
MCO of insulin leads to covalent aggregation through cross-linking via Michael addition to tyrosine oxidation products.
Electronic supplementary material
The online version of this article (doi:10.1007/s11095-012-0755-z) contains supplementary material, which is available to authorized users.
doi:10.1007/s11095-012-0755-z
PMCID: PMC3399080  PMID: 22572797
human insulin; oxidation; Michael addition; aggregation; fragmentation
6.  Detection and Characterization of Subvisible Aggregates of Monoclonal IgG in Serum 
Pharmaceutical Research  2012;29(8):2202-2212.
Purpose
To detect and characterize the aggregation of therapeutic monoclonal antibodies in undiluted biological fluids.
Methods
Fluorescently labeled subvisible IgG aggregates formed by applying either heat stress or by pH-shift were investigated immediately after addition to human serum, and after 24 h. Unstressed and stressed IgG formulations were analyzed by fluorescence single particle tracking, confocal laser scanning microscopy and flow cytometry.
Results
Unstressed formulations remained free from subvisible aggregates in serum, whereas heat-stressed and pH-shift stressed formulations showed dissimilar aggregation behaviors. The aggregation profile of the heat-stressed formulation diluted in serum remained practically the same as the one diluted in buffer, even after the 24 h incubation period. The pH-shift stressed formulation had strikingly smaller and more numerous subvisible aggregates immediately after dilution in serum compared to buffer. These aggregates became noticeably larger in both diluents after 24 h, but in serum they appeared to be formed by other types of constituents than the labeled protein itself.
Conclusion
These results show that subvisible therapeutic protein aggregates may undergo changes in number, type and size distribution upon contact with human serum. This emphasizes the importance of analytical strategies for monitoring aggregation in undiluted biological fluids.
Electronic supplementary material
The online version of this article (doi:10.1007/s11095-012-0749-x) contains supplementary material, which is available to authorized users.
doi:10.1007/s11095-012-0749-x
PMCID: PMC3399096  PMID: 22467219
confocal laser scanning microscopy; flow cytometry; monoclonal antibody; serum; subvisible protein aggregates; fluorescence single particle tracking
7.  PLGA, PLGA-TMC and TMC-TPP Nanoparticles Differentially Modulate the Outcome of Nasal Vaccination by Inducing Tolerance or Enhancing Humoral Immunity 
PLoS ONE  2011;6(11):e26684.
Development of vaccines in autoimmune diseases has received wide attention over the last decade. However, many vaccines showed limited clinical efficacy. To enhance vaccine efficacy in infectious diseases, biocompatible and biodegradable polymeric nanoparticles have gained interest as antigen delivery systems. We investigated in mice whether antigen-encapsulated PLGA (poly-lactic-co-glycolic acid), PLGA-TMC (N-trimethyl chitosan) or TMC-TPP (tri-polyphosphate) nanoparticles can also be used to modulate the immunological outcome after nasal vaccination. These three nanoparticles enhanced the antigen presentation by dendritic cells, as shown by increased in vitro and in vivo CD4+ T-cell proliferation. However, only nasal PLGA nanoparticles were found to induce an immunoregulatory response as shown by enhanced Foxp3 expression in the nasopharynx associated lymphoid tissue and cervical lymph nodes. Nasal administration of OVA-containing PLGA particle resulted in functional suppression of an OVA-specific Th-1 mediated delayed-type hypersensitivity reaction, while TMC-TPP nanoparticles induced humoral immunity, which coincided with the enhanced generation of OVA-specific B-cells in the cervical lymph nodes. Intranasal treatment with Hsp70-mB29a peptide-loaded PLGA nanoparticles suppressed proteoglycan-induced arthritis, leading to a significant reduction of disease. We have uncovered a role for PLGA nanoparticles to enhance CD4+ T-cell mediated immunomodulation after nasal application. The exploitation of this differential regulation of nanoparticles to modulate nasal immune responses can lead to innovative vaccine development for prophylactic or therapeutic vaccination in infectious or autoimmune diseases.
doi:10.1371/journal.pone.0026684
PMCID: PMC3206834  PMID: 22073184
8.  Immunogenicity of Therapeutic Proteins: The Use of Animal Models 
Pharmaceutical Research  2011;28(10):2379-2385.
