Publications by authors named "Patrick Garidel"

99 Publications

Hydrolytic polysorbate 20 degradation - Sensitive detection of free fatty acids in biopharmaceuticals via UPLC-QDa analytics with isolator column.

J Chromatogr B Analyt Technol Biomed Life Sci 2021 Apr 21;1174:122717. Epub 2021 Apr 21.

Boehringer Ingelheim Pharma GmbH & Co. KG, Innovation Unit, Birkendorfer Straße 65, 88400 Biberach an der Riss, Germany. Electronic address:

The enzymatic hydrolysis of polysorbates, e.g. induced by specific host cell proteins in biologics, is a known risk factor regarding the potential particle formation in the product over time. One of the root causes for this observation is an increase in free fatty acids (FA) within the formulation, which indicates the need for convenient monitoring of FA release. This study presents a novel UPLC-QDa based method to evaluate the content of the FAs esterified to polysorbate 20 (PS20) after hydrolysis. The presented method is label-free, i.e. independent of elaborate fluorophore-labeling and able to directly measure the ionized FAs. Furthermore, the method allows the determination of released FAs as percentage of ester bond hydrolysis and as absolute concentration expressed in ng/mL. Additionally, we describe for the first time in FA analytics the application of an isolator column, to remove trace levels of FAs present in the eluents to improve the sensitivity of the method. Lastly, the capabilities of the newly developed method are proven in case studies with three different monoclonal antibodies, which display characteristic FA release patterns in PS20-containing formulations. In summary, we developed a reliable, sensitive method for FA quantification in biologics, which could also be used as a predictive tool, considering FA solubility, regarding the formation of particles.
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http://dx.doi.org/10.1016/j.jchromb.2021.122717DOI Listing
April 2021

HP-β-CD for the formulation of IgG and Ig-based biotherapeutics.

Int J Pharm 2021 Mar 26;601:120531. Epub 2021 Mar 26.

Boehringer Ingelheim Pharma GmbH & Co. KG, Innovation Unit, Pharmaceutical Development Biologicals, Biberach an der Riss, Germany.

The main challenge to develop HCF for IgG and Ig-based therapeutics is to achieve essential solubility, viscosity and stability of these molecules in order to maintain product quality and meet regulatory requirement during manufacturing, production, storage, shipment and administration processes. The commonly used and FDA approved excipients for IgG and Ig -based therapeutics may no longer fulfil the challenge of HCF development for these molecules to certain extent, especially for some complex Ig-based platforms. 2-Hydroxypropyl beta-cyclodextrin (HP-β-CD) is one of the promising excipients applied recently for HCF development of IgG and Ig-based therapeutics although it has been used for formulation of small synthesized chemical drugs for more than thirty years. This review describes essential aspects about application of HP-β-CD as excipient in pharmaceutical formulation, including physico-chemical properties of HP-β-CD, supply chain, regulatory, patent landscape, marketed drugs with HP-β-CD, analytics and analytical challenges, stability and control strategies, and safety concerns. It also provides an overview of different studies, and outcomes thereof, regarding formulation development for IgGs and Ig-based molecules in liquid and solid (lyophilized) dosage forms with HP-β-CD. The review specifically highlights the challenges for formulation manufacturing of IgG and Ig-based therapeutics with HP-β-CD and identifies areas for future work in pharmaceutical and formulation development.
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http://dx.doi.org/10.1016/j.ijpharm.2021.120531DOI Listing
March 2021

Oil-Immersion Flow Imaging Microscopy for Quantification and Morphological Characterization of Submicron Particles in Biopharmaceuticals.

AAPS J 2021 01 4;23(1):13. Epub 2021 Jan 4.

Coriolis Pharma Research GmbH, Fraunhoferstr. 18 b, 82152, Martinsried, Germany.

Flow imaging microscopy (FIM) is widely used to analyze subvisible particles starting from 2 μm in biopharmaceuticals. Recently, an oil-immersion FIM system emerged, the FlowCam Nano, designed to enable the characterization of particle sizes even below 2 μm. The aim of our study was to evaluate oil-immersion FIM (by using FlowCam Nano) in comparison to microfluidic resistive pulse sensing and resonant mass measurement for sizing and counting of particles in the submicron range. Polystyrene beads, a heat-stressed monoclonal antibody formulation and a silicone oil emulsion, were measured to assess the performance on biopharmaceutical relevant samples, as well as the ability to distinguish particle types based on instrument-derived morphological parameters. The determination of particle sizes and morphologies suffers from inaccuracies due to a low image contrast of small particles and light-scattering effects. The ill-defined measured volume impairs an accurate concentration determination. Nevertheless, FlowCam Nano in its current design complements the limited toolbox of submicron particle analysis of biopharmaceuticals by providing particle images in a size range that was previously not accessible with commercial FIM instruments.
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http://dx.doi.org/10.1208/s12248-020-00547-9DOI Listing
January 2021

Particle Detection and Characterization for Biopharmaceutical Applications: Current Principles of Established and Alternative Techniques.

Pharmaceutics 2020 Nov 19;12(11). Epub 2020 Nov 19.

Innovation Unit, PDB, Boehringer Ingelheim Pharma GmbH & Co. KG, 88397 Biberach an der Riss, Germany.

Detection and characterization of particles in the visible and subvisible size range is critical in many fields of industrial research. Commercial particle analysis systems have proliferated over the last decade. Despite that growth, most systems continue to be based on well-established principles, and only a handful of new approaches have emerged. Identifying the right particle-analysis approach remains a challenge in research and development. The choice depends on each individual application, the sample, and the information the operator needs to obtain. In biopharmaceutical applications, particle analysis decisions must take product safety, product quality, and regulatory requirements into account. Biopharmaceutical process samples and formulations are dynamic, polydisperse, and very susceptible to chemical and physical degradation: improperly handled product can degrade, becoming inactive or in specific cases immunogenic. This article reviews current methods for detecting, analyzing, and characterizing particles in the biopharmaceutical context. The first part of our article represents an overview about current particle detection and characterization principles, which are in part the base of the emerging techniques. It is very important to understand the measuring principle, in order to be adequately able to judge the outcome of the used assay. Typical principles used in all application fields, including particle-light interactions, the Coulter principle, suspended microchannel resonators, sedimentation processes, and further separation principles, are summarized to illustrate their potentials and limitations considering the investigated samples. In the second part, we describe potential technical approaches for biopharmaceutical particle analysis as some promising techniques, such as nanoparticle tracking analysis (NTA), micro flow imaging (MFI), tunable resistive pulse sensing (TRPS), flow cytometry, and the space- and time-resolved extinction profile (STEP) technology.
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http://dx.doi.org/10.3390/pharmaceutics12111112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7699340PMC
November 2020

An update on endotoxin neutralization strategies in Gram-negative bacterial infections.

