Enzymatic degradation pattern of polysorbate 20 impacts interfacial properties of monoclonal antibody formulations.

Polysorbate 20 (PS20) is widely used to maintain protein stability in biopharmaceutical formulations. However, PS20 is susceptible to hydrolytic degradation catalyzed by trace amounts of residual host cell proteins present in monoclonal antibody (mAb) formulations. The resulting loss of intact surfactant and the presence of PS20 degradation products, such as free fatty acids (FFAs), may impair protein stability. In this study, two hydrolytically-active immobilized lipases, which primarily targeted either monoester or higher-order ester species in PS20, were used to generate partially-degraded PS20. The impact of PS20 degradation pattern on critical micelle concentration (CMC), surface tension, interfacial rheology parameters and agitation protection was assessed. CMC was slightly increased upon monoester degradation, but significantly increased upon higher-order ester degradation. The PS20 degradation pattern also significantly impacted the dynamic surface tension of a mAb formulation, whereas changes in the equilibrium surface tension were mainly caused by the adsorption of FFAs onto the air-water interface. In an agitation protection study, monoester degradation resulted in the formation of soluble mAb aggregates and proteinaceous particles, suggesting that preferential degradation of PS20 monoester species can significantly impair mAb stability. Additional mAbs should be tested in the future to assess the impact of the protein format.

Using Peptide Nucleic Acid Hybridization Probes for Qualitative and Quantitative Analysis of Nucleic Acid Therapeutics by Capillary Electrophoresis.

The space of advanced therapeutic modalities is currently evolving in rapid pace necessitating continuous improvement of analytical quality control methods. In order to evaluate the identity of nucleic acid species in gene therapy products, we propose a capillary electrophoresis-based gel free hybridization assay in which fluorescently labeled peptide nucleic acids (PNAs) are applied as affinity probes. PNAs are engineered organic polymers that share the base pairing properties with DNA and RNA but have an uncharged peptide backbone. In the present study, we conduct various proof-of-concept studies to identify the potential of PNA probes for advanced analytical characterization of novel therapeutic modalities like oligonucleotides, plasmids, mRNA, and DNA released by recombinant adeno-associated virus. For single-stranded nucleic acids up to 1000 nucleotides, the method is an excellent choice that proved to be highly specific by detecting DNA traces in complex samples, while having a limit of quantification in the picomolar range when multiple probes are used. For double-stranded samples, only fragments that are similar in size to the probe could be quantified. This limitation can be circumvented when target DNA is digested and multiple probes are used opening an alternative to quantitative PCR.

Hydrogel microspheres evading alveolar macrophages for sustained pulmonary protein delivery.

Pulmonary delivery is a highly attractive alternative to injections for biologics such as therapeutic proteins. However, bioavailabilities generally suffer from the presence of phagocytic cells that clear particulate matter entering the lung. In this study, microgel particles were developed using an all-aqueous two-phase system approach and evaluated for their efficacy as an inhalable controlled release system. Norbornene- and thiol-modified four- and eight-armed poly (ethylene glycol) with an average molecular mass of 10,000 Da were prepared as macromonomers for microgel formation. Emulsions of precursor solution droplets containing macromonomers and Irgacure 2959 as photocatalyst were prepared in a dextran solution. Irradiation with UV light was used to covalently crosslink the droplets by triggering the thiol-ene reaction. The resulting microgels were processed to dry powder inhaler formulations, and respirable aerodynamic sizes were assessed in vitro. Microgels were loaded with the model proteins lysozyme and bovine serum albumin, with encapsulation efficiencies of 51.5% and 73.6%, respectively. Depending on the macromonomer type, protein-loaded microgels released their cargo over a 6-14 day period. In an MTT assay, the particles did not show significant cytotoxicity, and their recognition by alveolar macrophages was considerably lower than for polystyrene control particles. This makes the microgels a promising pulmonary delivery system for proteins and other biologics.

Interaction of functionalized nanoparticles with serum proteins and its impact on colloidal stability and cargo leaching.