ABSTRACT
Immunogenicity of therapeutic proteins lowers patient well-being and drastically increases therapeutic costs. Preventing immunogenicity is an important issue to consider when developing novel therapeutic proteins and applying them in the clinic. Animal models are increasingly used to study immunogenicity of therapeutic proteins. They are employed as predictive tools to assess different aspects of immunogenicity during drug development and have become vital in studying the mechanisms underlying immunogenicity of therapeutic proteins. However, the use of animal models needs critical evaluation. Because of species differences, predictive value of such models is limited, and mechanistic studies can be restricted. This review addresses the suitability of animal models for immunogenicity prediction and summarizes the insights in immunogenicity that they have given so far.
doi:10.1007/s11095-011-0523-5
PMCID: PMC3170476  PMID: 21744171
animal models; immunogenicity; therapeutic proteins
9.  Taylor Dispersion Analysis Compared to Dynamic Light Scattering for the Size Analysis of Therapeutic Peptides and Proteins and Their Aggregates 
Pharmaceutical Research  2011;28(9):2302-2310.
ABSTRACT
Purpose
To evaluate Taylor dispersion analysis (TDA) as a novel method for determination of hydrodynamic radius of therapeutic peptides and proteins in non-stressed and stressed formulations and to compare it with dynamic light scattering (DLS).
Methods
The hydrodynamic radius of oxytocin, bovine serum albumin, various monoclonal antibodies (type IgG) and etanercept at concentrations between 0.05 and 50 mg/ml was determined by TDA and DLS. IgGs and etanercept were stressed (elevated temperatures) and analyzed by TDA, DLS and HP-SEC.
Results
TDA and DLS were comparable in sizing non-stressed peptides and proteins in a concentration range of about 0.5 to 50 mg/ml. TDA performed well even at lower concentrations, where DLS tends to provide theoretically high values of the Z-average radius. However, because of differences in the detection physics, DLS was more weighted towards the detection of aggregates in stressed formulations than TDA. Advantageously, TDA was also able to size the small peptide oxytocin, which was not feasible by DLS.
Conclusion
TDA allows the accurate determination of the hydrodynamic radius of peptides and proteins over a wide concentration range, with little interference from excipients present in the sample. It is marginally less sensitive than DLS in detecting size increase for stressed protein samples.
doi:10.1007/s11095-011-0460-3
PMCID: PMC3151397  PMID: 21560019
dynamic light scattering; hydrodynamic size; protein aggregation; protein characterization; Taylor dispersion analysis
10.  Oxidized and Aggregated Recombinant Human Interferon Beta is Immunogenic in Human Interferon Beta Transgenic Mice 
Pharmaceutical Research  2011;28(10):2393-2402.
ABSTRACT
Purpose
To study the effect of oxidation on the structure of recombinant human interferon beta-1a (rhIFNβ-1a) and its immunogenicity in wild-type and immune-tolerant transgenic mice.
Methods
Untreated rhIFNβ-1a was degraded by metal-catalyzed oxidation, H2O2-mediated oxidation, and guanidine-mediated unfolding/refolding. Four rhIFNβ-1a preparations with different levels of oxidation and aggregation were injected intraperitoneally in mice 15× during 3 weeks. Both binding and neutralizing antibodies were measured.
Results
All rhIFNβ-1a preparations contained substantial amounts of aggregates. Metal-catalyzed oxidized rhIFNβ-1a contained high levels of covalent aggregates as compared with untreated rhIFNβ-1a. H2O2-treated rhIFNβ-1a showed an increase in oligomer and unrecovered protein content by HP-SEC; RP-HPLC revealed protein oxidation. Guanidine-treated rhIFNβ-1a mostly consisted of dimers and oligomers and some non-covalent aggregates smaller in size than those in untreated rhIFNβ-1a. All degraded samples showed alterations in tertiary protein structure. Wild-type mice showed equally high antibody responses against all preparations. Transgenic mice were discriminative, showing elevated antibody responses against both metal-catalyzed oxidized and H2O2-treated rhIFNβ-1a as compared to untreated and guanidine-treated rhIFNβ-1a.