Expert Rev Anti Infect Ther 2021 Apr 19;19(4):495-517. Epub 2020 Nov 19.

Martin-Luther-Universität Halle-Wittenberg, Institut für Chemie, Halle/Saale, Germany.

Introduction: Gram-negative bacterial infections represent still a severe problem of human health care, regarding the increase in multi-resistance against classical antibiotics and the lack of newly developed antimicrobials. For the fight against these germs, anti-infective agents must overcome and/or bind to the Gram-negative outer membrane consisting of a lipopolysaccharide (LPS, endotoxin) outer leaflet and an inner leaflet from phospholipids, with additional peripheral or integral membrane proteins (OMP's).

Areas Covered: The current article reviews data of existing therapeutic options and summarizes newer approaches for targeting and neutralizing endotoxins, ranging from in vitro over in vivo animal data to clinical applications by using databases such as Medline.

Expert Opinion: Conventional antibiotic treatment of the bacteria leads to their killing, but not necessary LPS neutralization, which may be a severe problem in particular for the systemic pathway. This is the reason why there is an increasing number of therapeutic approaches, which - besides combating whole bacteria - at the same time try to neutralize endotoxin within or outside the bacterial cells mainly responsible for the high inflammation induction in Gram-negative species.
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http://dx.doi.org/10.1080/14787210.2021.1834847DOI Listing
April 2021

Development and validation of a selective marker-based quantification of polysorbate 20 in biopharmaceutical formulations using UPLC QDa detection.

J Chromatogr B Analyt Technol Biomed Life Sci 2020 Nov 31;1157:122287. Epub 2020 Jul 31.

Boehringer Ingelheim GmbH & Co KG, Innovation Unit, Birkendorfer Straße 65, 88400 Biberach, Germany. Electronic address:

Polysorbates are widely used as non-ionic surfactant in biopharmaceutical formulations. Recently, the degradation of polysorbate moved into the focus of attention, because in several published studies it was described, that stability issues in polysorbate containing formulations were observed leading to the formation and appearance of sub-visible and visible particles. For this reason, monitoring of polysorbate and its degradation products is of importance throughout the development of parenterals. The aim of the study was to develop a method for the selective marker-based quantification of adequate polysorbate 20 components of interest without the need to apply derivatization or complex detection techniques. A single quadrupole mass (QDa) detector was used coupled to an ultra-high performance liquid chromatography (UPLC) system. Method development was based on a reversed phase-high performance liquid chromatography assay coupled to a charged aerosol detector (RP-HPLC CAD). Instead of a charged aerosol detector (CAD) a QDa detector was used in order to significantly improve the selectivity. The focus of this study is the development of the QDa based method for the analysis of polysorbate 20. Modifications of the mobile phase and the type of chromatography column allowed the separation of several components of polysorbate 20 from polar non-esterified to apolar higher order species. In addition, a multitude of components could be quantified by their individual m/z values. The peak assignment identified 676 compounds which originated from polysorbate 20. Some of these were selected and defined as marker components. It was shown that the developed method is capable to determine polysorbate 20 in different biopharmaceutical formulations. The proposed assay is based on a smart sample preparation as well as a unique calibration procedure that make the determination of several selected components achievable. Furthermore, it was successfully demonstrated that the analytical procedure is valid to reliably quantify several polysorbate 20 components at its 100% level (corresponds to 0.4 mg/mL intact polysorbate 20) and even at lower concentrations that occur e.g. in case of polysorbate 20 degradation. In conclusion, the method is beneficial to determine selected polysorbate 20 species during formulation development of biopharmaceuticals as well as during stability testing and trouble shooting.
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http://dx.doi.org/10.1016/j.jchromb.2020.122287DOI Listing
November 2020

An in-depth examination of fatty acid solubility limits in biotherapeutic protein formulations containing polysorbate 20 and polysorbate 80.

Int J Pharm 2020 Dec 13;591:119934. Epub 2020 Oct 13.

Boehringer Ingelheim Pharma GmbH & Co. KG, Innovation Unit, PDB, Birkendorfer Straße 65, 88397 Biberach an der Riss, Germany; Martin-Luther-University Halle-Wittenberg, Institute of Chemistry, Faculty of Physical and Theoretical Chemistry, Von-Danckelmann-Platz 4, 06120 Halle (Saale), Germany. Electronic address:

Two of the most widely used surfactants to stabilize biologicals against e.g. interfacial stress are polysorbate20 (PS20) and polysorbate 80 (PS80). In recent years, polysorbate degradation in biopharmaceutical formulations has been observed. Polysorbate (PS) is mainly composed of sorbitan and isosorbide fatty acid (FA) esters, varying in their FA composition. Especially hydrolysis, which can be induced chemically as well as enzymatically, leads to the release of FAs from PS. These FAs are poorly soluble in aqueous buffer systems due to their hydrophobic nature and therefore prone to precipitation and particle formation. Since the emergence of particles in liquid formulations has to be avoided, it is important to prevent their formation. This study evaluates the solubility limits of selected FAs, which are likely to be released during the degradation of PS20 and PS80 in the presence of defined PS concentrations. Our results show that the solubility is highly dependent on the pH, the temperature, the used PS concentration and the aliphatic chain of respective FAs. Solubility of FAs, such as palmitic and oleic acid under the conditions determined in this study, are in the range of 3-130 µg·ml (12-460 µM). Furthermore, the results allow making an estimation to which extent PS may degrade before particle formation in the drug product may be expected.
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http://dx.doi.org/10.1016/j.ijpharm.2020.119934DOI Listing
December 2020

Albumin displacement at the air-water interface by Tween (Polysorbate) surfactants.

Eur Biophys J 2020 Oct 11;49(7):533-547. Epub 2020 Sep 11.

