Development and Application of a High-Throughput Method for the Purification and Analysis of Surface Carbohydrates from Klebsiella pneumoniae.

Klebsiella pneumoniae (Kp) is a Gram-negative bacterium, and a leading cause of neonatal sepsis in low- and middle-income countries, often associated with anti-microbial resistance. Two types of polysaccharides are expressed on the Kp cell surface and have been proposed as key antigens for vaccine design: capsular polysaccharides (known as K-antigens, K-Ags) and O-antigens (O-Ags). Historically, Kp has been classified using capsule serotyping and although 186 distinct genotypes have been predicted so far based on sequence analysis, many structures are still unknown. In contrast, only 11 distinct OAg serotypes have been described. The characterization of emerging strains requires the development of a high-throughput purification method to obtain sufficient K- and O-Ag material to characterize the large collection of serotypes and gain insight on structural features and potential cross-reactivity that could allow vaccine simplification. Here, this was achieved by adapting our established method for the simple purification of O-Ags, using mild acetic acid hydrolysis performed directly on bacterial cells, followed by filtration and precipitation steps. The method was successfully applied to purify the surface carbohydrates from different Kp strains, thereby demonstrating the robustness and general applicability of the purification method developed. Further, antigen characterization showed that the purification method had no impact on the structural integrity of the polysaccharides and preserved labile substituents such as O-acetyl and pyruvyl groups. This method can be further optimized for scaling up and manufacturing to support the development of high-valency saccharide-based vaccines against Kp.

Quality by Design Framework Applied to GMMA Purification.

In recent years, Generalized Modules for Membrane Antigens (GMMA) have received increased attention as an innovative vaccine platform against bacterial pathogens, particularly attractive for low- and middle-income countries because of manufacturing simplicity. The assessment of critical quality attributes (CQAs), product-process interactions, identification of appropriate in process analytical methods, and process modeling is part of a robust quality by design (QbD) framework to support further development and control of manufacturing processes. QbD implementation in the context of the GMMA platform will ensure robust manufacturing of batches with desired characteristics, facilitating technical transfer to local manufacturers, regulatory approval, and commercialization of vaccines based on this technology. Here, we summarize the methodology suggested, applied to a first step of GMMA manufacturing process.

Comparison of Shigella GMMA and glycoconjugate four-component formulations in animals.

Shigellosis is leading bacterial cause of diarrhea with high prevalence in children younger than 5 years in low- and middle-income countries, and increasing number of reports of Shigella cases associated to anti-microbial resistance. No vaccines against Shigella are still licensed, but different candidates based on the O-antigen portion of lipopolysaccharides are in clinic. Generalized Modules for Membrane Antigens (GMMA) have been proposed as an alternative delivery system for the O-antigen, and a 4-component vaccine candidate (altSonflex1-2-3), containing GMMA from S. sonnei and S. flexneri 1b, 2a and 3a is being tested in a phase 1/2 clinical trial, with the aim to elicit broad protection against the most prevalent Shigella serotypes. Here, the 4-component GMMA vaccine candidate has been compared to a more traditional glycoconjugate formulation for the ability to induce functional antibodies in mice and rabbits. In mice, in the absence of Alhydrogel, GMMA induce higher IgG antibodies than glycoconjugates and stronger bactericidal titers against all Shigella serotypes. In the presence of Alhydrogel, GMMA induce O-antigen specific IgG levels similar to traditional glycoconjugates, but with a broader range of IgG subclasses, resulting in stronger bactericidal activity. In rabbits, GMMA elicit higher functional antibodies than glycoconjugates against S. sonnei, and similar responses to S. flexneri 1b, 2a and 3a, independently from the presence of Alhydrogel. Different O-antigen based vaccines against Shigella are now in clinical stage and it will be of particular interest to understand how the preclinical findings in the different animal models translate in humans.

Testing a Recombinant Form of Tetanus Toxoid as a Carrier Protein for Glycoconjugate Vaccines.

