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Objectives The process of 3D printing to produce microfluidic chips is becoming commonplace, due to its quality, versatility and newfound availability. In this study, a UV liquid crystal display (LCD) printer has been implemented to produce a progression of microfluidic chips for the purpose of liposomal synthesis. The emphasis of this research is to test the limitations of UV LCD printing in terms of resolution and print speed optimisation for the production of microfluidic chips. Key findings By varying individual channel parameters such as channel length and internal geometries, the essential channel properties for optimal liposomal formulation are being investigated to act as a basis for future experimentation including the encapsulation of active pharmaceutical ingredients. Using the uniquely designed chips, liposomes of ≈120 nm, with polydispersity index values of ≤0.12 are able to be reproducibly synthesised. Conclusions The influence of total flow rates and lipid choice is investigated in depth, to provide further clarification on how a microfluidic setup should be optimised. In-depth explanations of the importance of each channel parameter are also explained throughout, with reference to their importance for the properties of a successful liposome.

IntroductionCovid-19, alongside previous pandemics, have highlighted the need for the continued development of the technologies that are at our disposal. Emerging technologies are those that show true promise in achieving such a goal and have begun to form sturdy independent research areas. It’s the case that advancements in healthcare capacity must continually develop to ensure that the world is prepared for any future diseases that may ensue. As such, a strategic review into 39 manuscripts since 2019 has been conducted to determine the prominence of emerging technologies since the beginning of the Covid-19 pandemic. Areas coveredRelating to their use in a pandemic state, additive manufacturing (AM), biofabrication, microfluidics, biomedical microelectromechanical systems (BioMEMS), and artificial intelligence (AI) are described. Applications over the past 2-3 years, as well as future developments are considered throughout. Expert OpinionAll the technologies mentioned in this review are sure to develop further, having shown their importance and value during the covid-19 pandemic. As research continues within the area, their efficacy will increase to the point where it likely will become gold standard for pandemic control. Combining certain technologies mentioned has also proved to have had great success in improving the final results obtained.

Molecular crowding in highly concentrated monoclonal antibody (mAb) solutions results in significant increases in viscosity, which complicates fill-finish steps and patient administration by subcutaneous injection. As viscosity measurements for optimization of the mAb formulation require significant amounts of material not always available in early development, fluorescence correlation spectroscopy (FCS) is evaluated as a potential ultra-low volume technique for viscosity measurement of high concentration protein solutions assuming the Generalised Stokes Einstein relation (GSE) remains valid. Using like-charge fluorescent tracers of different sizes, FCS provided measurements of microviscosities which were compared to the macroviscosity. After parametrising the protein concentration dependence of the viscosity by the exponential coefficient (k) of a simple exponential model, FCS derived k-values of like-size tracer to the crowder followed the same ordering as the macroviscosity derived k-values with respect to solvent conditions. Furthermore, k and the diffusion-derived protein-protein interaction parameter, kD, are linked, and, attractive conditions for mAbs result in a stronger concentration dependence of the viscosity. For tracers and crowders of like-size, a key result is negative deviations from the GSE relation are observed in presence of strong attractive interactions between crowder molecules. These data demonstrate that FCS has application to the screening of high concentration mAb solutions for formulation selection.

Every biosensor, bioengineered scaffold or biomedical implant depends crucially on an ability to control protein adsorption at the material surface. Yet the adsorption of proteins to solid surfaces in aqueous media is a complex and poorly understood phenomenon. To gain further insights we study protein adsorption using the quartz crystal microbalance for 10 model globular proteins interacting with positive, negative, neutral, hydrophobic and mixed alkanethiol monolayers as well as silica, polystyrene and Teflon, equating to approximately 200 protein-surface combinations. The charge state of the materials in liquid was measured with atomic force microscopy using a colloidal probe and numerically solving the full non-linear Poisson-Boltzmann equation. This approach has allowed us to address some of the important questions surrounding the basic principles that govern protein adsorption including the relative importance of net charge and hydrophobicity and why some materials are protein resistant. With our set of mixed monolayer surfaces, we can modulate charge over a wide range whilst eliminating hydrophobic interactions and vice versa- thus permitting determination of the functional dependence of adsorption on these parameters. This has led us to develop two empirical predictive models with up to 90% accuracy that together encompass most materials relevant to biotechnological and biomedical applications.

The implementation of peptide-based molecules within the medical field has vast potential, owing to their unique nature and predictable physicochemical profiles. However, peptide therapeutic usage is hindered by delivery-related challenges, meaning that their formulations must be altered to overcome these limitations. This process could be propelled by applying microfluidics (MFs) due to its highly controllable and adaptable attributes; however, therapeutic research within this field is extremely limited. Peptides possess multifunctional roles within therapeutic formulations, ranging from enhancing target specificity to acting as the active component of the medicine. Diagnostically, MFs are well explored in the field of peptides, as MFs provide an unsullied platform to provide fast yet accurate examinations. The capacity to add attributes, such as integrated sensors and microwells, to the MF chip, only enhances the attractiveness of MFs as a diagnostic platform. The structural individuality of peptides makes them prime candidates for diagnostic purposes, for example, antigen detection and isolation. Therefore, this review provides a useful insight into the current applications of MFs for peptide-based therapy and diagnostics and highlights potential gaps in the field that are yet to be explored or optimized.