Boehringer Ingelheim Pharma GmbH and Co. KG, Innovation Unit, PDB, 88397, Biberach an der Riss, Germany.

Tween (polysorbate) 20 and 80 are surfactants used for the development of parenteral protein drugs, due to their beneficial safety profile and stabilisation properties. To elucidate the mechanism by which Tween 20 and 80 stabilise proteins in aqueous solutions, either by a "direct" protein to surfactant interaction and/or by an interaction with the protein film at the air-water interface, we used spectroscopic (Infrared Reflection Absorption Spectroscopy, IRRAS) and microscopic techniques (Brewster Angle Microscopy, BAM) in combination with surface pressure measurements. To this end, the impact of both types of Tweens with regard to the displacement of the protein from the air-water interface was studied. As a model protein, human serum albumin (HSA) was used. The results for the displacement of the adsorbed HSA films by Tweens 20 and 80 can partially be understood on the basis of an orogenic displacement mechanism, which depends on the critical surface pressure of the adsorbed protein film. With increasing concentration of Tween in the sub-phase, BAM images showed the formation of different domain morphologies. IRRA-spectra supported the finding that at high protein concentration in the sub-phase, the protein film could not be completely displaced by the surfactants. Comparing the impact of both surfactants, we found that Tween 20 adsorbed faster to the protein film than Tween 80. The adsorption kinetics of both Tweens and the speed of protein displacement increased with rising surfactant concentration. Tween 80 reached significant lower surface pressures than Tween 20, which led to an incomplete displacement of the observed HSA film.
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http://dx.doi.org/10.1007/s00249-020-01459-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7666296PMC
October 2020

Rational optimization of a monoclonal antibody improves the aggregation propensity and enhances the CMC properties along the entire pharmaceutical process chain.

MAbs 2020 Jan-Dec;12(1):1787121

Early Stage Pharmaceutical Development, Pharmaceutical Development Biologicals, Boehringer Ingelheim Pharma GmbH & Co. KG , Biberach/Riss, Germany.

The discovery of therapeutic monoclonal antibodies (mAbs) primarily focuses on their biological activity favoring the selection of highly potent drug candidates. These candidates, however, may have physical or chemical attributes that lead to unfavorable chemistry, manufacturing, and control (CMC) properties, such as low product titers, conformational and colloidal instabilities, or poor solubility, which can hamper or even prevent development and manufacturing. Hence, there is an urgent need to consider the developability of mAb candidates during lead identification and optimization. This work provides a comprehensive proof of concept study for the significantly improved developability of a mAb variant that was optimized with the help of sophisticated tools relative to its difficult-to-develop parental counterpart. Interestingly, a single amino acid substitution in the variable domain of the light chain resulted in a three-fold increased product titer after stable expression in Chinese hamster ovary cells. Microscopic investigations revealed that wild type mAb-producing cells displayed potential antibody inclusions, while the optimized variant-producing cells showed a rescued phenotype. Notably, the drug substance of the optimized variant contained substantially reduced levels of aggregates and fragments after downstream process purification. Finally, formulation studies unraveled a significantly enhanced colloidal stability of the optimized variant while its folding stability and potency were maintained. This study emphasizes that implementation of bioinformatics early in lead generation and optimization of biotherapeutics reduces failures during subsequent development activities and supports the reduction of project timelines and resources.
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http://dx.doi.org/10.1080/19420862.2020.1787121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7531517PMC
July 2020

Thermodynamic Unfolding and Aggregation Fingerprints of Monoclonal Antibodies Using Thermal Profiling.

Pharm Res 2020 Apr 1;37(4):78. Epub 2020 Apr 1.

Pharmaceutical Development Biologicals, Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorferstrasse 65, DE-88397, Biberach, Germany.

Purpose: Predicting thermal protein stability is of major interest in the development of protein-based biopharmaceuticals. Therefore, this study provides a predictive tool for determining transition enthalpies, which can be used for ranking different proteins according to their thermal stability.

Methods: Unfolding and aggregation profiles of eight different therapeutic monoclonal antibodies (mAbs) of type G, isotype 1 were investigated. The unfolding profiles were determined by intrinsic fluorescence (IF) spectroscopy and differential scanning calorimetry (DSC). A three-state unfolding fitting model was used to determine thermodynamic parameters for macromolecular multi-domain mAbs in IF experiments, like the van't Hoff enthalpy change (∆H) and the entropy change (∆S) of the unfolding event. The derived values were compared to thermodynamic parameters obtained directly by calorimetry. Moreover, differences in the Fab enthalpies were used to predict aggregation behavior and protein thermal stabilities. To do so, the liquid-formulated mAbs were investigated exemplarily by size exclusion chromatography (SEC) after accelerated thermal-induced stress conditions.

Results: Comparing the thermodynamic parameters derived from IF spectroscopy and DSC resulted in similar values. Data generated by thermal-induced stress at 40°C show similar stability ranking as postulated through the Fab enthalpies for mAbs in two different formulations, while at 25°C a meaningful ranking is not possible, because distinct differences in the thermal stability cannot be observed. The additional consideration of Fab enthalpies to predict the 40 °C SEC ranking seems to be more reliable compared to the use of exclusively the melting temperatures or aggregation onset temperatures and times.

Conclusion: We show that thermodynamic profiling can help predicting unfolding and aggregation properties of therapeutic mAbs at 40°C. Therefore, analyzing thermodynamic unfolding parameters is a useful and supportive tool discriminating thermal stability profiles of mAbs for further pharmaceutical development and clinical studies.
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http://dx.doi.org/10.1007/s11095-020-02792-1DOI Listing
April 2020

Fast pH-mediated changes of the viscosity of protein solutions studied with a voltage-modulated quartz crystal microbalance.

Biointerphases 2020 03 24;15(2):021004. Epub 2020 Mar 24.

Institute of Physical Chemistry, Clausthal University of Technology, Arnold-Sommerfeld-Str. 4, D-38678 Clausthal-Zellerfeld, Germany.

An electrochemical quartz crystal microbalance is described, which achieves a time resolution down to 100 μs. Accumulation and averaging over a few hours bring the noise down to about 30 mHz. The application examples are pH-driven viscosity changes in albumin solutions. The pH was switched with the electrode potential. The characteristic response time is in the millisecond range. The focus is on experimental aspects as well as advantages and limitations of the technique.
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http://dx.doi.org/10.1116/1.5140619DOI Listing
March 2020

Thermal and Chemical Unfolding of a Monoclonal IgG1 Antibody: Application of the Multistate Zimm-Bragg Theory.