Glycoconjugate vaccines play a major role in the prevention of infectious diseases worldwide, with significant impact on global health, enabling the polysaccharides to induce immunogenicity in infants and immunological memory. Tetanus toxoid (TT), a chemically detoxified bacterial toxin, is among the few carrier proteins used in licensed glycoconjugate vaccines. The recombinant full-length 8MTT was engineered in E. coli with eight individual amino acid mutations to inactivate three toxin functions. Previous studies in mice showed that 8MTT elicits a strong IgG response, confers protection, and can be used as a carrier protein. Here, we compared 8MTT to traditional carrier proteins TT and cross-reactive material 197 (CRM197), using different polysaccharides as models: Group A Streptococcus cell-wall carbohydrate (GAC), Salmonella Typhi Vi, and Neisseria meningitidis serogroups A, C, W, and Y. The persistency of the antibodies induced, the ability of the glycoconjugates to elicit booster response after re-injection at a later time point, the eventual carrier-induced epitopic suppression, and immune interference in multicomponent formulations were also evaluated. Overall, immunogenicity responses obtained with 8MTT glycoconjugates were compared to those obtained with corresponding TT and, in some cases, were higher than those induced by CRM197 glycoconjugates. Our results support the use of 8MTT as a good alternative carrier protein for glycoconjugate vaccines, with advantages in terms of manufacturability compared to TT.

A next-generation GMMA-based vaccine candidate to fight shigellosis.

Shigellosis is a leading cause of diarrheal disease in low-middle-income countries (LMICs). Effective vaccines will help to reduce the disease burden, exacerbated by increasing antibiotic resistance, in the most susceptible population represented by young children. A challenge for a broadly protective vaccine against shigellosis is to cover the most epidemiologically relevant serotypes among >50 Shigella serotypes circulating worldwide. The GMMA platform has been proposed as an innovative delivery system for Shigella O-antigens, and we have developed a 4-component vaccine against S. sonnei, S. flexneri 1b, 2a and 3a identified among the most prevalent Shigella serotypes in LMICs. Driven by the immunogenicity results obtained in clinic with a first-generation mono-component vaccine, a new S. sonnei GMMA construct was generated and combined with three S. flexneri GMMA in a 4-component Alhydrogel formulation (altSonflex1-2-3). This formulation was highly immunogenic, with no evidence of negative antigenic interference in mice and rabbits. The vaccine induced bactericidal antibodies also against heterologous Shigella strains carrying O-antigens different from those included in the vaccine. The Monocyte Activation Test used to evaluate the potential reactogenicity of the vaccine formulation revealed no differences compared to the S. sonnei mono-component vaccine, shown to be safe in several clinical trials in adults. A GLP toxicology study in rabbits confirmed that the vaccine was well tolerated. The preclinical study results support the clinical evaluation of altSonflex1-2-3 in healthy populations, and a phase 1-2 clinical trial is currently ongoing.

From an in vivo to an in vitro relative potency (IVRP) assay to fully characterize a multicomponent O-antigen based vaccine against Shigella.

Outer membrane vesicles (OMV) represent an innovative platform for the design of polysaccharide based vaccines. Generalized Modules for Membrane Antigens (GMMA), OMV released from engineered Gram-negative bacteria, have been proposed for the delivery of the O-Antigen, key target for protective immunity against several pathogens including Shigella. altSonflex1-2-3 is a GMMA based vaccine, including S. sonnei and S. flexneri 1b, 2a and 3a O-Antigens, with the aim to elicit broad protection against the most prevalent Shigella serotypes, especially affecting children in low-middle income countries. Here we developed an In Vitro Relative Potency assay, based on recognition of O-Antigen by functional monoclonal antibodies selected to bind the key epitopes of the different O-Antigen active ingredients, directly applied to our Alhydrogel-formulated vaccine. Heat-stressed altSonflex1-2-3 formulations were generated and extensively characterized. The impact of detected biochemical changes in in vivo and in vitro potency assays was assessed. The overall results showed how the in vitro assay can replace the use of animals, overcoming the inherently high variability of in vivo potency studies. The entire panel of physico-chemical methods developed will contribute to detect suboptimal batches and will be valuable to perform stability studies. The work on Shigella vaccine candidate can be easily extended to other O-Antigen based vaccines.

Exploring the Role of GMMA Components in the Immunogenicity of a 4-Valent Vaccine against Shigella.