Obstacle avoidance is an important task in robotics as the autonomous robot's aim is to reach the destination without collision. One type of autonomous robot that can detect obstacles and edges and take alternative paths free of obstacles and edges is a real-time obstacle avoiding edge detection robot. This paper proposes a robotic Robot with intelligence built into it that guides itself whenever an obstacle comes along its way by bug algorithm. This robotic Robot is constructed using AT mega 8 families' micro-controller (Arduino Uno R3). The ultrasonic sensor is used to detect any obstacle with edges and sends a command to the microcontroller. The micro-controller, based on the received input signal, redirects the robot to push in an alternative direction by actuating the motors that are interfaced with it via a motor driver. Depending on the situation the robot is able to choose the correct path [1]. A decision-making process of obstacle avoiding edge detection occurs spontaneously here. This robot was designed to think about its day-today potentialities.

Protein-protein interactions are commonly measured in terms of the second osmotic virial coefficient, B22 from static light scattering (SLS) or the interaction parameter, kD from dynamic light scattering (DLS). Often these measurements are carried out at high co-solvent compositions, where correction factors are required for the light scattering analysis. For lysozyme in aqueous solutions containing the co-solvents NaCl, arginine chloride, urea, sucrose or guanidine chloride, we show that B22 determination requires using in the light scattering equation the refractive index increment of the protein measured at constant solvent chemical potential. Because the increment decreases with increasing co-solvent composition, using a constant value can lead to mis-interpretation of protein-protein interaction trends deduced from the B22 measurements. Furthermore, there is a contribution to the intensity auto-correlation function measured by dynamic light scattering due to co-solvents. This effect is removed by including longer delay times when fitting the cumulant analysis to determine the diffusion coefficients. We show that an experimentally observed correlation between B22 and kD is recovered once these correction factors have been applied. The findings are particularly relevant to biopharmaceutical industry, where B22 and kD measurements are used for screening excipient effects in liquid formulations.

Therapeutic protein candidates should exhibit favorable properties that render them suitable to become drugs. Nevertheless, there are no well-established guidelines for the efficient selection of proteinaceous molecules with desired features during early stage development. Such guidelines can emerge only from a large body of published research that employs orthogonal techniques to characterize therapeutic proteins in different formulations. In this work, we share a study on a diverse group of proteins, including their primary sequences, purity data, and computational and biophysical characterization at different pH and ionic strength. We report weak linear correlations between many of the biophysical parameters. We suggest that a stability comparison of diverse therapeutic protein candidates should be based on a computational and biophysical characterization in multiple formulation conditions, as the latter can largely determine whether a protein is above or below a certain stability threshold. We use the presented data set to calculate several stability risk scores obtained with an increasing level of analytical effort and show how they correlate with protein aggregation during storage. Our work highlights the importance of developing combined risk scores that can be used for early stage developability assessment. We suggest that such scores can have high prediction accuracy only when they are based on protein stability characterization in different solution conditions.

Peptides play an important role in human physiology, and peptide deficiencies or dysfunction can lead to disease or illness. Therefore, the role that peptides play in disease treatment goes back many years, starting with the use of insulin in patients with type 1 diabetes almost a century ago. The range of targets available for peptide therapeutics and the limitations associated with peptide delivery and therapeutic effect require consideration. This chapter will review the history of the use of peptides as therapeutics, address some challenges in clinical use, and touch on major advances in the use of peptides as drugs. It will also address issues related with formulation and formulation development; peptide bond formation methods; the use of excipients; the synthesis of peptides, as well as aggregation, separation, and purification; and characterisation and stability testing. It further explores the pharmacokinetics of peptides and various routes of drug delivery and challenges related to peptide delivery.

The encapsulation of biologic molecules using a microfluidic platform is a procedure that has been understudied but shows great promise from initial reported studies. The study focusses upon the encapsulation of bovine serum albumin (BSA) under various parameters and using multiple phospholipids to identify optimal conditions for the manufacturing of protein loaded lipid nanoparticles. Additionally, encapsulation of the enzyme trypsin (TRP) has been investigated to show the eligibility of the system to other biological medications. All liposomes were subject to rigorous physicochemical characterisation, including differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopy (FTIR), to document the successful synthesis of the liposomes. Drug-loaded liposome stability was investigated over a 28-day period at 5 °C and 37 °C, which showed encouraging results for 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) at all concentrations of BSA used. The sample containing 1 mg/ml BSA grew by only 10% over the study, which considering liposomes should be affected highly by biologic adsorption, shows great promise for the formulations. Encapsulation and in vitro release studies showed improved loading capacity for BSA compared to conventional methods, whilst maintaining a concise controlled release of the active pharmaceutical ingredient (API).