Biophys J 2020 03 16;118(5):1067-1075. Epub 2020 Jan 16.

Biozentrum, University of Basel, Klingelbergstrasse 50/70, Basel, Switzerland. Electronic address:

The thermal unfolding of a recombinant monoclonal antibody IgG1 (mAb) was measured with differential scanning calorimetry (DSC). The DSC thermograms reveal a pretransition at 72°C with an unfolding enthalpy of ΔH ∼200-300 kcal/mol and a main transition at 85°C with an enthalpy of ∼900-1000 kcal/mol. In contrast to small single-domain proteins, mAb unfolding is a complex reaction that is analyzed with the multistate Zimm-Bragg theory. For the investigated mAb, unfolding is characterized by a cooperativity parameter σ ∼6 × 10 and a Gibbs free energy of unfolding of g ∼100 cal/mol per amino acid. The enthalpy of unfolding provides the number of amino acid residues ν participating in the unfolding reaction. On average, ν∼220 ± 50 amino acids are involved in the pretransition and ν∼850 ± 30 in the main transition, accounting for ∼90% of all amino acids. Thermal unfolding was further studied in the presence of guanidineHCl. The chemical denaturant reduces the unfolding enthalpy ΔH and lowers the midpoint temperature T. Both parameters depend linearly on the concentration of denaturant. The guanidineHCl concentrations needed to unfold mAb at 25°C are predicted to be 2-3 M for the pretransition and 5-7 M for the main transition, varying with pH. GuanidineHCl binds to mAb with an exothermic binding enthalpy, which partially compensates the endothermic mAb unfolding enthalpy. The number of guanidineHCl molecules bound upon unfolding is deduced from the DSC thermograms. The bound guanidineHCl-to-unfolded amino acid ratio is 0.79 for the pretransition and 0.55 for the main transition. The pretransition binds more denaturant molecules and is more sensitive to unfolding than the main transition. The current study shows the strength of the Zimm-Bragg theory for the quantitative description of unfolding events of large, therapeutic proteins, such as a monoclonal antibody.
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http://dx.doi.org/10.1016/j.bpj.2019.12.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7063443PMC
March 2020

Acidic and alkaline hydrolysis of polysorbates under aqueous conditions: Towards understanding polysorbate degradation in biopharmaceutical formulations.

Eur J Pharm Sci 2020 Mar 10;144:105211. Epub 2020 Jan 10.

Boehringer Ingelheim Pharma GmbH & Co. KG, Innovation Unit, PDB, Birkendorfer Strasse 65, 88397, Biberach an der Riss, Germany. Electronic address:

Polysorbate is one of the most commonly employed non-ionic surfactant in protein containing biological formulations, whereby, it can stabilize these biomolecules under different stress conditions. Despite the fact that polysorbates are present in almost 70% of currently marketed parenteral biological drugs, polysorbate degradation in biopharmaceutical formulations has emerged as a specific quality concern. Different degradation pathways have been explored in the recent years with the aim of understanding the root cause for polysorbate degradation in biopharmaceutical formulations. In an attempt to explore hydrolytic degradation of polysorbates in accelerated degradation conditions, we studied extreme pH conditions. We investigated specific polysorbate degradation profiles depending on acidic or alkaline solution conditions. The acidic and alkaline hydrolysis of polysorbate is monitored for the total content using a fluorescence micelle assay (FMA). Additionally, the compositional changes in polysorbates were detected using reversed phase high performance liquid chromatography coupled to a charged aerosol detector (RP-HPLC-CAD). We show that the stability of polysorbate against chemical hydrolysis is dependent upon selected pH condition and differ for polysorbate 20 and polysorbate 80. Additionally, we were able to show that a degradation pathway dependent fingerprint may support the identification of the degradation root cause.
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http://dx.doi.org/10.1016/j.ejps.2019.105211DOI Listing
March 2020

Taking Subvisible Particle Quantitation to the Limit: Uncertainties and Statistical Challenges With Ophthalmic Products for Intravitreal Injection.

J Pharm Sci 2020 01 2;109(1):505-514. Epub 2019 Nov 2.

Coriolis Pharma Research GmbH, Fraunhoferstr. 18 b, 82152 Martinsried, Germany. Electronic address:

Subvisible particles are a critical quality attribute of pharmaceutical products. The reliability of particle quantitation increases with the number of particles in the analyzed sample volume. However, for analyses of low-volume drug products, such as ophthalmic products for intravitreal injection or biopharmaceuticals in general, sample volumes as small as possible should be used to avoid pooling and consequently, the contamination with foreign particles. The aim of our study was to evaluate the variability of particle concentrations obtained by light obscuration measurements to define the minimum required analyzed sample volume to achieve statistically meaningful results by using conditions that are practically feasible. Statistical evaluation suggests that for particle concentrations close to a predefined limit, large sample volumes (a multiple of typical intravitreal product volumes) would be required for a high probability to correctly classify samples with respect to the predefined limit. Below a minimum analyzed volume, even a measurement result of 0 particles does not allow to conclude compliance with the respective particle concentration limit with sufficient certainty. A small analyzed volume could be justified as long as the measurement uncertainty remains acceptable compared with the predefined limit.
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http://dx.doi.org/10.1016/j.xphs.2019.10.061DOI Listing
January 2020

Electrostatic interactions of alkaline earth cations with 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid (DMPA) model membranes at neutral and acidic pH.

Eur Biophys J 2019 Dec 26;48(8):757-772. Epub 2019 Oct 26.

Institute of Chemistry-Physical Chemistry, Martin-Luther-University Halle-Wittenberg, von-Danckelmann-Platz 4, 06120, Halle (Saale), Germany.