Shigellosis is the leading cause of diarrheal disease, especially in children of low- and middle-income countries, and is often associated with anti-microbial resistance. Currently, there are no licensed vaccines widely available against Shigella, but several candidates based on the O-antigen (OAg) portion of lipopolysaccharides are in development. We have proposed Generalized Modules for Membrane Antigens (GMMA) as an innovative delivery system for OAg, and a quadrivalent vaccine candidate containing GMMA from S. sonnei and three prevalent S. flexneri serotypes (1b, 2a and 3a) is moving to a phase II clinical trial, with the aim to elicit broad protection against Shigella. GMMA are able to induce anti-OAg-specific functional IgG responses in animal models and healthy adults. We have previously demonstrated that antibodies against protein antigens are also generated upon immunization with S. sonnei GMMA. In this work, we show that a quadrivalent Shigella GMMA-based vaccine is able to promote a humoral response against OAg and proteins of all GMMA types contained in the investigational vaccine. Proteins contained in GMMA provide T cell help as GMMA elicit a stronger anti-OAg IgG response in wild type than in T cell-deficient mice. Additionally, we observed that only the trigger of Toll-like Receptor (TLR) 4 and not of TLR2 contributed to GMMA immunogenicity. In conclusion, when tested in mice, GMMA of a quadrivalent Shigella vaccine candidate combine both adjuvant and carrier activities which allow an increase in the low immunogenic properties of carbohydrate antigens.

Methods for Assessment of OMV/GMMA Quality and Stability.

Outer membrane vesicles (OMV) represent a promising platform for the development of vaccines against bacterial pathogens. More recently, bacteria have been genetically modified to increase OMV yield and modulate the design of resulting particles, also named generalized modules for membrane antigens (GMMA). OMV/GMMA resemble the bacterial surface of the pathogen, where key antigens to elicit a protective immune response are and contain pathogen-associated molecular patterns (e.g., lipopolysaccharides, lipoproteins) conferring self-adjuvanticity. On the other hand, OMV/GMMA are quite complex molecules and a comprehensive panel of analytical methods is needed to ensure quality, consistency of manufacture and to follow their stability over time. Here, we describe several procedures that can be used for OMV/GMMA characterization as particles and for analysis of key antigens displayed on their surface.

Comparison and Optimization of Quantification Methods for Shigella flexneri Serotype 6 O-antigen Containing Galacturonic Acid and Methyl-Pentose.

Shigella is a leading diarrheal cause of morbidity and mortality worldwide, especially in low- and middle-income countries and in children under five years of age. Increasing levels of antimicrobial resistance make vaccine development an even higher global health priority. S. flexneri serotype 6 is one of the targets of many multicomponent vaccines in development to ensure broad protection against Shigella. The O-antigen (OAg) is a key active ingredient and its content is a critical quality attribute for vaccine release in order to monitor their stability and to ensure appropriate immune response. Here, the optimization of two methods to quantify S. flexneri 6 OAg is reported together with the characterization of their performances. The optimized Dische colorimetric method allows a tenfold increment of the sensitivity with respect to the original method and is useful for fast analysis detecting selectively methyl-pentoses, as rhamnose in S. flexneri 6 OAg. Also, a more specific HPAEC-PAD method was developed, detecting the dimer galacturonic acid-galactosamine (GalA-GalN) coming from S. flexneri 6 OAg acid hydrolysis. These methods will facilitate characterization of S. flexneri 6 OAg based vaccines. The colorimetric method can be used for quantification of other polysaccharide containing methyl-pentoses, and the HPAEC-PAD could be extended to other polysaccharides containing uronic acids.

Novel Simple Conjugation Chemistries for Decoration of GMMA with Heterologous Antigens.

Outer Membrane Vesicles (OMV) constitute a promising platform for the development of efficient vaccines. OMV can be decorated with heterologous antigens (proteins or polysaccharides), becoming attractive novel carriers for the development of multicomponent vaccines. Chemical conjugation represents a tool for linking antigens, also from phylogenetically distant pathogens, to OMV. Here we develop two simple and widely applicable conjugation chemistries targeting proteins or lipopolysaccharides on the surface of Generalized Modules for Membrane Antigens (GMMA), OMV spontaneously released from Gram-negative bacteria mutated to increase vesicle yield and reduce potential reactogenicity. A Design of Experiment approach was used to identify optimal conditions for GMMA activation before conjugation, resulting in consistent processes and ensuring conjugation efficiency. Conjugates produced by both chemistries induced strong humoral response against the heterologous antigen and GMMA. Additionally, the use of the two orthogonal chemistries allowed to control the linkage of two different antigens on the same GMMA particle. This work supports the further advancement of this novel platform with great potential for the design of effective vaccines.