Lipidation is a powerful strategy to improve the stability in vivo of peptide drugs. Attachment of a lipid chain to a hydrophilic peptide leads to amphiphilicity and the potential for surfactant-like self-assembly. Here, the self-assembly and conformation of three lipidated derivatives of the gastrointestinal peptide hormone PYY3-36 is examined using a comprehensive range of spectroscopic, scattering, and electron microscopy methods and compared to those of the parent PYY3-36 peptide. The peptides are lipidated at Ser(11), Arg(17), or Arg(23) in the peptide; the former is within the β-turn domain (based on the published solution NMR structure), and the latter two are both within the α-helical domain. We show that it is possible to access a remarkable diversity of nanostructures ranging from micelles to nanotapes and fibrillar hydrogels by control of assembly conditions (concentration, pH, and temperature). All of the lipopeptides self-assemble above a critical aggregation concentration (cac), determined through pyrene fluorescence probe measurements, and they all have predominantly α-helical secondary structure at their native pH. The pH and temperature dependence of the α-helical conformation were probed via circular dichroism spectroscopy experiments. Lipidation was found to provide enhanced stability against changes in temperature and pH. The self-assembled structures were investigated using small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cryo-TEM). Distinct differences in nanostructure were observed for lipidated and unlipidated peptides, also depending on the position of lipidation. Remarkably, micelles containing lipopeptides with α-helical peptide conformation were observed. Gelation was observed at higher concentrations in certain pH intervals for the lipidated peptides, but not for unlipidated PYY3-36. Thus, lipidation, in addition to enhancing stability against pH and temperature variation, also provides a route to prepare PYY peptide hydrogels. These findings provide important insights into the control of PYY3-36 conformation and aggregation by lipidation, relevant to the development of future therapeutics based on this peptide hormone, for example, in treatments for obesity.

Conformational fluctuations within scFv antibodies are characterized by a novel perturbation-response decomposition of molecular dynamics trajectories. Both perturbation and response profiles are stratified into stabilizing and destabilizing conditions. The linker between the VH and VL domains exhibits the dominant dynamical response by being coupled to nearly the entire protein, responding to both stabilizing and destabilizing perturbations. Perturbations within complementarity-determining regions (CDR) induce rich behavior in dynamic response. Among many effects, stabilizing any CDR loop in the VH domain triggers a destabilizing response in all CDR loops in the VL domain and vice versa. Destabilizing residues within the VL domain are likely to stabilize all CDR loops in the VH domain, and, when these residues are not buried, the CDR loops in the VL domain are also likely to be stabilized. These effects, described by shifts in normal mode characteristics, initiate a propensity for dynamic allostery with possible functional implications in bispecific antibodies.

The use of peptides as therapeutic agents is undergoing a renaissance with the expectation of new drugs with enhanced levels of efficacy and safety. Their clinical potential will be only fully realised once their physicochemical and pharmacokinetic properties have been precisely controlled. Here we demonstrate a reversible peptide self-assembly strategy to control and prolong the bioactivity of a native peptide hormone in vivo. We show that oxyntomodulin, a peptide with potential to treat obesity and diabetes, self-assembles into a stable nanofibril formulation which subsequently dissociates to release active peptide and produces a pharmacological effect in vivo. The subcutaneous administration of the nanofibrils in rats results in greatly prolonged exposure, with a constant oxyntomodulin bioactivity detectable in serum for at least 5 days as compared to free oxyntomodulin which is undetectable after only 4 h. Such an approach is simple, cost-efficient and generic in addressing the limitations of peptide therapeutics., The clinical potential of peptide therapeutic agents can only be fully realised once their physicochemical and pharmacokinetic properties are precisely controlled. Here the authors show a reversible peptide self-assembly strategy to control and prolong the bioactivity of a native peptide hormone in vivo.

Characterisation of particulates in therapeutic monoclonal antibody (mAb) formulations is routinely extended to the sub-visible size-range (0.1–10 μm). Additionally, with the increased use of pre-filled syringes (PFS), particle differentiation is required between proteinaceous and non-proteinaceous particles such as silicone-oil droplets. Here, three orthogonal techniques: Raster Image Correlation Spectroscopy (RICS), Resonance Mass Measurements (RMM) and Micro-Flow Imaging (MFI), were evaluated with respect to their sub-visible particle measurement and characterisation capabilities. Particle formation in mAb PFS solutions was evaluated with increasing polysorbate-20 (PS-20) concentrations. All three techniques provided complementary but distinct information on protein aggregate and silicone-oil droplet presence. PS-20 limited the generation of mAb aggregates during agitation, while increasing the number of silicone-oil droplets (PS-20 concentration dependant). MFI and RMM revealed PS-20 lead to the formation of larger micron-sized droplets, with RICS revealing an increase in smaller sub-micron droplets. Subtle differences in data sets complicate the apparent correlation between silicone-oil sloughing and mAb aggregates’ generation. RICS (though the use of a specific dye) demonstrates an improved selectivity for mAb aggregates, a broader measurement size-range and smaller sample volume requirement. Thus, RICS is proposed to add value to the currently available particle measurement techniques and enable informed decisions during mAb formulation development.

Aggregation and amyloid fibril formation of peptides and proteins is a widespread phenomenon. It has serious implications in a range of areas from biotechnological and pharmaceutical applications to medical disorders. The aim of this study was to develop a better understanding of the mechanism of aggregation and amyloid fibrillation of an important pharmaceutical, human glucagon-like peptide-1 (GLP-1). GLP-1 is a 31-residue hormone peptide that plays an important role regulating blood glucose levels, analogues of which are used for treatment of type 2 diabetes. Amyloid fibril formation of GLP-1 was monitored using thioflavin T fluorescence as a function of peptide concentration between pH 7.5 and 8.2. Results from these studies establish that there is a highly unusual pH-induced switch in GLP-1 aggregation kinetics. At pH 8.2, the kinetics are consistent with a nucleation-polymerization mechanism for fibril formation. However, at pH 7.5, highly unusual kinetics are observed, where the lag time increases with increasing peptide concentration. We attribute this result to the formation of off-pathway species together with an initial slow, unimolecular step where monomer converts to a different monomeric form that forms on-pathway oligomers and ultimately fibrils. Estimation of the pKa values of all the ionizable groups in GLP-1 suggest it is the protonation/deprotonation of the N-terminus that is responsible for the switch with pH. In addition, a range of biophysical techniques were used to characterize (1) the start point of the aggregation reaction and (2) the structure and stability of the fibrils formed. These results show that the off-pathway species form under conditions where GLP-1 is most prone to form oligomers.