The binding of alkaline earth cations Mg, Ca, and Sr (M) to unilamellar 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid (DMPA) vesicles was analysed by pH potentiometry, differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC) and FT-IR spectroscopy. The binding of alkaline earth cations induces deprotonation of the DMPA headgroup even at very low concentration of divalent cations (~ 100 µM). The amount of deprotonated DMPA was measured by pH potentiometry as a function of divalent cation concentration. The thermotropic phase behaviour of DMPA:M complexes was studied by DSC and FT-IR as a function of pH of the dispersion (pH 7 and pH 3-5). The formation of metastable phases was observed, especially for Ca and Sr at pH 3-5. In unbuffered solutions, the divalent cations bind to single and/or double negatively charged DMPA, leading to the formation of different complexes and changes in the mixing behaviour of the two complexes. At pH 7, all three equimolar lipid/cation mixtures form a very stable, highly ordered 1:1 DMPA:M complex. At lower divalence, the presence of a mixture of 2:1 and 1:1 complexes was observed. FT-IR spectroscopy experiments indicated an ordering of the acyl chains of DMPA after ion binding even in the liquid-crystalline phase and the induction of the dissociation of the second proton from the headgroup induced by Ca or Sr binding at pH 7. With ITC, the binding enthalpy ΔH of Mg, Ca, and Sr to DMPA model membranes in the gel and in the liquid-crystalline phase was measured. Evidence for dehydration of hydrophobic surfaces due to cation binding was derived from changes in heat capacity.
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http://dx.doi.org/10.1007/s00249-019-01402-2DOI Listing
December 2019

Adaption of human antibody λ and κ light chain architectures to CDR repertoires.

Protein Eng Des Sel 2019 12;32(3):109-127

Switch Laboratory, VIB Center for Brain and Disease Research, Herestraat 49, Leuven, Belgium.

Monoclonal antibodies bind with high specificity to a wide range of diverse antigens, primarily mediated by their hypervariable complementarity determining regions (CDRs). The defined antigen binding loops are supported by the structurally conserved β-sandwich framework of the light chain (LC) and heavy chain (HC) variable regions. The LC genes are encoded by two separate loci, subdividing the entity of antibodies into kappa (LCκ) and lambda (LCλ) isotypes that exhibit distinct sequence and conformational preferences. In this work, a diverse set of techniques were employed including machine learning, force field analysis, statistical coupling analysis and mutual information analysis of a non-redundant antibody structure collection. Thereby, it was revealed how subtle changes between the structures of LCκ and LCλ isotypes increase the diversity of antibodies, extending the predetermined restrictions of the general antibody fold and expanding the diversity of antigen binding. Interestingly, it was found that the characteristic framework scaffolds of κ and λ are stabilized by diverse amino acid clusters that determine the interplay between the respective fold and the embedded CDR loops. In conclusion, this work reveals how antibodies use the remarkable plasticity of the beta-sandwich Ig fold to incorporate a large diversity of CDR loops.
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http://dx.doi.org/10.1093/protein/gzz012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6908821PMC
December 2019

Lipid-based nanoparticle formulations for small molecules and RNA drugs.

Expert Opin Drug Deliv 2019 11;16(11):1205-1226

Boehringer Ingelheim Pharma GmbH & Co. KG, Innovation Unit, Pharmaceutical Development Biologicals , Biberach an der Riss , Germany.

: Liposomes and lipid-based nanoparticles (LNPs) effectively deliver cargo molecules to specific tissues, cells, and cellular compartments. Patients benefit from these nanoparticle formulations by altered pharmacokinetic properties, higher efficacy, or reduced side effects. While liposomes are an established delivery option for small molecules, Onpattro® (Sanofi Genzyme, Cambridge, MA) is the first commercially available LNP formulation of a small interfering ribonucleic acid (siRNA). : This review article summarizes key features of liposomal formulations for small molecule drugs and LNP formulations for RNA therapeutics. We describe liposomal formulations that are commercially available or in late-stage clinical development and the most promising LNP formulations for ASOs, siRNAs, saRNA, and mRNA therapeutics. : Similar to liposomes, LNPs for RNA therapeutics have matured but still possess a niche application status. RNA therapeutics, however, bear an immense hope for difficult to treat diseases and fuel the imagination for further applications of RNA drugs. LNPs face similar challenges as liposomes including limitations in biodistribution, the risk to provoke immune responses, and other toxicities. However, since properties of RNA molecules within the same group are very similar, the entire class of therapeutic molecules would benefit from improvements in a few key parameters of the delivery technology.
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http://dx.doi.org/10.1080/17425247.2019.1669558DOI Listing
November 2019

Concentration Effects in the Interaction of Monoclonal Antibodies (mAbs) with their Immediate Environment Characterized by EPR Spectroscopy.

Molecules 2019 Jul 10;24(14). Epub 2019 Jul 10.

Institute of Chemistry, Martin Luther University Halle-Wittenberg, D06120 Halle (Saale), Germany.

Monoclonal antibodies (mAbs) are often needed and applied in high concentration solutions, >100 mg/mL. Due to close intermolecular distances between mAbs at high concentrations (~10-20 nm at 200 mg/mL), intermolecular interactions between mAbs and mAbs and solvent/co-solute molecules become non-negligible. Here, EPR spectroscopy is used to study the high-concentration solutions of mAbs and their effect on co-solvated small molecules, using EPR "spin probing" assay in aqueous and buffered solutions. Such, information regarding the surrounding environments of mAbs at high concentrations were obtained and comparisons between EPR-obtained micro-viscosities (rotational correlation times) and macroscopic viscosities measured by rheology were possible. In comparison with highly viscous systems like glycerol-water mixtures, it was found that up to concentrations of 50 mg/mL, the mAb-spin probe systems have similar trends in their macro- (rheology) and micro-viscosities (EPR), whereas at very high concentrations they deviate strongly. The charged spin probes sense an almost unchanged aqueous solution even at very high concentrations, which in turn indicates the existence of large solvent regions that despite their proximity to large mAbs essentially offer pure water reservoirs for co-solvated charged molecules. In contrast, in buffered solutions, amphiphilic spin probes like TEMPO interact with the mAb network, due to slight charge screening. The application of EPR spectroscopy in the present work has enabled us to observe and discriminate between electrostatic and hydrophobic kinds of interactions and depict the potential underlying mechanisms of network formation at high concentrations of mAbs. These findings could be of importance as well for the development of liquid-liquid phase separations often observed in highly concentrated protein solutions.
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http://dx.doi.org/10.3390/molecules24142528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6680867PMC
July 2019

Structure of a Therapeutic Full-Length Anti-NPRA IgG4 Antibody: Dissecting Conformational Diversity.

Biophys J 2019 05 5;116(9):1637-1649. Epub 2019 Apr 5.