The majority of effort in the area of polymeric nanocarriers is aimed at providing controlled drug delivery in vivo. Therefore, it is essential to understand the delicate interplay of polymeric NPs with serum proteins in order to forecast their performance in a biological system. In this study, the interaction of serum proteins with functionalized polymeric colloids as a function of particle charge and hydrophobicity was investigated. Moreover, impact on NP stability and cargo leaching was assessed. The hard protein corona of polymeric NPs with either uncharged methoxy groups (methoxy-NPs), positively charged amine groups (amine-NPs), negatively charged carboxylic acid groups (carboxyl-NPs) or zwitterionic NPs decorated with amine and carboxylic acid groups (zwitterion-NPs) was quantitatively and qualitatively analyzed and correlated with the respective colloidal stability using fluorescence resonance energy transfer. Positively charged amine-NPs displayed an enhanced interaction with serum proteins via electrostatic interactions resulting in a hard corona consisting of diverse protein components. As revealed by FRET and agarose gel electrophoresis, the enhanced adsorption of proteins onto the colloidal surface significantly altered the NP identity and severely impaired the colloidal integrity as the lipophilic cargo was continuously leached out of the hydrophobic NP core. These results highlight the importance of generating a profound knowledge of the bio-nano interface as adherence of biomolecules can severely compromise the performance of a colloidal drug delivery system by changing its identity and integrity.

Ligand Density and Linker Length are Critical Factors for Multivalent Nanoparticle-Receptor Interactions.

Although there are a large number of studies available for the evaluation of the therapeutic efficacy of targeted polymeric nanoparticles, little is known about the critical attributes that can further influence their uptake into target cells. In this study, varying cRGD ligand densities (0-100% surface functionalization) were combined with different poly(ethylene glycol) (PEG) spacer lengths (2/3.5/5 kDa), and the specific receptor binding of targeted core-shell structured poly(lactic- co-glycolic acid)/poly(lactic acid)-PEG nanoparticles was evaluated using αvß3 integrin-overexpressing U87MG glioblastoma cells. Nanoparticles with 100% surface functionalization and short PEG2k linkers displayed a high propensity to form colloidal clusters, allowing for the cooperative binding to integrin receptors on the cellular membrane. In contrast, the high flexibility of longer PEG chains enhanced the chance of ligand entanglement and shrouding, decreasing the number of ligand-receptor binding events. As a result, the combination of short PEG2k linkers and a high cRGD surface modification synergistically increased the uptake of nanoparticles into target cells. Even though to date, the nanoparticle size and its degree of functionalization are considered to be the major determinants for controlling the uptake efficiency of targeted colloids, these results strongly suggest that the role of the linker length should be carefully taken into consideration for the design of targeted drug delivery formulations to maximize the therapeutic efficacy and minimize adverse side effects.

Fabrication of antibody-loaded microgels using microfluidics and thiol-ene photoclick chemistry.

Reducing burst effects, providing controlled release, and safeguarding biologics against degradation are a few of several highly attractive applications for microgels in the field of controlled release. However, the incorporation of proteins into microgels without impairing stability is highly challenging. In this proof of concept study, the combination of microfluidics and thiol-ene photoclick chemistry was evaluated for the fabrication of antibody-loaded microgels with narrow size distribution. Norbornene-modified eight-armed poly(ethylene glycol) with an average molecular mass of 10,000 Da, 20,000 Da, or 40,000 Da were prepared as macromonomers for microgel formation. For functionalization, either hydrolytically cleavable ester or stable amide bonds were used. A microfluidic system was employed to generate precursor solution droplets containing macromonomers, the cross-linker dithiothreitol and the initiator Eosin-Y. Irradiation with visible light was used to trigger thiol-ene reactions which covalently cross-linked the droplets. For all bond-types, molecular masses, and concentrations gelation was very rapid (<20 s) and a plateau for the complex shear modulus was reached after only 5 min. The generated microgels had a rod-like shape and did not show considerable cellular toxicity. Stress conditions during the fabrication process were simulated and it could be shown that fabrication did not impair the activity of the model proteins lysozyme and bevacizumab. It was confirmed that the average hydrogel network mesh size was similar or smaller than the hydrodynamic diameter of bevacizumab which is a crucial factor for restricting diffusion and delaying release. Finally, microgels were loaded with bevacizumab and a sustained release over a period of 30 ±â€¯4 and 47 ±â€¯7 days could be achieved in vitro.

Controlled Antibody Release from Degradable Thermoresponsive Hydrogels Cross-Linked by Diels-Alder Chemistry.

Amine-modified four- and eight-armed poloxamines were prepared and subsequently functionalized with maleimide or furyl groups. Aqueous solutions of these polymers exhibited an immediate gelation at a temperature above 37 °C. Concomitantly, Diels-Alder reactions gradually cross-linked and cured the gels. Different ratios between four- and eight-armed macromonomers were used to tune hydrogel stability and mechanical properties. In this way, hydrogel stability could be precisely controlled in the range of 14 to 329 days. Controlled release of the model antibody bevacizumab was achieved over a period of 7, 21, and 115 days. Release profiles were triphasic with a low burst; approximately 87% of the released antibody was intact and displayed functional binding. The hydrogels presented in this study are degradable, nontoxic, rapidly gelling, stable, and provide controlled antibody release. They can be tailored to match the demands of various applications and present an attractive platform for antibody delivery.