Interoperable Blockchains for Highly-Integrated Supply Chains in Collaborative Manufacturing.

Blockchain technology plays a pivotal role in the undergoing fourth industrial revolution or Industry 4.0. It is considered a tremendous boost to company digitalization; thus, considerable investments in blockchain are being made. However, there is no single blockchain technology, but various solutions exist, and they cannot interoperate with one each other. The ecosystem envisioned by the Industry 4.0 does not have centralized management or leading organization, so a single blockchain solution cannot be imposed. The various organizations hold their own blockchains, which must interoperate seamlessly. Despite some solutions for blockchain interoperability being proposed, the problem is still open. This paper aims to devise a secure solution for blockchain interoperability. The proposed approach consists of a relay scheme based on Trusted Execution Environment to provide higher security guarantees than the current literature. In particular, the proposed solution adopts an off-chain secure computation element invoked by a smart contract on a blockchain to securely communicate with its peered counterpart. A prototype has been implemented and used for the performance assessment, e.g., to measure the latency increase due to cross-blockchain interactions. The achieved and reported experimental results show that the proposed security solution introduces an additional latency that is entirely tolerable for transactions. At the same time, the usage of the Trusted Execution Environment imposes a negligible overhead.

Stability of Outer Membrane Vesicles-Based Vaccines, Identifying the Most Appropriate Methods to Detect Changes in Vaccine Potency.

Ensuring the stability of vaccines is crucial to successfully performing global immunization programs. Outer Membrane Vesicles (OMV) are receiving great attention as vaccine platforms. OMV are complex molecules and few data have been collected so far on their stability. OMV produced by bacteria, genetically modified to increase their spontaneous release, simplifying their production, are also known as Generalized Modules for Membrane Antigens (GMMA). We have performed accelerated stability studies on GMMA from different pathogens and verified the ability of physico-chemical and immunological methods to detect possible changes. High-temperature conditions (100 °C for 40 min) did not affect GMMA stability and immunogenicity in mice, in contrast to the effect of milder temperatures for a longer period of time (37 °C or 50 °C for 4 weeks). We identified critical quality attributes to monitor during stability assessment that could impact vaccine efficacy. In particular, specific recognition of antigens by monoclonal antibodies through competitive ELISA assays may replace in vivo tests for the potency assessment of GMMA-based vaccines.

O-Antigen Extraction, Purification, and Chemical Conjugation to a Carrier Protein.

A variety of bacterial infections have been tackled by glycoconjugates over the recent years, and more vaccines are either under development at preclinical level or in clinical trials. So far, licensed glycoconjugate vaccines have made use of capsular polysaccharides or derived fragments. Today, many glycoconjugates are making use of other classes of sugars, in particular, the O-antigen portion of lipopolysaccharide molecules. Here, we report a simplified method for O-antigen extraction and purification that avoids the step of lipopolysaccharide isolation. Also, a selective chemistry for terminal linkage of O-antigen chains to a carrier protein is described, together with analytical methods for intermediates and final conjugate characterization.

Short Vi-polysaccharide abrogates T-independent immune response and hyporesponsiveness elicited by long Vi-CRM197 conjugate vaccine.

Polysaccharide-protein conjugates have been developed to overcome the T-independent response, hyporesponsiveness to repeated vaccination, and poor immunogenicity in infants of polysaccharides. To address the impact of polysaccharide length, typhoid conjugates made with short- and long-chain fractions of Vi polysaccharide with average sizes of 9.5, 22.8, 42.7, 82.0, and 165 kDa were compared. Long-chain-conjugated Vi (165 kDa) induced a response in both wild-type and T cell-deficient mice, suggesting that it maintains a T-independent response. In marked contrast, short-chain Vi (9.5 to 42.7 kDa) conjugates induced a response in wild-type mice but not in T cell-deficient mice, suggesting that the response is dependent on T cell help. Mechanistically, this was explained in neonatal mice, in which long-chain, but not short-chain, Vi conjugate induced late apoptosis of Vi-specific B cells in spleen and early depletion of Vi-specific B cells in bone marrow, resulting in hyporesponsiveness and lack of long-term persistence of Vi-specific IgG in serum and IgG+ antibody-secreting cells in bone marrow. We conclude that while conjugation of long-chain Vi generates T-dependent antigens, the conjugates also retain T-independent properties, leading to detrimental effects on immune responses. The data reported here may explain some inconsistencies observed in clinical trials and help guide the design of effective conjugate vaccines.