For a therapeutic monoclonal antibody (mAb) to reach the clinic, the molecule must be produced at an appropriate yield and quality, then formulated to maintain efficacy and stability. The formation of subvisible particles (SVPs) can impact product stability and is monitored during formulation development; however, the potential of a mAb to form such species can be influenced throughout the whole bioprocess. The levels of intracellular endoplasmic reticulum (ER) stress perceived by Chinese hamster ovary (CHO) cell lines, the day of mAb harvest, and the relationship with subsequent product stability of two mAbs (denoted A and B), as determined by the SVP content after accelerated stability studies, are reported here. Here, it is shown that the propensity of mAb A to form SVPs can be predicted by transcript expression of biomarkers of cellular ER stress, heavy/light-chain transcript and polypeptide amounts, and harvest day. Further, mAb A material harvested on day 9 of culture was more stable, in terms of SVP formation, than material harvested on day 13. These data suggest that ER stress perceived by CHO cells can reflect the stability of a mAb, and that biomarkers of such stress could help define culture harvest time as a tool to control SVP formation in formulated mAbs.

Purpose Anti-drug antibodies can impair the efficacy of therapeutic proteins and, in some circumstances, induce adverse health effects. Immunogenicity can be promoted by aggregation; here we examined the ability of recombinant mouse heat shock protein 70 (rmHSP70) - a common host cell impurity - to modulate the immune responses to aggregates of two therapeutic mAbs in mice. Methods Heat and shaking stress methods were used to generate aggregates in the sub-micron size range from two human mAbs, and immunogenicity assessed by intraperitoneal exposure in BALB/c mice. Results rmHSP70 was shown to bind preferentially to aggregates of both mAbs, but not to the native, monomeric proteins. Aggregates supplemented with 0.1% rmHSP70 induced significantly enhanced IgG2a antibody responses compared with aggregates alone but the effect was not observed for monomeric mAbs. Dendritic cells pulsed with mAb aggregate showed enhanced IFNγ production on co-culture with T cells in the presence of rmHSP70. Conclusion The results indicate a Th1-skewing of the immune response by aggregates and show that murine rmHSP70 selectively modulates the immune response to mAb aggregates, but not monomer. These data suggest that heat shock protein impurities can selectively accumulate by binding to mAb aggregates and thus influence immunogenic responses to therapeutic proteins. Electronic supplementary material The online version of this article (10.1007/s11095-019-2586-7) contains supplementary material, which is available to authorized users.

Assessing how excipients affect the self-association of monoclonal antibodies (mAbs) requires informative and direct in situ measurements for highly concentrated solutions, without sample dilution or perturbation. This study explores the application of solution nuclear magnetic resonance (NMR) spectroscopy for characterization of typical mAb behavior in formulations containing arginine glutamate. The data show that the analysis of signal intensities in 1D 1H NMR spectra, when compensated for changes in buffer viscosity, is invaluable for identifying conditions where protein-protein interactions are minimized. NMR-derived molecular translational diffusion rates for concentrated solutions are less useful than transverse relaxation rates as parameters defining optimal formulation. Furthermore, NMR reports on the solution viscosity and mAb aggregation during accelerated stability study assessment, generating data consistent with that acquired by size-exclusion chromatography. The methodology developed here offers NMR spectroscopy as a new tool providing complementary information useful to formulation development of mAbs and other large therapeutic proteins.

A mechanical perturbation method that locally restricts conformational entropy along the protein backbone is used to identify putative allosteric sites in a series of antibody fragments. The method is based on a distance constraint model that integrates mechanical and thermodynamic viewpoints of protein structure wherein mechanical clamps that mimic substrate or cosolute binding are introduced. Across a set of six single chain-Fv fragments of the anti-lymphotoxin-β receptor antibody, statistically significant responses are obtained by averaging over 10 representative structures sampled from a molecular dynamics simulation. As expected, the introduced clamps locally rigidify the protein, but long-ranged increases in both rigidity and flexibility are also frequently observed. Expanding our analysis to every molecular dynamics frame demonstrates that the allosteric responses are modulated by fluctuations within the hydrogen-bond network where the native ensemble is comprised of conformations that both are, and are not, affected by the perturbation in question. Population shifts induced by the mutations alter the allosteric response by adjusting which hydrogen-bond networks are the most probable. These effects are compared using response maps that track changes across each single chain-Fv fragment, thus providing valuable insight into how sensitive allosteric mechanisms are to mutations.

The effect of protein concentration on solution viscosity in a commercially available biopharmaceutical formulation of recombinant albumin (rAlbumin) was studied. The level of protein aggregation with concentration and its impact on solution viscosity was investigated. Theoretical models predicting viscosity with concentration were applied to these data, and a model that accounts for multiple protein species in solution provided the best fit. The results highlight the need to account for heterogeneity in the level of aggregation when addressing the increase of viscosity observed at high concentrations of protein solutions, a significant issue for the manufacture and use of protein-based therapeutics.