Dynamic NHERF interaction with TRPC4/5 proteins is required for channel gating by diacylglycerol.

The activation mechanism of the classical transient receptor potential channels TRPC4 and -5 via the Gq/11 protein-phospholipase C (PLC) signaling pathway has remained elusive so far. In contrast to all other TRPC channels, the PLC product diacylglycerol (DAG) is not sufficient for channel activation, whereas TRPC4/5 channel activity is potentiated by phosphatidylinositol 4,5-bisphosphate (PIP2) depletion. As a characteristic structural feature, TRPC4/5 channels contain a C-terminal PDZ-binding motif allowing for binding of the scaffolding proteins Na+/H+ exchanger regulatory factor (NHERF) 1 and 2. PKC inhibition or the exchange of threonine for alanine in the C-terminal PDZ-binding motif conferred DAG sensitivity to the channel. Altogether, we present a DAG-mediated activation mechanism for TRPC4/5 channels tightly regulated by NHERF1/2 interaction. PIP2 depletion evokes a C-terminal conformational change of TRPC5 proteins leading to dynamic dissociation of NHERF1/2 from the C terminus of TRPC5 as a prerequisite for DAG sensitivity. We show that NHERF proteins are direct regulators of ion channel activity and that DAG sensitivity is a distinctive hallmark of TRPC channels.

Polyanions effectively prevent protein conjugation and activity loss during hydrogel cross-linking.

In situ encapsulation is a frequently used method to prepare hydrogels loaded with high quantities of therapeutic proteins. However, many cross-linking reactions, such as Michael-type addition or Diels-Alder (DA) reaction are not tolerant toward nucleophiles; therefore, side-reactions with proteins can occur during cross-linking. This may lead to undesired protein conjugation, activity loss and incomplete protein release. In this study, a number of polyanions, namely alginate, dextran sulfate, hyaluronic acid, heparin, and poly(acrylic acid), were screened for their capability to protect proteins during covalent cross-linking. To this end, lysozyme was incubated with furyl- and maleimide-substituted methoxy poly(ethylene glycol); different pH values were tested. The degree of PEGylation and the residual activity of lysozyme were investigated. Without polyanions, 61.1% of the total lysozyme amount was PEGylated at pH7.4; the residual activity was 20.3% of the initial activity. With the most effective polyanion (dextran sulfate), PEGylation could be completely suppressed; the residual activity was 98.4%. The protective effect of polyanions was attributed to electrostatic interactions with proteins; the "shielding" could be reversed by adding high salt concentrations. Furthermore, the protective effect was dependent on the concentration and molecular mass of the polyanion, but almost independent of the protein concentration. As a proof of concept, hydrogels were loaded with lysozyme and bevacizumab during cross-linking via DA reaction. Without polyanions, a large fraction of the protein was covalently bound to the polymer network resulting in degradation-controlled release; the residual activity of lysozyme was 50.0%. With polyanions, the protein molecules were mobile and their release was diffusion-controlled. The residual activity of lysozyme was 88.9%; the released bevacizumab was structurally intact. Polyanions can, therefore, be used as protective additive to prevent chemical protein modification during hydrogel cross-linking.

Design of hydrogels for delayed antibody release utilizing hydrophobic association and Diels-Alder chemistry in tandem.

Biodegradable hydrogels were prepared from furan- and maleimide-functionalized eight-armed poly(ethylene glycol) with an average molecular mass of 40 000 Da (8armPEG40k-furan and 8armPEG40k-maleimide) using the Diels-Alder (DA) reaction as a cross-linking mechanism. Hydrophobic 6-aminohexanoic acid (C6) and 12-aminododecanoic acid (C12) spacers were introduced between the polymer backbone and the functional end-groups; the influence on the gel properties was studied. Modification with C6 and C12 spacers induced hydrophobic interactions between the macromonomers leading to association and increased viscosity of the polymer solutions; both effects were influenced by the spacer length. In combination with DA cross-linking, hydrophobic derivatives of 8armPEG40k-furan and 8armPEG40k-maleimide led to hydrogels with improved properties. Upon introduction of C12 spacers, gelation of 8armPEG40k hydrogels occurred twice as fast. Interestingly, no effect was observed when only one of the two components had been modified. Our experiments suggest that the association of macromonomers by hydrophobic interactions facilitates chemical cross-linking via DA chemistry. This hypothesis is supported by calculations of the network mesh size and the Young's modulus of compression, which showed an increased cross-linking density of hydrophobically modified hydrogels. As a consequence of the increased cross-linking density, the degradation stability of C12-modified hydrogels increased by a factor of 4. Moreover, hydrophobic modification improved the hydrolytic resistance of maleimides; this also contributes to gel stability. The in vitro release of bevacizumab, which served as a model antibody, could be delayed for almost 60 days using modification with C12. Similar trends were observed for C6-modified 8armPEG40k hydrogels; however, the effects were considerably weaker. In summary, utilizing hydrophobic association and chemical cross-linking in tandem is a promising approach to create biodegradable hydrogels for delayed antibody release.