Innovation Unit, Pharmaceutical Development Biologics, Biberach (Riss), Germany. Electronic address:

We report the x-ray crystal structure of intact, full-length human immunoglobulin (IgG4) at 1.8 Å resolution. The data for IgG4 (S228P), an antibody targeting the natriuretic peptide receptor A, show a previously unrecognized type of Fab-Fc orientation with a distorted λ-shape in which one Fab-arm is oriented toward the Fc portion. Detailed structural analysis by x-ray crystallography and molecular simulations suggest that this is one of several conformations coexisting in a dynamic equilibrium state. These results were confirmed by small angle x-ray scattering in solution. Furthermore, electron microscopy supported these findings by preserving molecule classes of different conformations. This study fosters our understanding of IgG4 in particular and our appreciation of antibody flexibility in general. Moreover, we give insights into potential biological implications, specifically for the interaction of human anti-natriuretic peptide receptor A IgG4 with the neonatal Fc receptor, Fcγ receptors, and complement-activating C1q by considering conformational flexibility.
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http://dx.doi.org/10.1016/j.bpj.2019.03.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6506711PMC
May 2019

Synthetic Anti-lipopolysaccharide Peptides (SALPs) as Effective Inhibitors of Pathogen-Associated Molecular Patterns (PAMPs).

Adv Exp Med Biol 2019 ;1117:111-129

Brandenburg Antiinfektiva GmbH, c/o Forschungszentrum Borstel, Borstel, Germany.

Antimicrobial peptides (AMPs) are in the focus of scientific research since the 1990s. In most cases, the main aim was laid on the design of AMP to kill bacteria effectively, with particular emphasis on broadband action and independency on antibiotic resistance. However, so far no approved drug on the basis of AMP has entered the market.Our approach of constructing AMP, called synthetic anti-lipopolysaccharide peptides (SALPs), on the basis of inhibiting the inflammatory action of lipopolysaccharide (LPS, endotoxin) from Gram-negative bacteria was focused on the neutralization of the decisive toxins. These are, beside LPS from Gram-negative bacteria, the lipoproteins (LP) from Gram-positive origin. Although some of the SALPs have an antibacterial action, the most important property is the high-affinity binding to LPS and LP, whether as constituent of the bacteria or in free form which prevents the damaging inflammation, that could otherwise lead to life-threatening septic shock. Most importantly, the SALP may inhibit inflammation independently of the resistance status of the bacteria, and so far the repeated use of the peptides apparently does not cause resistance of the attacking pathogens.In this chapter, an overview is given over the variety of possible applications in the field of fighting against severe bacterial infections, from the use in systemic infection/inflammation up to various topical applications such as anti-biofilm action and severe skin and soft tissue infections.
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http://dx.doi.org/10.1007/978-981-13-3588-4_8DOI Listing
August 2019

Polysorbate degradation in biotherapeutic formulations: Identification and discussion of current root causes.

Int J Pharm 2018 Dec 6;552(1-2):422-436. Epub 2018 Oct 6.

Boehringer Ingelheim Pharma GmbH & Co. KG, Innovation Unit, PDB, Birkendorfer Strasse 65, 88397 Biberach an der Riss, Germany. Electronic address:

Biotherapeutic protein formulations are often high concentration liquid protein solutions, which are required to be stable under pharmaceutically relevant storage conditions and presence of external stress. Non-ionic detergents like polysorbate have been the most commonly used detergent to maintain formulation stability. Recently, particle formation in polysorbate containing biotherapeutic formulations has arisen as a major quality concern and potential patient risk factor. In this review, we provide a general overview into (i) degradation of polysorbates, (ii) polysorbate analytics, (iii) particle formation induced by polysorbate degradation and root causes thereof, (iv) particle composition and (v) various influencing factors that might lead to particle formation. Consequently, we explore the role of polysorbate degradation in particle formation. Additionally, various degradation pathways and the current discussed root causes are reviewed.
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http://dx.doi.org/10.1016/j.ijpharm.2018.10.008DOI Listing
December 2018

Spectroscopic methods for assessing the molecular origins of macroscopic solution properties of highly concentrated liquid protein solutions.

Anal Biochem 2018 11 20;561-562:70-88. Epub 2018 Sep 20.

Institute of Chemistry, Martin Luther University Halle-Wittenberg, Von-Danckelmann-Platz 4, 06120, Halle/Saale, Germany; Boehringer Ingelheim Pharma GmbH & Co. KG, Protein Science, Birkerndorfer Str. 65, 88397, Biberach/Riß, Germany. Electronic address:

In cases of subcutaneous injection of therapeutic monoclonal antibodies, high protein concentrations (>50 mg/ml) are often required. During the development of these high concentration liquid formulations (HCLF), challenges such as aggregation, gelation, opalescence, phase separation, and high solution viscosities are more prone compared to low concentrated protein formulations. These properties can impair manufacturing processes, as well as protein stability and shelf life. To avoid such unfavourable solution properties, a detailed understanding about the nature of these properties and their driving forces are required. However, the fundamental mechanisms that lead to macroscopic solution properties, as above mentioned, are complex and not fully understood, yet. Established analytical methods for assessing the colloidal stability, i.e. the ability of a native protein to remain dispersed in solution, are restricted to dilute conditions and provide parameters such as the second osmotic virial coefficient, B, and the diffusion interaction coefficient, k. These parameters are routinely applied for qualitative estimations and identifications of proteins with challenging solution behaviours, such as high viscosities and aggregation, although the assays are prepared for low protein concentration conditions, typically between 0.1 and 20 mg/ml ("ideal" solution conditions). Quantitative analysis of samples of high protein concentration is difficult and it is hard to obtain information about the driving forces of such solution properties and corresponding protein-protein self-interactions. An advantage of using specific spectroscopic methods is the potential of directly analysing highly concentrated protein solutions at different solution conditions. This allows for collecting/gaining valuable information about the fundamental mechanisms of solution properties of the high protein concentration regime. In addition, the derived parameters might be more predictive as compared to the parameters originating from assays which are optimized for the low protein concentration range. The provided information includes structural data, molecular dynamics at various timescales and protein-solvent interactions, which can be obtained at molecular resolution. Herein, we provide an overview about spectroscopic techniques for analysing the origins of macroscopic solution behaviours in general, with a specific focus on pharmaceutically relevant high protein concentration and formulation conditions.
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http://dx.doi.org/10.1016/j.ab.2018.09.013DOI Listing
November 2018

Inhibition of Lipopolysaccharide- and Lipoprotein-Induced Inflammation by Antitoxin Peptide Pep19-2.5.