Lipidation is a powerful strategy to improve the stability in vivo of peptide drugs. Attachment of a lipid chain to a hydrophilic peptide leads to amphiphilicity and the potential for surfactant-like self-assembly. Here, the self-assembly and conformation of three lipidated derivatives of the gastrointestinal peptide hormone PYY

A critical step in monoclonal antibody (mAb) screening and formulation selection is the ability of the mAb to resist aggregation following exposure to environmental stresses. Regulatory authorities welcome not only information on the presence of micron-sized particles, but often any information on sub-visible particles in the size range obtained by orthogonal sizing techniques. The present study demonstrates the power of combining established techniques such as dynamic light scattering (DLS) and micro-flow imaging (MFI), with novel analyses such as raster image correlation spectroscopy (RICS) that offer to bridge existent particle sizing gaps in this area. The influence of thermal and freeze-thaw stress treatments on particle size and morphology was assessed for a bi-specific antibody (mAb2). Aggregation of mAb2 was confirmed to be concentration- and treatment-dependent following thermal stress and freeze-thaw cycling. Particle size and count data show concentration- and treatment-dependent behaviour of aggregate counts, morphological descriptors and particle size distributions. Complementarity in particle size output was observed between all approaches utilised, where RICS bridged the analytical size gap (∼0.5-5 μm) between DLS and MFI. Overall, this study highlights the potential of orthogonal image analyses such as RICS (analytical size gap) and MFI (particle morphology) for formulation screening.

Predicting the concentrated solution behavior for monoclonal antibodies requires developing and using minimal models to describe their shape and interaction potential. Toward this end, the small-angle X-ray scattering (SAXS) profiles for a monoclonal antibody (COE-03) have been measured under solution conditions chosen to produce weak self-association. The experiments are complemented with molecular simulations of a three-bead antibody model with and without interbead attraction. The scattering profile is extracted directly from the molecular simulation to avoid using the decoupling approximation. We examine the ability of the three-bead model to capture features of the scattering profile and the dependence of compressibilty on protein concentration. The three-bead model is able to reproduce generic features of the experimental structure factor as a function of wave vector S(k) including a well-defined shoulder, which is a consequence of the planar structure of the antibody, and a well-defined minimum in S(k) at k ∼ 0.025 Å

Ionic excipients are commonly used in aqueous therapeutic monoclonal antibody (mAb) formulations. Novel excipients are of industrial interest, with a recent focus on Arg salt forms and their application as viscosity reducing and stabilizing additives. Here, we report that the calcium salt of dipicolinic acid (DPA, pyridine-2,6-dicarboxylic acid), uniquely present in nature in the core of certain bacterial spores, reduces the viscosity of a mAb formulated at 150mg/mL, below that achieved by Arg hydrochloride at the same concentration (10mM). DPA also reduced the reversible phase separation of the same formulation, which characteristically occurs for this mAb upon cooling to 4°C. Differential scanning calorimetry and differential scanning fluorimetry did not reveal a conformation destabilisation of the mAb in the presence of 10mM DPA, or by the related quinolinic acid (QA, pyridine-2,3-dicarboxylic acid). However, fluorescence spectrophotometry did reveal localised (aromatic) conformational changes to the mAb attributed to DPA, dependent on the salt form. While precise mechanisms of action remain to be identified, our preliminary data suggest that these DPA salts are worthy of further investigation as novel ionic excipient for biologics formulation.

Characterizing the influence of fragment crystallization (Fc) and antigen-binding fragment (Fab) on monoclonal antibody (mAb) adsorption at the air/water interface is an important step to understanding liquid mAb drug product stability during manufacture, shipping, and storage. Here, neutron reflection is used to study the air/water adsorption of a mAb and its Fc and Fab fragments. By varying the isotopic contrast, the adsorbed amount, thickness, orientation, and immersion of the adsorbed layers could be determined unambiguously. While Fc adsorption reached saturation within the hour, its surface adsorbed amount showed little variation with bulk concentration. In contrast, Fab adsorption was slower and the adsorbed amount was concentration dependent. The much higher Fc adsorption, as compared to Fab, was linked to its lower surface charge. Time and concentration dependence of mAb adsorption was dominated by Fab behavior, although both Fab and Fc behaviors contributed to the amount of mAb adsorbed. Changing the pH from 5.5 to 8.8 did not much perturb the adsorbed amount of Fc, Fab, or mAb. However, a small decrease in adsorption was observed for the Fc over pH 8-8.8 and vice versa for the Fab and mAb, consistent with a dominant Fab behavior. As bulk concentration increased from 5 to 50 ppm, the thicknesses of the Fc layers were almost constant at 40 Å, while Fab and mAb layers increased from 45 to 50 Å. These results imply that the adsorbed mAb, Fc, and Fab all retained their globular structures and were oriented with their short axial lengths perpendicular to the interface.