Intra-Laboratory Evaluation of Luminescence Based High-Throughput Serum Bactericidal Assay (L-SBA) to Determine Bactericidal Activity of Human Sera against Shigella.

Despite the huge decrease in deaths caused by Shigella worldwide in recent decades, shigellosis still causes over 200,000 deaths every year. No vaccine is currently available, and the morbidity of the disease coupled with the rise of antimicrobial resistance renders the introduction of an effective vaccine extremely urgent. Although a clear immune correlate of protection against shigellosis has not yet been established, the demonstration of the bactericidal activity of antibodies induced upon vaccination may provide one means of the functionality of antibodies induced in protecting against Shigella. The method of choice to evaluate the complement-mediated functional activity of vaccine-induced antibodies is the Serum Bactericidal Assay (SBA). Here we present the development and intra-laboratory characterization of a high-throughput luminescence-based SBA (L-SBA) method, based on the detection of ATP as a proxy of surviving bacteria, to evaluate the complement-mediated killing of human sera. We demonstrated the high specificity of the assay against a homologous strain without any heterologous aspecificity detected against species-related and non-species-related strains. We assessed the linearity, repeatability and reproducibility of L-SBA on human sera. This work will guide the bactericidal activity assessment of clinical sera raised against S. sonnei. The method has the potential of being applicable with similar performances to determine the bactericidal activity of any non-clinical and clinical sera that rely on complement-mediated killing.

Development of a Specific and Sensitive HPAEC-PAD Method for Quantification of Vi Polysaccharide Applicable to other Polysaccharides Containing Amino Uronic Acids.

Typhoid fever is a major cause of morbidity and mortality in developing countries. Vaccines based on the Vi capsular polysaccharide are licensed or in development against typhoid fever. Vi content is a critical quality attribute for vaccines release, to monitor their stability and to ensure appropriate immune response. Vi polysaccharide is a homopolymer of α-1,4-N-acetylgalactosaminouronic acid, O-acetylated at the C-3 position, resistant to the commonly used acid hydrolysis for sugar chain depolymerization before monomer quantification. We previously developed a quantification method based on strong alkaline hydrolysis followed by High Performance Anion Exchange Chromatography-Pulsed Amperometric Detection analysis, but with low sensitivity and use for quantification of an unknown product coming from polysaccharide depolymerization. Here we describe the development of a method for Vi polysaccharide quantification based on acid hydrolysis with concomitant use of trifluoroacetic and hydrochloric acids. A Design of Experiment approach was used for the identification of the optimal hydrolysis conditions. The method is 100-fold more sensitive than the previous one, and specifically, resulting in the formation of a known product, confirmed to be the Vi monomer both de-O- and de-N-acetylated by mono- and bidimensional Nuclear Magnetic Resonance spectroscopy and mass spectrometry. Accuracy and precision were determined, and chromatographic conditions were improved to result in reduced time of analysis. This method will facilitate characterization of Vi-based vaccines. Furthermore, a similar approach has the potential to be extended to other polysaccharides containing 2-amino uronic acids, as already verified here for Shigella sonnei O-antigen, Streptococcus pneumoniae serotype 12F, and Staphylococcus aureus types 5 and 8 capsular polysaccharides.

The Advent of the Internet of Things in Airfield Lightning Systems: Paving the Way from a Legacy Environment to an Open World.

This paper discusses the design and prototype implementation of a software solution facilitating the interaction of third-party developers with a legacy monitoring and control system in the airfield environment. By following the Internet of Things (IoT) approach and adopting open standards and paradigms such as REpresentational State Transfer (REST) and Advanced Message Queuing Protocol (AMQP) for message dispatching, the work aims at paving the way towards a more open world in the airfield industrial sector. The paper also presents performance results achieved by extending legacy components to support IoT standards. Quantitative results not only demonstrate the feasibility of the proposed solution, but also its suitability in terms of prompt message dispatching and increased fault tolerance.

Characterization of the Salmonella Typhimurium core oligosaccharide and its reducing end 3-deoxy-d-manno-oct-2-ulosonic acid used for conjugate vaccine production.