Conclusions
Oxidation-mediated aggregation increased the immunogenicity of rhIFNβ-1a in transgenic mice, whereas aggregated preparations devoid of measurable oxidation levels were hardly immunogenic.
doi:10.1007/s11095-011-0451-4
PMCID: PMC3170469  PMID: 21544687
antibodies; immunogenicity; oxidation; protein aggregates; recombinant human interferon beta
11.  A New Strategy to Stabilize Oxytocin in Aqueous Solutions: I. The Effects of Divalent Metal Ions and Citrate Buffer 
The AAPS Journal  2011;13(2):284-290.
In the current study, the effect of metal ions in combination with buffers (citrate, acetate, pH 4.5) on the stability of aqueous solutions of oxytocin was investigated. Both monovalent metal ions (Na+ and K+) and divalent metal ions (Ca2+, Mg2+, and Zn2+) were tested all as chloride salts. The effect of combinations of buffers and metal ions on the stability of aqueous oxytocin solutions was determined by RP-HPLC and HP-SEC after 4 weeks of storage at either 4°C or 55°C. Addition of sodium or potassium ions to acetate- or citrate-buffered solutions did not increase stability, nor did the addition of divalent metal ions to acetate buffer. However, the stability of aqueous oxytocin in aqueous formulations was improved in the presence of 5 and 10 mM citrate buffer in combination with at least 2 mM CaCl2, MgCl2, or ZnCl2 and depended on the divalent metal ion concentration. Isothermal titration calorimetric measurements were predictive for the stabilization effects observed during the stability study. Formulations in citrate buffer that had an improved stability displayed a strong interaction between oxytocin and Ca2+, Mg2+, or Zn2+, while formulations in acetate buffer did not. In conclusion, our study shows that divalent metal ions in combination with citrate buffer strongly improved the stability of oxytocin in aqueous solutions.
doi:10.1208/s12248-011-9268-7
PMCID: PMC3085697  PMID: 21448747
citrate buffer; divalent metal ions; improved stability; oxytocin
12.  A New Strategy to Stabilize Oxytocin in Aqueous Solutions: I. The Effects of Divalent Metal Ions and Citrate Buffer 
The AAPS Journal  2011;13(2):284-290.
In the current study, the effect of metal ions in combination with buffers (citrate, acetate, pH 4.5) on the stability of aqueous solutions of oxytocin was investigated. Both monovalent metal ions (Na+ and K+) and divalent metal ions (Ca2+, Mg2+, and Zn2+) were tested all as chloride salts. The effect of combinations of buffers and metal ions on the stability of aqueous oxytocin solutions was determined by RP-HPLC and HP-SEC after 4 weeks of storage at either 4°C or 55°C. Addition of sodium or potassium ions to acetate- or citrate-buffered solutions did not increase stability, nor did the addition of divalent metal ions to acetate buffer. However, the stability of aqueous oxytocin in aqueous formulations was improved in the presence of 5 and 10 mM citrate buffer in combination with at least 2 mM CaCl2, MgCl2, or ZnCl2 and depended on the divalent metal ion concentration. Isothermal titration calorimetric measurements were predictive for the stabilization effects observed during the stability study. Formulations in citrate buffer that had an improved stability displayed a strong interaction between oxytocin and Ca2+, Mg2+, or Zn2+, while formulations in acetate buffer did not. In conclusion, our study shows that divalent metal ions in combination with citrate buffer strongly improved the stability of oxytocin in aqueous solutions.
doi:10.1208/s12248-011-9268-7
PMCID: PMC3085697  PMID: 21448747
citrate buffer; divalent metal ions; improved stability; oxytocin
13.  Fluorescence Single Particle Tracking for the Characterization of Submicron Protein Aggregates in Biological Fluids and Complex Formulations 
Pharmaceutical Research  2011;28(5):1112-1120.