Liquid-liquid phase separation (LLPS) of monoclonal antibody (mAb) formulations involves spontaneous separation into dense (protein-rich) and diluted (protein-lean) phases and should be avoided in the final drug product. Understanding the factors leading to LLPS and ways to predict and prevent it would therefore be highly beneficial. Here we describe the link between LLPS behavior of an IgG1 mAb (mAb5), its solubility, and parameters extracted using

The aggregation propensities for a series of single-chain variable fragment (scFv) mutant proteins containing supercharged sequences, salt bridges and lysine/arginine-enriched motifs were characterised as a function of pH and ionic strength to isolate the electrostatic contributions. Recent improvements in aggregation predictors rely on using knowledge of native-state protein-protein interactions. Consistent with previous findings, electrostatic contributions to native protein-protein interactions correlate with aggregate growth pathway and rates. However, strong reversible self-association observed for selected mutants under native conditions did not correlate with aggregate growth, indicating 'sticky' surfaces that are exposed in the native monomeric state are inaccessible when aggregates grow. We find that even though similar native-state protein-protein interactions occur for the arginine and lysine-enriched mutants, aggregation propensity is increased for the former and decreased for the latter, providing evidence that lysine suppresses interactions between partially folded states under these conditions. The supercharged mutants follow the behaviour observed for basic proteins under acidic conditions; where excess net charge decreases conformational stability and increases nucleation rates, but conversely reduces aggregate growth rates due to increased intermolecular electrostatic repulsion. The results highlight the limitations of using conformational stability and native-state protein-protein interactions as predictors for aggregation propensity and provide guidance on how to engineer stabilizing charged mutations.

Assessing how excipients affect the self-association of monoclonal antibodies (mAbs) requires informative and direct in situ measurements for highly concentrated solutions, without sample dilution or perturbation. This study explores the application of solution nuclear magnetic resonance (NMR) spectroscopy for characterization of typical mAb behavior in formulations containing arginine glutamate. The data show that the analysis of signal intensities in 1D 1H NMR spectra, when compensated for changes in buffer viscosity, is invaluable for identifying conditions where protein-protein interactions are minimized. NMR-derived molecular translational diffusion rates for concentrated solutions are less useful than transverse relaxation rates as parameters defining optimal formulation. Furthermore, NMR reports on the solution viscosity and mAb aggregation during accelerated stability study assessment, generating data consistent with that acquired by size-exclusion chromatography. The methodology developed here offers NMR spectroscopy as a new tool providing complementary information useful to formulation development of mAbs and other large therapeutic proteins.

Poly(ethylene glycol) (PEG) may be covalently conjugated to peptide drugs to overcome their rapid clearance but in doing so their potency can be lost. Here, a non-covalent approach was used to conjugate PEG bearing a terminal cholanic moiety (mPEG5kDa-cholane) to a 28 amino acid peptide, vasoactive intestinal peptide (VIP). Palmitoylation of the peptide was essential to facilitate physical interaction via a single binding site involving two mPEG5kDa-cholane molecules with an affinity constant of ~3·10(4)M(-1); these calorimetry data corroborating Scatchard analysis of dissolution data. The peptide/polymer complex (below 10-12nm diameter) provided for up to 5000-fold greater solubility of the peptide at pH7.4 (4μg/mL) and markedly increased peptide solution stability at 25°C over 30days. Mannitol enabled the complex to be lyophilized to yield a freeze-dried formulation which was efficiently reconstituted albeit with an ~10% decrease in solubility. The predominantly α-helical conformation of the peptide alone at pH5-6.5 was lost at pH7.4 but fully recovered with 2 molar equivalents of mPEG5kDa-cholane. After lyophilization and reconstitution an ~10% loss of α-helical conformation was observed, which may reflect the equivalent decrease in solubility. Pharmacokinetic studies following subcutaneous administration of the peptide (0.1mg/Kg) alone and with 2 molar equivalents of polymer showed that mPEG5kDa-cholane dramatically increased peptide concentration in the systemic circulation. This is the first demonstration of non-covalent PEGylation of acylated peptides, an important biologic class, which improves in vitro and in vivo properties, and thereby may prove an alternative to covalent PEGylation strategies.

Protein products come into contact with many surfaces of differing wettability during bioprocessing, formulation and delivery, but corresponding data for the adsorbed conformations and the associated force of adhesion (F(ad)) is sparse. Here we have generated a series of hydrophilic and hydrophobic surfaces through silanization of silica with various terminal groups, characterizing the surface energies and droplet contact angles. F(ad) measured by atomic force microscopy for oriented monolayers of a human monoclonal antibody (mAb-1) clearly distinguished hydrophobic surfaces (low F(ad) values) from hydrophilic surfaces (high F(ad) values). High F(ad) for a methoxy capped polyethylene glycol (1000 MW) surface supports the interaction of mAb-1 with buried ethylene oxide groups, consistent with mAb-1 compression into a distorted brush border. Solid state circular dichroism showed that mAb-1 (β-sheet) or albumin (α-helical) adsorbed to bare silica beads largely retained their secondary structures. However, the extent of structural loss upon protein adsorption to functionalized silica beads could not be simply correlated to hydrophilic/hydrophobic surface interaction as seen for the F(ad) measurements. For example, of the hydrophilic surfaces mAb-1 unfolded notably more when adsorbed to the aminopropyl surface, and of the hydrophobic surfaces both mAb-1 and albumin retained most secondary structure when adsorbed to the perfluorooctyl surface (consistent with the lipophobic perfluorinated moiety limiting exposure of the protein hydrophobic core). The data show that F(ad) values are not necessarily predictive of the subsequent extent of structural relaxation, and that significant structural loss is evident for proteins adsorbed to both hydrophilic and hydrophobic surfaces.