Front Immunol 2018 26;9:1704. Epub 2018 Jul 26.

Biophysics, Research Center Borstel, Borstel, Germany.

The most potent cell wall-derived inflammatory toxins ("pathogenicity factors") of Gram-negative and -positive bacteria are lipopolysaccharides (LPS) (endotoxins) and lipoproteins (LP), respectively. Despite the fact that the former signals toll-like receptor 4 (TLR4) and the latter TLR2, the physico-chemistry of these compounds exhibits considerable similarity, an amphiphilic molecule with a polar and charged backbone and a lipid moiety. While the exterior portion of the LPS (i.e., the O-chain) represents the serologically relevant structure, the inner part, the lipid A, is responsible for one of the strongest inflammatory activities known. In the last years, we have demonstrated that antimicrobial peptides from the Pep19-2.5 family, which were designed to bind to LPS and LP, act as anti-inflammatory agents against sepsis and endotoxic shock caused by severe bacterial infections. We also showed that this anti-inflammatory activity requires specific interactions of the peptides with LPS and LP leading to exothermic reactions with saturation characteristics in calorimetry assays. Parallel to this, peptide-mediated neutralization of LPS and LP involves changes in various physical parameters, including both the gel to liquid crystalline phase transition of the acyl chains and the three-dimensional aggregate structures of the toxins. Furthermore, the effectivity of neutralization of pathogenicity factors by peptides was demonstrated in several models together with the finding that a peptide-based therapy sensitizes bacteria (also antimicrobial resistant) to antibiotics. Finally, a significant step in the understanding of the broad anti-inflammatory function of Pep19-2.5 was the demonstration that this compound is able to block the intracellular endotoxin signaling cascade.
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http://dx.doi.org/10.3389/fimmu.2018.01704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6070603PMC
September 2019

Hydrogel formulations for biologicals: current spotlight from a commercial perspective.

Ther Deliv 2018 02;9(3):221-230

Boehringer Ingelheim Pharma GmbH & Co. KG, Innovation Unit, Biologicals Development Germany, D-88397 Biberach an der Riss, Germany.

Hydrogels are, from a commercial perspective especially because of their ease of production, attractive sustained-release systems for high potent immunoglobulins with short circulation half-lives. Hydrogel formulations can reduce the dosing frequency while maintaining therapeutically relevant drug concentrations locally as well as systemically. However, hydrogels have only limited loading capacities and release hydrophilic immunoglobulins typically within hours or days, whereas weeks or months would be more preferable. Despite an evident medical need, the call for novel depot formulations seems to go unheard. This special report explores sought-after hydrogel properties, discusses arguments for using established versus novel excipients and provides selected examples for hydrogel formulations of biologicals that have proceeded into clinical development.
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http://dx.doi.org/10.4155/tde-2017-0085DOI Listing
February 2018

Characterizing protein-protein-interaction in high-concentration monoclonal antibody systems with the quartz crystal microbalance.

Phys Chem Chem Phys 2017 Dec;19(48):32698-32707

Boehringer Ingelheim Pharma GmbH and Co. KG, Protein Science, Birkendorfer Str. 65, 88397 Biberach/Riss, Germany.

Making use of a quartz crystal microbalance (QCM), concentrated solutions of therapeutic antibodies were studied with respect to their behavior under shear excitation with frequencies in the MHz range. At high protein concentration and neutral pH, viscoelastic behavior was found in the sense that the storage modulus, G', was nonzero. Fits of the frequency dependence of G'(ω) and G''(ω) (G'' being the loss modulus) using the Maxwell-model produced good agreement with the experimental data. The fit parameters were the relaxation time, τ, and the shear modulus at the inverse relaxation time, G* (at the "cross-over frequency" ω = 1/τ). The influence of two different pharmaceutical excipients (histidine and citrate) was studied at variable concentrations of the antibody and variable pH. In cases, where viscoelasticity was observed, G* was in the range of a few kPa, consistent with entropy-driven interactions. τ was small at low pH, where the antibody carries a positive charge. τ increased with increasing pH. The relaxation time τ was found to be correlated with other parameters quantifying protein-protein interactions, namely the steady shear viscosity (η), the second osmotic virial coefficient as determined with both self-interaction chromatography (B) and static light scattering (B), and the diffusion interaction parameter as determined with dynamic light scattering (k). While B and k describe protein-protein interactions in diluted samples, the QCM can be applied to concentrated solutions, thereby being sensitive to higher-order protein-protein interactions.
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http://dx.doi.org/10.1039/c7cp05711cDOI Listing
December 2017

Improved Solution-State Properties of Monoclonal Antibodies by Targeted Mutations.

J Phys Chem B 2017 12 29;121(48):10818-10827. Epub 2017 Nov 29.

Theoretical Chemistry, Ruhr-University Bochum , D-44780 Bochum, Germany.

Monoclonal antibody (mAb)-based therapeutics often require high-concentration formulations. Unfortunately, highly concentrated antibody solutions often have biophysical properties that are disadvantageous for therapeutic development, such as high viscosity, solubility limitations, precipitation issues, or liquid-liquid phase separation. In this work, we present a computational rational design principle for improving the thermodynamic stability of mAb solutions through targeted point mutations. Two publicly available IgG1 monoclonal antibodies that exhibit high viscosity at high concentrations were used as model systems. Guided by a computationally efficient approach that combines molecular dynamics simulations with three-dimensional reference interaction site model theory, point mutations of charged residues were introduced in the variable Fv regions in such a manner that the hydration free energy was optimized. Two selected point mutants were then produced by transient expression and characterized experimentally. Both engineered mAbs have reduced viscosity at high concentration, less negative second virial coefficient, and improved solubility compared to the respective wild-types. The results obtained with the suggested straightforward design principle underline the relevance of solvation effects for understanding, and ultimately optimizing, the properties of highly concentrated mAb solutions, with possible implications also for other biomolecular systems.
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http://dx.doi.org/10.1021/acs.jpcb.7b09126DOI Listing
December 2017

Proteasome impairment by α-synuclein.