ABSTRACT
Purpose
To evaluate the potential of fluorescence single particle tracking (fSPT) for the characterization of submicron protein aggregates in human serum, plasma and formulations containing human serum albumin (HSA).
Methods
A monoclonal IgG was covalently labeled with a fluorescent dye and cross-linked with glutaraldehyde. IgG aggregates and fluorescent beads of 0.1 μm (control) were diluted in buffer, serum and plasma, and their size distributions were analyzed by fSPT and nanoparticle tracking analysis (NTA). In a separate experiment, IgG and HSA, fluorescently labeled with different dyes, were mixed and subjected to heat stress. The stressed sample was analyzed by fSPT using a dual color mode and by NTA.
Results
The accuracy and precision of fSPT proved to be comparable to NTA. fSPT was able to successfully measure all the samples in buffer, serum and plasma. The average size of the cross-linked protein aggregates showed a slight increase in biological fluids. Moreover, fSPT analysis showed that a significant proportion of the aggregates formed by subjecting an IgG/HSA mixture to heat stress were composed of both proteins.
Conclusion
fSPT is a powerful technique for the characterization of submicron protein aggregates in biological fluids and complex formulations.
Electronic Supplementary Material
The online version of this article (doi:10.1007/s11095-011-0374-0) contains supplementary material, which is available to authorized users.
doi:10.1007/s11095-011-0374-0
PMCID: PMC3073042  PMID: 21298328
fluorescence single particle tracking; HSA; IgG; protein aggregates; serum and plasma
14.  Strategies for the Assessment of Protein Aggregates in Pharmaceutical Biotech Product Development 
Pharmaceutical Research  2010;28(4):920-933.
ABSTRACT
Within the European Immunogenicity Platform (EIP) (http://www.e-i-p.eu), the Protein Characterization Subcommittee (EIP-PCS) has been established to discuss and exchange experience of protein characterization in relation to unwanted immunogenicity. In this commentary, we, as representatives of EIP-PCS, review the current state of methods for analysis of protein aggregates. Moreover, we elaborate on why these methods should be used during product development and make recommendations to the biotech community with regard to strategies for their application during the development of protein therapeutics.
doi:10.1007/s11095-010-0297-1
PMCID: PMC3063870  PMID: 20972611
aggregation; aggregates; analytical characterization; biotech products; commentary; drug development; particles; proteins; recommendation
15.  Quality of Original and Biosimilar Epoetin Products 
Pharmaceutical Research  2010;28(2):386-393.
ABSTRACT
Purpose
To compare the quality of therapeutic erythropoietin (EPO) products, including two biosimilars, with respect to content, aggregation, isoform profile and potency.
Methods
Two original products, Eprex (epoetin alfa) and Dynepo (epoetin delta), and two biosimilar products, Binocrit (epoetin alfa) and Retacrit (epoetin zeta), were compared using (1) high performance size exclusion chromatography, (2) ELISA, (3) SDS-PAGE, (4) capillary zone electrophoresis and (5) in-vivo potency.
Results
Tested EPO products differed in content, isoform composition, and potency.
Conclusion
Of the tested products, the biosimilars have the same or even better quality as the originals. Especially, the potency of originals may significantly differ from the value on the label.
doi:10.1007/s11095-010-0288-2
PMCID: PMC3026707  PMID: 20886265
biosimilar; immunogenicity; protein characterization; recombinant human erythropoietin
16.  Mass Spectrometric Analysis of Intact Human Monoclonal Antibody Aggregates Fractionated by Size-Exclusion Chromatography 
Pharmaceutical Research  2010;27(10):2197-2204.
ABSTRACT
Purpose
The aim of this study was to develop a method to characterize intact soluble monoclonal IgG1 antibody (IgG) oligomers by mass spectrometry.
Methods
IgG aggregates (dimers, trimers, tetramers and high-molecular-weight oligomers) were created by subjecting an IgG formulation to several pH jumps. Protein oligomer fractions were isolated by high performance size exclusion chromatography (HP-SEC), dialyzed against ammonium acetate pH 6.0 (a mass spectrometry-compatible volatile buffer), and analyzed by native electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS).