Terahertz time-domain spectroscopy (THz-TDS) has been shown to detect overlapping extended hydration layers around proteins. Here, we used THz-TDS to detect modulation of the extended hydration layer around monoclonal antibodies (mAbs) by the introduction of commonly used excipients. Proline and sucrose altered the hydration layer around a mAb (mAb1), which was observed as a negative shift in the plateau in absorbance above ~100 mg/mL mAb1 (~70,000 water molecules per mAb); arginine had no effect. At lower concentrations of ~10 mg/mL mAb1 (~700,000 water molecules per mAb) proline and arginine modulated the hydration layer, which was observed as a negative shift in the relative absorbance, whereas sucrose had no effect. The changes in the extended hydration layer were not translated to shifts in the thermal stability or protein:protein interaction parameter. The hydration layer of a second mAb (mAb2) was further shown to be modulated by more complex formulations composed of two or more excipients; although the differences in terahertz absorbance were not predictive of viscosity or long-term stability. THz-TDS promises to be a useful tool for understanding a protein's interaction with excipients in solution and the challenge will be to determine how to apply this knowledge to protein formulation.

A rotating disk shear device was used to study the effect of interfacial shear on the structural integrity of human monoclonal antibodies of IgG4 isotype. Factors associated with the solution conditions (pH, ionic strength, surfactant concentration, temperature) and the interface (surface roughness) were studied for their effect on the rate of IgG4 monomer loss under high shear conditions. The structural integrity of the IgG4 was probed after exposure to interfacial shear effects by SDS-PAGE, IEF, dynamic light scattering, and peptide mapping by LC-MS. This analysis revealed that the main denaturation pathway of IgG4 exposed to these effects was the formation of large insoluble aggregates. Soluble aggregation, breakdown in primary structure, and chemical modifications were not detected. The dominant factors found to affect the rate of IgG4 monomer loss under interfacial shear conditions were found to be pH and the nanometer-scale surface roughness associated with the solid-liquid interface. Interestingly, temperature was not found to be a significant factor in the range tested (15-45 degrees C). The addition of surfactant was found to have a significant stabilizing effect at concentrations up to 0.02% (w/v). Implications of these findings for the bioprocessing of this class of therapeutic protein are briefly discussed. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 1499-1507, 2009

The effects of an equimolar mixture of l-arginine and l-glutamate (Arg·Glu) on cell viability and cellular stress using in vitro cell culture systems are examined with reference to NaCl, in the context of monoclonal antibody formulation. Cells relevant to subcutaneous administration were selected: the human monocyte cell line THP-1, grown as a single cell suspension, and adherent human primary fibroblasts. For THP-1 cells, the mechanism of cell death caused by relatively high salt concentrations was investigated and effects on cell activation/stress assessed as a function of changes in membrane marker and cytokine (interleukin-8) expression. These studies demonstrated that Arg·Glu does not have any further detrimental effects on THP-1 viability in comparison to NaCl at equivalent osmolalities, and that both salts at higher concentrations cause cell death by apoptosis; there was no significant effect on measures of THP-1 cellular stress/activation. For adherent fibroblasts, both salts caused significant toxicity at ~ 400 mOsm/kg, although Arg·Glu caused a more precipitous subsequent decline in viability than did NaCl. These data indicate that Arg·Glu is of equivalent toxicity to NaCl and that the mechanism of toxicity is such that cell death is unlikely to trigger inflammation upon subcutaneous injection in vivo., Highlights • The effect of Arg·Glu on cell viability was examined with reference to NaCl. • Arg·Glu is of equivalent toxicity to NaCl. • The mechanism of cell death is via apoptosis and is thus not pro-inflammatory. • Arg·Glu as a subcut excipient is not expected to result in atypical inflammation.

Interrogation of the protein hydration layer in the context of the rational design of high concentration monoclonal antibody (mAb) formulations has not yet been reported. Here, terahertz time domain spectroscopy (THz-TDS) was used to show that the hydration layer for ‘mAb1’ up to 140 mg/ml was perturbed by the addition of 200 mM proline or arginine but not 200 mM sucrose. The hydration layer of ‘mAb2’ was also shown to be modulated by more complex formulations composed of two or more excipients in buffer. Thus, THz-TDS promises to be a useful tool for protein formulation by providing an improved understanding of solution behaviour at high concentrations and associated mechanisms of control by the addition of excipients.