PLoS One 2017 25;12(9):e0184040. Epub 2017 Sep 25.

Neurology Department, Ulm University, Ulm, Germany.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder worldwide and characterized by the loss of dopaminergic neurons in the patients' midbrains. Both the presence of the protein α-synuclein in intracellular protein aggregates in surviving neurons and the genetic linking of the α-synuclein encoding gene point towards a major role of α-synuclein in PD etiology. The exact pathogenic mechanisms of PD development are not entirely described to date, neither is the specific role of α-synuclein in this context. Previous studies indicate that one aspect of α-synuclein-related cellular toxicity might be direct proteasome impairment. The 20/26S proteasomal machinery is an important instrument of intracellular protein degradation. Thus, direct proteasome impairment by α-synuclein might explain or at least contribute to the formation of intracellular protein aggregates. Therefore this study investigates direct proteasomal impairment by α-synuclein both in vitro using recombinant α-synuclein and isolated proteasomes as well as in living cells. Our experiments demonstrate that the impairment of proteasome activity by α-synuclein is highly dependent upon the cellular background and origin. We show that recombinant α-synuclein oligomers and fibrils scarcely affect 20S proteasome function in vitro, neither does transient α-synuclein expression in U2OS ps 2042 (Ubi(G76V)-GFP) cells. However, stable expression of both wild-type and mutant α-synuclein in dopaminergic SH-SY5Y and PC12 cells results in a prominent impairment of the chymotrypsin-like 20S/26S proteasomal protein cleavage. Thus, our results support the idea that α-synuclein in a specific cellular environment, potentially present in dopaminergic cells, cannot be processed by the proteasome and thus contributes to a selective vulnerability of dopaminergic cells to α-synuclein pathology.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0184040PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5612461PMC
October 2017

High-concentration protein formulations: How high is high?

Eur J Pharm Biopharm 2017 Oct 6;119:353-360. Epub 2017 Jul 6.

Boehringer Ingelheim Pharma GmbH & Co. KG, Corporate Division Biopharmaceuticals, Process Science, Protein Science, Biberach an der Riss, Germany.

High-concentration protein formulation (HCPF) is a term that is used to describe protein formulations, mostly monoclonal antibody (mAb) drugs, at high protein concentration. The concentration is rarely defined, with typical ranges varying between 50 and 150mg/ml for mAbs. The term HCPF is meant to include and express specific solution properties of formulations that are prone to appear at high protein concentrations such as high viscosity, high opalescence, phase separation, gel formation or the increased propensity for protein particle formation. Thus the term HCPF can be understood as a descriptor of protein formulations, usually at high protein (monoclonal antibody) concentrations, which have specific solution, stability and colloidal properties that differ from formulations at low protein concentration (e.g. at 10mg/ml). The current paper highlights in brief the development challenges that might occur for high-concentration protein/monoclonal antibody formulations. In particular, the maximum concentration regimes achievable in HCPF remained unclear. Based on geometrical considerations involving packing of monoclonal antibodies in a lattice we map out a maximum concentration range that might be theoretically achievable. Different geometrical assumptions and packing models are compared and their relevance is critically discussed, in particular concerning the influence of the physicochemical properties of the monoclonal antibodies on their solubility, which is neglected in the simple geometrical model. According to our estimates, monoclonal antibody concentration above 500mg/ml will be very challenging to achieve. Our results have implications for setting up realistic drug product development strategies and for preparing convincing drug target product profiles for development.
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http://dx.doi.org/10.1016/j.ejpb.2017.06.029DOI Listing
October 2017

Prediction and Reduction of the Aggregation of Monoclonal Antibodies.

J Mol Biol 2017 04 18;429(8):1244-1261. Epub 2017 Mar 18.

VIB Switch Laboratory, Herestraat 49, B-3000 Leuven, Belgium; Department of Cellular and Molecular Medicine, KU Leuven, Herestraat 49, PO 802, B-3000 Leuven, Belgium. Electronic address:

Protein aggregation remains a major area of focus in the production of monoclonal antibodies. Improving the intrinsic properties of antibodies can improve manufacturability, attrition rates, safety, formulation, titers, immunogenicity, and solubility. Here, we explore the potential of predicting and reducing the aggregation propensity of monoclonal antibodies, based on the identification of aggregation-prone regions and their contribution to the thermodynamic stability of the protein. Although aggregation-prone regions are thought to occur in the antigen binding region to drive hydrophobic binding with antigen, we were able to rationally design variants that display a marked decrease in aggregation propensity while retaining antigen binding through the introduction of artificial aggregation gatekeeper residues. The reduction in aggregation propensity was accompanied by an increase in expression titer, showing that reducing protein aggregation is beneficial throughout the development process. The data presented show that this approach can significantly reduce liabilities in novel therapeutic antibodies and proteins, leading to a more efficient path to clinical studies.
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http://dx.doi.org/10.1016/j.jmb.2017.03.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5397608PMC
April 2017

Liquid-liquid phase separation of a monoclonal antibody at low ionic strength: Influence of anion charge and concentration.

Biophys Chem 2017 Jan 2;220:7-19. Epub 2016 Oct 2.

Boehringer Ingelheim Pharma GmbH & Co. KG, Protein Science, Birkendorfer Strasse 65, 88397 Biberach/Riss, Germany; Martin-Luther-University Halle-Wittenberg, Germany. Electronic address:

Liquid-liquid phase separation (LLPS) of a monoclonal antibody solution was investigated at low ionic strength in the presence of oligovalent anions, such as citrate, trimellitate, pyromellitate and mellitate. Phase separation was observed at the isoelectric point of the antibody at pH8.7 as well as in more acidic pH regions in the presence of the tested oligovalent ions. This can be attributed to charge neutralization via binding of the oligovalent anions to the positively charged antibody. The influence of the anion concentration on liquid-liquid phase separation with respect to the net charge of the antibody was examined. Similarities to the formation of a complex coacervate were shown to apply. These findings enable us to understand the usage of excipients to rationally induce or avoid liquid-liquid phase separation at low ionic strength. Furthermore we present a method to directly examine the competition of different ions for the solvation shell, called buffer equilibration.
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http://dx.doi.org/10.1016/j.bpc.2016.08.003DOI Listing
January 2017