Results
Monomeric and aggregated IgG fractions in the stressed IgG formulation were successfully isolated by HP-SEC. ESI-TOF MS analysis enabled us to determine the molecular weight of the monomeric IgG as well as the aggregates, including dimers, trimers and tetramers. HP-SEC separation and sample preparation proved to be necessary for good quality signal in ESI-TOF MS. Both the HP-SEC protocol and the ESI-TOF mass spectrometric technique were shown to leave the IgG oligomers largely intact.
Conclusions
ESI-TOF MS is a useful tool complementary to HP-SEC to identify and characterize small oligomeric protein aggregates.
doi:10.1007/s11095-010-0224-5
PMCID: PMC2939344  PMID: 20680668
HP-SEC; IgG1; monoclonal antibody; native ESI-TOF mass spectrometry; protein aggregation
17.  Microneedle-Based Transcutaneous Immunisation in Mice with N-Trimethyl Chitosan Adjuvanted Diphtheria Toxoid Formulations 
Pharmaceutical Research  2010;27(9):1837-1847.
ABSTRACT
Purpose
The purpose of this study was to gain insight into the delivery and immunogenicity of N-trimethyl chitosan (TMC) adjuvanted diphtheria toxoid (DT) formulations applied transcutaneously with microneedles.
Methods
Mice were vaccinated with DT-loaded TMC nanoparticles, a solution of TMC and DT (TMC/DT) or DT alone. The formulations were applied onto the skin before or after microneedle treatment with two different 300-µm-long microneedle arrays and also injected intradermally (ID). As a positive control, alum-adjuvanted DT (DT-alum) was injected subcutaneously (SC). Ex vivo confocal microscopy studies were performed with rhodamine-labelled TMC.
Results
Independent of the microneedle array used and the sequence of microneedle treatment and vaccine application, transcutaneous immunisation with the TMC/DT mixture elicited 8-fold higher IgG titres compared to the TMC nanoparticles or DT solution. The toxin-neutralising antibody titres from this group were similar to those elicited by SC DT-alum. After ID immunisation, both TMC-containing formulations induced enhanced titres compared to a DT solution. Confocal microscopy studies revealed that transport of the TMC nanoparticles across the microneedle conduits was limited compared to a TMC solution.
Conclusions
In conclusion, TMC has an adjuvant function in transcutaneous immunisation with microneedles, but only if applied in a solution.
Electronic Supplementary Material
The online version of this article (doi:10.1007/s11095-010-0182-y) contains supplementary material, which is available to authorized users.
doi:10.1007/s11095-010-0182-y
PMCID: PMC2920068  PMID: 20559701
diphtheria toxoid; microneedles; nanoparticles; transcutaneous immunisation; trimethyl chitosan (TMC)
18.  Aggregated Recombinant Human Interferon Beta Induces Antibodies but No Memory in Immune-Tolerant Transgenic Mice 
Pharmaceutical Research  2010;27(9):1812-1824.
ABSTRACT
Purpose
To study the influence of protein aggregation on the immunogenicity of recombinant human interferon beta (rhIFNβ) in wild-type mice and transgenic, immune-tolerant mice, and to evaluate the induction of immunological memory.
Methods
RhIFNβ-1b and three rhIFNβ-1a preparations with different aggregate levels were injected intraperitoneally in mice 15× during 3 weeks, and the mice were rechallenged with rhIFNβ-1a. The formation of binding (BABs) and neutralizing antibodies (NABs) was monitored.
Results
Bulk rhIFNβ-1a contained large, mainly non-covalent aggregates and stressed rhIFNβ-1a mainly covalent, homogeneous (ca. 100 nm) aggregates. Reformulated rhIFNβ-1a was essentially aggregate-free. All products induced BABs and NABs in wild-type mice. Immunogenicity in the transgenic mice was product dependent. RhIFNβ-1b showed the highest and reformulated rhIFNβ-1a the lowest immunogenicity. In contrast with wild-type mice, transgenic mice did not show NABs, nor did they respond to the rechallenge.