The effects of somatic mutations that transform polyspecific germline (GL) antibodies to affinity mature (AM) antibodies with monospecificity are compared among three GL-AM Fab pairs. In particular, changes in conformational flexibility are assessed using a Distance Constraint Model (DCM). We have previously established that the DCM can be robustly applied across a series of antibody fragments (VL to Fab), and subsequently, the DCM was combined with molecular dynamics (MD) simulations to similarly characterize five thermostabilizing scFv mutants. The DCM is an ensemble based statistical mechanical approach that accounts for enthalpy/entropy compensation due to network rigidity, which has been quite successful in elucidating conformational flexibility and Quantitative Stability/Flexibility Relationships (QSFR) in proteins. Applied to three disparate antibody systems changes in QSFR quantities indicate that the VH domain is typically rigidified, whereas the VL domain and CDR L2 loop become more flexible during affinity maturation. The increase in CDR H3 loop rigidity is consistent with other studies in the literature. The redistribution of conformational flexibility is largely controlled by nonspecific changes in the H-bond network, although certain Arg to Asp salt bridges create highly localized rigidity increases. Taken together, these results reveal an intricate flexibility/rigidity response that accompanies affinity maturation., Author Summary Antibodies are protective proteins used by the immune system to recognize and neutralize foreign objects through interactions with a specific part of the target, called an antigen. Antibody structures are Y-shaped, contain multiple protein chains, and include two antigen-binding sites. The binding sites are located at the end of the Fab fragments, which are the upward facing arms of the Y-structure. The Fab fragments maintain binding affinity by themselves, and are thus often used as surrogates to student antibody-antigen interactions. High affinity antibodies are produced during the course of an immune response by successive mutations to germline gene-encoded antibodies. Germline antibodies are more likely to be polyspecific, whereas the affinity maturation process yields monoclonal antibodies that bind specifically to the target antigen. In this work, we use a computational Distance Constraint Model to characterize how mechanical properties change as three disparate germline antibodies are converted to affinity mature. Our results reveal a rich set of mechanical responses throughout the Fab structure. Nevertheless, increased rigidity in the VH domain is consistently observed, which is consistent with the transition from polyspecificity to monospecificity. That is, flexible antibody structures are able to recognize multiple antigens, while increased affinity and specificity is achieved—in part—by structural rigidification.

Background: Shoulder disorder has a high occurrence in physically active and adult population can be due to most prevalent shoulder impingement. Objective: This study was conducted to compare effectiveness of manual therapy vs ultrasound therapy for shoulder pain due to secondary impingement. Methodology: A double blind study done through the interview and assessment of 24 subjects among adult population from different occupation with unilateral shoulder pain. Subjects were randomly allocated in experimental and control group to receive prescribed treatment for continuous 5 weeks with informed consent. Result: The experimental group showed the better improvement in all aspects than the control group. From the measurement the difference of standard deviation of Visual Analogue Scale was 1.31, abduction was 13.46, external rotation was 2.23 and disability score was 1.67 in control group whereas in the experimental group the VAS was 0.83, abduction was 15.83, external rotation was 4.51 and disability score was 1.82. Conclusion: As a whole manual therapy with other common item of treatment for both groups can be more effective but further large scale research should be done in this respect.

Background: Worldwide most adult population would suffer acute low back pain with some disabilities at some stage of life almost without specific cause but it is essential to manage pain to prevent transition to chronic pain which presents a significant individual, social and economic burden. Objective: To compare effectiveness of manual therapy, electrotherapy and core stability exercise vs electrotherapy, traction and traditional exercise for acute low back pain. Methodology: It was a double blind study in which 52 subjects of 46 year mean age were randomized in control and intervention groups. All the subjects were selected from different occupation through assessment, interview, who was suffering central, unilateral or bilateral low back pain less than three months. Subjects of both groups were agreed to receive the allocated treatment for continuous four weeks and follow up the instructions. To measure the effectiveness of this study numerical point box scale for pain, modified Schober method for flexion, extension range and Quebec Back Pain Disability Scale was used. Result: From the measurement, the intervention group showed better improvement and performance in all aspects than the control group. The measurements showed the difference of deviation pain reduction box scale 2.99, flexion 0.28, extension 0.82, disability scale 14.21 was in control group and pain reduction 5.20, flexion 1.47, extension 1.30; disability scale 22.83 was in intervention group. Conclusion: Manual therapy, core stability exercises with other treatments can be effective for acute low back pain but further research should be done in this respect.

Better predictive ability of salt and buffer effects on protein-protein interactions requires separating out contributions due to ionic screening, protein charge neutralization by ion binding, and salting-in(out) behavior. We have carried out a systematic study by measuring protein-protein interactions for a monoclonal antibody over an ionic strength range of 25 to 525 mM at 4 pH values (5, 6.5, 8, and 9) in solutions containing sodium chloride, calcium chloride, sodium sulfate, or sodium thiocyante. The salt ions are chosen so as to represent a range of affinities for protein charged and noncharged groups. The results are compared to effects of various buffers including acetate, citrate, phosphate, histidine, succinate, or tris. In low ionic strength solutions, anion binding affinity is reflected by the ability to reduce protein-protein repulsion, which follows the order thiocyanate > sulfate > chloride. The sulfate specific effect is screened at the same ionic strength required to screen the pH dependence of protein-protein interactions indicating sulfate binding only neutralizes protein charged groups. Thiocyanate specific effects occur over a larger ionic strength range reflecting adsorption to charged and noncharged regions of the protein. The latter leads to salting-in behavior and, at low pH, a nonmonotonic interaction profile with respect to sodium thiocyanate concentration. The effects of thiocyanate can not be rationalized in terms of only neutralizing double layer forces indicating the presence of an additional short-ranged protein-protein attraction at moderate ionic strength. Conversely, buffer specific effects can be explained through a charge neutralization mechanism, where buffers with greater valency are more effective at reducing double layer forces at low pH. Citrate binding at pH 6.5 leads to protein charge inversion and the formation of attractive electrostatic interactions. Throughout the report, we highlight similarities in the measured protein-protein interaction profiles with previous studies of globular proteins and of antibodies providing evidence that the behavior will be common to other protein systems.