The Diels-Alder reaction: A powerful tool for the design of drug delivery systems and biomaterials.

Click reactions have the potential to greatly facilitate the development of drug delivery systems and biomaterials. These reactions proceed under mild conditions, give high yields, and form only inoffensive by-products. The Diels-Alder cycloaddition is one of the click reactions that do not require any metal catalyst; it is one of the most useful reactions in synthetic organic chemistry and material design. Herein, we highlight possible applications of the Diels-Alder reaction in pharmaceutics and biomedical engineering. Particular focus is placed on the synthesis of polymers and dendrimers for drug delivery, the preparation of functionalized surfaces, bioconjugation techniques, and applications of the Diels-Alder reaction in nanotechnology. Moreover, applications of the reaction for the preparation of hydrogels for drug delivery and tissue engineering are reviewed. A general introduction to the Diels-Alder reaction is presented, along with a discussion of potential pitfalls and challenges. At the end of the article, we provide a set of tools that may facilitate the application of the Diels-Alder reaction to solve important pharmaceutical or biomedical problems.

Diels-Alder hydrogels with enhanced stability: First step toward controlled release of bevacizumab.

Eight-armed PEG was functionalized with furyl and maleimide groups (8armPEG20k-Fur and 8armPEG20k-Mal); degradable hydrogels were obtained by cross-linking via Diels-Alder chemistry. To increase the stability to degradation, the macromonomers were modified by introducing a hydrophobic 6-aminohexanoic acid spacer between PEG and the reactive end-groups (8armPEG20k-Ahx-Fur and 8armPEG20k-Ahx-Mal). In an alternative approach, the number of reactive groups per macromonomer was increased by branching the terminal ends of eight-armed PEG with lysine (Lys) and Ahx residues (8armPEG20k-Lys-Ahx-Fur2 and 8armPEG20k-Lys-Ahx-Mal2). The hydrolytic resistance of the synthesized macromonomers was determined by UV spectroscopy; the obtained hydrogels were characterized by rheology and degradation studies. The degradation time of 5% (w/v) 8armPEG20k-Ahx hydrogels (28days) was twice as long as the degradation time of 5% (w/v) 8armPEG20k hydrogels (14days); this is explained by increased hydrolytic resistance of the maleimide group. Using dendritic 8armPEG20k-Lys-Ahx macromonomers substantially increased the stability of the resulting hydrogels; degradation of 5% (w/v) 8armPEG20k-Lys-Ahx hydrogels occurred after 34 weeks. 8armPEG20k hydrogels had the largest mesh size of all tested hydrogels, while hydrogels made from dendritic 8armPEG20k-Lys-Ahx macromonomers showed the smallest value. To evaluate their potential for the controlled release of therapeutic antibodies, the hydrogels were loaded with bevacizumab. The incorporated bevacizumab was released over 10 days (8armPEG20k) and 42days (8armPEG20k-Ahx), respectively; release from 8armPEG20k-Lys-Ahx hydrogels was not completed after 105 days. In summary, we believe that 8armPEG20k-Ahx or 8armPEG20k-Lys-Ahx hydrogels could serve as controlled release system for therapeutic antibodies such as bevacizumab.

How subvisible particles become invisible-relevance of the refractive index for protein particle analysis.

The aim of the present study was to quantitatively assess the relevance of transparency and refractive index (RI) on protein particle analysis by the light-based techniques light obscuration (LO) and Micro-Flow Imaging (MFI). A novel method for determining the RI of protein particles was developed and provided an RI of 1.41 for protein particles from two different proteins. An increased RI of the formulation by high protein concentration and/or sugars at pharmaceutically relevant levels was shown to lead to a significant underestimation of the subvisible particle concentration determined by LO and MFI. An RI match even caused particles to become "invisible" for the system, that is, not detectable anymore by LO and MFI. To determine the influence of formulation RI on particle measurements, we suggest the use of polytetrafluoroethylene (PTFE) particles to test a specific formulation for RI effects. In case of RI influences, we recommend also using a light-independent technique such as resonant mass measurement (RMM) (Archimedes) for subvisible particle analysis in protein formulations.