Conclusions
The immunogenicity of the products in transgenic mice, unlike in wild-type mice, varied. In the transgenic mice, neither NABs nor immunological memory developed. The immunogenicity of rhIFNβ in a model reflecting the human immune system depends on the presence and the characteristics of aggregates.
doi:10.1007/s11095-010-0172-0
PMCID: PMC2916121  PMID: 20499141
antibodies; immunogenicity; immunological memory; protein aggregates; recombinant human interferon beta
19.  The Molecular Chaperone α-Crystallin as an Excipient in an Insulin Formulation 
Pharmaceutical Research  2010;27(7):1337-1347.
ABSTRACT
Purpose
To investigate insulin fibrillation under accelerated stress conditions in the presence of a novel excipient, the molecular chaperone α-crystallin, in comparison with common excipients.
Methods
To induce fibrillation, recombinant human insulin (0.58 mg ml−1) formulations without excipient or with bovine α-crystallin (0.01–0.2 mg ml−1), human serum albumin (1–5 mg ml−1), sucrose (10–100 mg ml−1) or polysorbate 80 (0.075–0.3 mg ml−1) were subjected to stirring stress in a fluorescence well plate reader and formulation vials. Protein fibrillation was monitored by thioflavin T. The formulations were further characterized by size-exclusion chromatography, light obscuration, UV/Vis and circular dichroism spectroscopy.
Results
In both methods, insulin formed thioflavin T-binding species, most likely fibrils. Addition of α-crystallin in the well plate assay greatly improved insulin’s resistance to fibrillation, measured as a 6-fold increase in fibrillation lag time for the lowest and 26-fold for the highest concentration used, whereas all other excipients showed only a marginal increase in lag time. The stabilizing effect of α-crystallin was shown by all characterization techniques used.
Conclusions
The effect of α-crystallin on insulin’s physical stability outperforms that of commonly used excipients. α-Crystallin is proposed to bind specifically to pre-fibrillation species, thereby inhibiting fibrillation. This makes α-crystallin an interesting excipient for proteins with propensity to fibrillate.
doi:10.1007/s11095-010-0116-8
PMCID: PMC2883933  PMID: 20333453
fibrillation; formulation design; insulin; protein excipients; α-crystallin
20.  Transcutaneous Immunization Studies in Mice Using Diphtheria Toxoid-Loaded Vesicle Formulations and a Microneedle Array 
Pharmaceutical Research  2010;28(1):145-158.
ABSTRACT
Purpose
To determine the immunogenicity of diphtheria toxoid (DT) formulated in two types of vesicles following transcutaneous immunization (TCI) of mice onto microneedle array-treated skin.
Methods
DT-containing cationic liposomes or anionic surfactant-based vesicles were prepared by extrusion and sonication. The physicochemical properties were characterized in terms of size, ζ-potential, vesicle elasticity and antigen association. TCI was performed by applying formulations onto intact or microneedle array-pretreated mice skin, using cholera toxin as an adjuvant. Subcutaneous and intradermal immunizations were as control. Immune responses were evaluated by IgG and neutralizing antibody titers, and the immune-stimulatory properties were assessed using cultured dendritic cells.
Results
Stable DT-containing cationic liposomes (∼150 nm) and anionic vesicles (∼100 nm) were obtained. Incorporation of Span 80 increased liposome elasticity. About 90% and 77% DT was associated with liposomes and vesicles, respectively. TCI of all formulations resulted in substantial antibody titers only if microneedle pretreatment was applied. Co-administration of cholera toxin further augmented the immune responses of TCI. However, vesicle formulations didn’t enhance the immunogenicity on either intact or microneedle-treated skin and showed low stimulatory activity on dendritic cells.
Conclusions
Microneedle pretreatment and cholera toxin, but not antigen association to vesicles, enhances the immunogenicity of topically applied DT.
doi:10.1007/s11095-010-0093-y
PMCID: PMC3003783  PMID: 20237826
cholera toxin; diphtheria toxoid; microneedle array; transcutaneous immunization; vesicles
21.  Critical Evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the Measurement of Nanoparticles and Protein Aggregates 
Pharmaceutical Research  2010;27(5):796-810.