One of the strategies adopted for the development of a bivalent conjugate vaccine against invasive nontyphoidal Salmonella consists of linking the O-antigen component of S. Typhimurium and S. Entertidis lipopolysaccharides to the carrier protein CRM197, a non-toxic variant of diphtheria toxin. The conjugation reaction uses the reducing end residue 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) of the core to which the O-antigen chain is bound (OAg-core). OAg-core chains are cleaved from the lipid A directly in the fermentation broth by mild acid treatment. Kdo has been reported to undergo structural changes under these conditions and therefore the Kdo at the reducing end was thoroughly analysed to verify its structural integrity. For this purpose, low molecular mass OAg-core chains extracted from S. Typhimurium wild type bacteria and core oligosaccharides extracted from S. Typhimurium bacteria mutated not to produce O-antigen repeats were characterized by GLC-MS, MALDI-TOF-MS and NMR spectroscopy. Moreover, a combination of 1H-1H and 1H-13C experiments confirmed the linkage positions, sequence and structure of the octasaccharide core with 5-linked Kdo present at the reducing end in its native structure: α-GlcpNAc-(1→2)-α-Glcp-(1→2)-α-Galp-(1→3)-[α-Galp-(1→6)]-α-Glcp-(1→3)-[α-Hepp-(1→7)]-α-Hepp-(1→3)-α-Hepp-(1→5)-Kdo.

A Middleware Solution for Wireless IoT Applications in Sparse Smart Cities.

The spread of off-the-shelf mobile devices equipped with multiple wireless interfaces together with sophisticated sensors is paving the way to novel wireless Internet of Things (IoT) environments, characterized by multi-hop infrastructure-less wireless networks where devices carried by users act as sensors/actuators as well as network nodes. In particular, the paper presents Real Ad-hoc Multi-hop Peer-to peer-Wireless IoT Application (RAMP-WIA), a novel solution that facilitates the development, deployment, and management of applications in sparse Smart City environments, characterized by users willing to collaborate by allowing new applications to be deployed on their smartphones to remotely monitor and control fixed/mobile devices. RAMP-WIA allows users to dynamically configure single-hop wireless links, to manage opportunistically multi-hop packet dispatching considering that the network topology (together with the availability of sensors and actuators) may abruptly change, to actuate reliably sensor nodes specifically considering that only part of them could be actually reachable in a timely manner, and to upgrade dynamically the nodes through over-the-air distribution of new software components. The paper also reports the performance of RAMP-WIA on simple but realistic cases of small-scale deployment scenarios with off-the-shelf Android smartphones and Raspberry Pi devices; these results show not only the feasibility and soundness of the proposed approach, but also the efficiency of the middleware implemented when deployed on real testbeds.

Multiple Techniques for Size Determination of Generalized Modules for Membrane Antigens from Salmonella typhimurium and Salmonella enteritidis.

In the last years, outer membrane vesicles have attracted a lot of attention for the development of vaccines against bacterial pathogens. Extracellular vesicles can be obtained in high yields by genetic mutations, resulting in generalized modules for membrane antigens (GMMA). Methods to check the quality, consistency of production, and stability of GMMA vaccines are of fundamental importance. In this context, analytical methods for size distribution determination and verifying the integrity and possible aggregation of GMMA particles are strongly needed. Herein, GMMA particle size distribution has been evaluated by means of three different techniques. Dynamic light scattering (DLS), multiangle light scattering (MALS) coupled with high-performance liquid chromatography-size exclusion chromatography (SEC), and nanoparticle tracking analysis (NTA) have been compared to characterize GMMA from different mutants of Salmonella typhimurium and Salmonella enteritidis strains. We found that the presence of O-antigen chains on GMMA determined higher Z-average diameters by DLS compared to size estimation by MALS and that the hydrodynamic diameter increased with the number of O-antigen chains per GMMA particle. In the case of SEC-MALS, the size of the whole population better reflects the size of the most abundant particles, whereas DLS diameter is more influenced by the presence of larger particles in the sample. SEC-MALS and NTA are preferable to DLS for the analysis of bimodal samples, as they better distinguish populations of different size. MALS coupled to a size exclusion chromatography module also allows checking the purity of GMMA preparations, allowing determination of generally occurring contaminants such as soluble proteins and DNA. NTA permits real-time visualization with simultaneous tracking and counting of individual particles, but it is deeply dependent on the choice of data analysis parameters. All of the three techniques have provided complementary information leading to a more complete characterization of GMMA particles.