Purpose
To evaluate the nanoparticle tracking analysis (NTA) technique, compare it with dynamic light scattering (DLS) and test its performance in characterizing drug delivery nanoparticles and protein aggregates.
Methods
Standard polystyrene beads of sizes ranging from 60 to 1,000 nm and physical mixtures thereof were analyzed with NTA and DLS. The influence of different ratios of particle populations was tested. Drug delivery nanoparticles and protein aggregates were analyzed by NTA and DLS. Live monitoring of heat-induced protein aggregation was performed with NTA.
Results
NTA was shown to accurately analyze the size distribution of monodisperse and polydisperse samples. Sample visualization and individual particle tracking are features that enable a thorough size distribution analysis. The presence of small amounts of large (1,000 nm) particles generally does not compromise the accuracy of NTA measurements, and a broad range of population ratios can easily be detected and accurately sized. NTA proved to be suitable to characterize drug delivery nanoparticles and protein aggregates, complementing DLS. Live monitoring of heat-induced protein aggregation provides information about aggregation kinetics and size of submicron aggregates.
Conclusion
NTA is a powerful characterization technique that complements DLS and is particularly valuable for analyzing polydisperse nanosized particles and protein aggregates.
doi:10.1007/s11095-010-0073-2
PMCID: PMC2852530  PMID: 20204471
dynamic light scattering; liposomes; nanoparticles; nanoparticle tracking analysis; protein aggregates
22.  Fluorescent Molecular Rotors as Dyes to Characterize Polysorbate-Containing IgG Formulations 
Pharmaceutical Research  2009;27(2):314-326.
Purpose
The aim was to evaluate fluorescent molecular rotors (DCVJ and CCVJ), which are mainly sensitive to viscosity, for the characterization of polysorbate-containing IgG formulations and compare them to the polarity-sensitive dyes ANS, Bis-ANS and Nile Red.
Methods
IgG formulations with polysorbate 20 or 80 were stressed below the aggregation temperature and analyzed by steady-state and time-resolved fluorescence and by HP-SEC with UV and fluorescent dye detection (Bis-ANS and CCVJ). Furthermore, commercial protein preparations of therapeutic proteins (Enbrel®50 mg, Humira®40 mg and MabThera®100 mg) were aggregated accordingly and analyzed with CCVJ fluorescence and HP-SEC.
Results
Contrarily to (Bis-)ANS and Nile Red, the molecular rotors DCVJ and CCVJ showed low background fluorescence in polysorbate-containing buffers. Time-resolved fluorescence experiments confirmed the steady-state fluorescence data. Both DCVJ and CCVJ showed enhanced fluorescence intensity for aggregated IgG formulations and were suitable for the characterization of polysorbate-containing IgG formulations in steady-state fluorescence and HP-SEC with dye detection (CCVJ). CCVJ was capable of detecting thermally induced aggregation in the commercial polysorbate-containing products Enbrel®50 mg, Humira®40 mg and MabThera®100 mg.
Conclusion
Fluorescent molecular rotors are suitable probes to detect aggregation in polysorbate-containing IgG formulations.
Electronic supplementary material
The online version of this article (doi:10.1007/s11095-009-0020-2) contains supplementary material, which is available to authorized users.
doi:10.1007/s11095-009-0020-2
PMCID: PMC2812426  PMID: 20041280
aggregation; fluorescence; fluorescent molecular rotors; monoclonal antibodies; size-exclusion chromatography
23.  Towards Heat-stable Oxytocin Formulations: Analysis of Degradation Kinetics and Identification of Degradation Products 
Pharmaceutical Research  2009;26(7):1679-1688.
Purpose
To investigate degradation kinetics of oxytocin as a function of temperature and pH, and identify the degradation products.
Materials and Methods
Accelerated degradation of oxytocin formulated at pH 2.0, 4.5, 7.0 and 9.0 was performed at 40, 55, 70 and 80°C. Degradation rate constants were determined from RP-HPLC data. Formulations were characterized by HP-SEC, UV absorption and fluorescence spectroscopy. Degradation products were identified by ESI-MS/MS.
Results
The loss of intact oxytocin in RP-HPLC was pH- and temperature-dependent and followed (pseudo) first order kinetics. Degradation was fastest at pH 9.0, followed by pH 7.0, pH 2.0 and pH 4.5. The Arrhenius equation proved suitable to describe the kinetics, with the highest activation energy (116.3 kJ/mol) being found for pH 4.5 formulations. At pH 2.0 deamidation of Gln4, Asn5, and Gly9-NH2, as well as combinations thereof were found. At pH 4.5, 7.0 and 9.0, the formation of tri- and tetrasulfide-containing oxytocin as well as different types of disulfide and dityrosine-linked dimers were found to occur. Beta-elimination and larger aggregates were also observed. At pH 9.0, mono-deamidation of Gln4, Asn5, and Gly9-NH2 additionally occurred.
Conclusions
Multiple degradation products of oxytocin have been identified unequivocally, including various deamidated species, intramolecular oligosulfides and covalent aggregates. The strongly pH dependent degradation can be described by the Arrhenius equation.
doi:10.1007/s11095-009-9878-2
PMCID: PMC2689356  PMID: 19343484
aggregation; Arrhenius kinetics; degradation; mass spectrometry; oxytocin
24.  Extrinsic Fluorescent Dyes as Tools for Protein Characterization 
Pharmaceutical Research  2008;25(7):1487-1499.
Noncovalent, extrinsic fluorescent dyes are applied in various fields of protein analysis, e.g. to characterize folding intermediates, measure surface hydrophobicity, and detect aggregation or fibrillation. The main underlying mechanisms, which explain the fluorescence properties of many extrinsic dyes, are solvent relaxation processes and (twisted) intramolecular charge transfer reactions, which are affected by the environment and by interactions of the dyes with proteins. In recent time, the use of extrinsic fluorescent dyes such as ANS, Bis-ANS, Nile Red, Thioflavin T and others has increased, because of their versatility, sensitivity and suitability for high-throughput screening. The intention of this review is to give an overview of available extrinsic dyes, explain their spectral properties, and show illustrative examples of their various applications in protein characterization.
doi:10.1007/s11095-007-9516-9
PMCID: PMC2440933  PMID: 18172579
extrinsic fluorescent dyes; fluorescence spectroscopy; protein aggregation; protein characterization; protein folding
25.  Characterization of a cyclosporine solid dispersion for inhalation 
The AAPS Journal  2007;9(2):E190-E199.
For lung transplant patients, a respirable, inulin-based solid dispersion containing cyclosporine A (CsA) has been developed. The solid dispersions were prepared by spray freezedrying. The solid dispersion was characterized by water vapor uptake, specific surface area analysis, and particle size analysis. Furthermore, the mode of inclusion of CsA in the dispersion was investigated with Fourier transform infrared spectroscopy. Finally, the dissolution behavior was determined and the aerosol that was formed by the powder was characterized. The powder had large specific surface areas (∼160 m2). The water vapor uptake was dependent linearly on the drug load. The type of solid dispersion was a combination of a solid solution and solid suspension. At a 10% drug load, 55% of the CsA in the powder was in the form of a solid solution and 45% as solid suspension. At 50% drug load, the powder contained 90% of CsA as solid suspension. The powder showed excellent dispersion characteristics as shown by the high emitted fraction (95%), respirable fraction (75%), and fine-particle fraction (50%). The solid dispersions consisted of relatively large (x50≈7 μm), but low-density particles (ρ≈0.2 g/cm3). The solid dispersions dissolved faster than the physical mixture, and inulin dissolved faster than CsA. The spray freeze-drying with inulin increased the specific surface area and wettability of CsA. In conclusion, the developed powder seems suitable for inhalation in the local treatment of lung transplant patients.
doi:10.1208/aapsj0902021
PMCID: PMC2751408  PMID: 17614361
DPI; Cyclosporine A; solid dispersion; FTIR; aerosol; large porous particles

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