Avoiding implant-related complications in medically compromised patients with or without unhealthy lifestyle/Elevated oxidative stress.

Increased human life expectancy broadens the alternatives for missing teeth and played a role in the widespread use of dental implants and related augmentation procedures for the aging population. Though, many of these patients may have one or more diseases. These systemic conditions may directly lead to surgical complications, compromise implant/bone healing, or influence long-term peri-implant health and its response to biologic nuisances. Offering patients credible expectations regarding intra- and postoperative complications and therapeutic prognosis is an ethical and legal obligation. Clear identification of potential types of adverse effects, complications, or errors is important for decision-making processes as they may be related to different local, systemic, and technical aspects. Therefore, the present review structures the underlying biological mechanisms, clinical evidence, and clinical recommendations for the most common systemic risk factors for implant-related complications.

Toward in silico CMC: An industrial collaborative approach to model-based process development.

The Third Modeling Workshop focusing on bioprocess modeling was held in Kenilworth, NJ in May 2019. A summary of these Workshop proceedings is captured in this manuscript. Modeling is an active area of research within the biotechnology community, and there is a critical need to assess the current state and opportunities for continued investment to realize the full potential of models, including resource and time savings. Beyond individual presentations and topics of novel interest, a substantial portion of the Workshop was devoted toward group discussions of current states and future directions in modeling fields. All scales of modeling, from biophysical models at the molecular level and up through large scale facility and plant modeling, were considered in these discussions and are summarized in the manuscript. Model life cycle management from model development to implementation and sustainment are also considered for different stages of clinical development and commercial production. The manuscript provides a comprehensive overview of bioprocess modeling while suggesting an ideal future state with standardized approaches aligned across the industry.

Polyclonal and monoclonal IgG binding on protein A resins-Evidence of competitive binding effects.

Protein A (ProA) chromatography is used extensively in the biopharmaceutical industry for the selective capture of both polyclonal and monoclonal antibodies (mAbs). This work provides a comparison of the adsorptive behavior of a highly heterogeneous polyclonal hIgG versus that of a mAb as well as the behavior of their mixtures on representative ProA resins. Both pH gradient elution and frontal loading experiments using human polyclonal IgG (hIgG) reveal a distribution of IgG-ProA binding strengths likely associated with multiple IgG subclasses and the heterogeneity of the variable region. pH gradient analysis of fractions collected along the breakthrough curve demonstrate a clear progression from weaker binding (higher pH eluting) to stronger binding (lower pH eluting) IgG species leaving the column suggesting the possibility of stronger binding species displacing the weaker binding ones. Displacement is directly observed by visualizing the adsorption of fluorescently labeled mAb and hIgG using confocal laser scanning microscopy (CLSM). Here, the displacement of hIgG results in a broad adsorption front compared to the sharp, "shrinking core" behavior typically observed with mAbs. Sequential CLSM adsorption experiments with a mAb and hIgG confirm that stronger or equivalent-binding hIgG species are able to displace and desorb bound mAb molecules. These phenomena are examined using a variety of ProA resins including CaptivA PriMAB, MabSelect, and MabSelect SuRe to understand the effect of different ligand properties on binding strength and competition among different IgG species. The results of these comparisons suggest that the competition kinetics are slower with ligands that have a single-point covalent attachment to the base matrix compared to a multi-point attachment. Biotechnol. Bioeng. 2017;114: 1803-1812. © 2017 Wiley Periodicals, Inc.

Structural and functional characteristics of virgin and fouled Protein A MabSelect resin cycled in a monoclonal antibody purification process.

The structural and functional characteristics of the Protein A MabSelect resin are determined for a virgin sample and for samples removed from a column that had been operated in an antibody capture process which had shown losses in product recovery over fewer than 20 cycles. Compared to the virgin resin, the cycled samples show reduced porosity and apparent pore size based on inverse size exclusion chromatography while transmission electron microscopy (TEM) shows accumulation of foulants on the cycled resin. Adsorption isotherms, batch adsorption kinetics, and batch desorption kinetics, obtained using the antibody in purified form, show that the cycled samples have about 10% lower binding capacity and slower mass transfer. Confocal scanning laser microscopy shows, however, that different degrees of fouling exist for different beads in the cycled samples, which may correspond to the existence of areas exposed to minimal or no flow in the process column. Replacing the standard cleaning procedure with an improved multi-step cleaning protocol prevented the accumulation of foulants in the resin beads, as evident from TEM, and resulted in a stable operation with high recovery.

Nature of foulants and fouling mechanism in the Protein A MabSelect resin cycled in a monoclonal antibody purification process.

The composition and origin of foulants and their spatial distribution within the particles of the Protein A MabSelect resin cycled in a mAb purification process are determined using electron and confocal microscopy techniques with gold and fluorescently labeled protein probes that associate with the foulants. The results show that the foulants are primarily related to the mAb product, are heterogeneously dispersed both on the outer surface and in the interior of the resin beads, and accumulate only when loading the conditioned CHO cell culture supernatant. Insignificant accumulation is seen if the process is run with purified mAb or with the null cell culture supernatant. When bound to the Protein A ligand, the mAb responsible for the observed fouling behavior is shown to associate with BSA and α-lactalbumin. This property is exploited using labeled versions of these lipophilic proteins to assess the effectiveness of improved resin cleaning processes and to elucidate the fouling mechanism. Resin fouling for this mAb appears to be consistent with the occurrence of conformational changes that occur upon binding, which, in turn, facilitate association of lipophilic proteins with the mAb. Upon desorption at low pH, these destabilized mAb complexes are deposited on and within the resin growing with each cycle and eventually leading to significant degradation of process performance.

Effects of polymer graft properties on protein adsorption and transport in ion exchange chromatography: a multiscale modeling study.

Multiscale simulation is used to study the adsorption of lysozyme onto ion exchangers obtained by grafting charged polymers into a porous matrix, in systems with various polymer properties and strengths of electrostatic interaction. Molecular dynamics simulations show that protein partitioning into the polymer-filled pore space increases with the overall charge content of the polymers, while the diffusivity in the pore space decreases. However, the combination of greatly increased partitioning and modestly decreased diffusion results in macroscopic transport rates that increase as a function of charge content, as the large concentration driving force due to enhanced pore space partitioning outweighs the reduction in the pore space diffusivity. Matrices having greater charge associated with the grafted polymers also exhibit more diffuse intraparticle concentration profiles during transient adsorption. In systems with a high charge content per polymer and a low protein loading, the polymers preferentially partition toward the surface due to favorable interactions with the surface-bound protein. These results demonstrate the potential of multiscale modeling to illuminate qualitative trends between molecular properties and the adsorption equilibria and kinetic properties observable on macroscopic scales.

Toolboxes for a standardised and systematic study of glycans.

BACKGROUND: Recent progress in method development for characterising the branched structures of complex carbohydrates has now enabled higher throughput technology. Automation of structure analysis then calls for software development since adding meaning to large data collections in reasonable time requires corresponding bioinformatics methods and tools. Current glycobioinformatics resources do cover information on the structure and function of glycans, their interaction with proteins or their enzymatic synthesis. However, this information is partial, scattered and often difficult to find to for non-glycobiologists. METHODS: Following our diagnosis of the causes of the slow development of glycobioinformatics, we review the "objective" difficulties encountered in defining adequate formats for representing complex entities and developing efficient analysis software. RESULTS: Various solutions already implemented and strategies defined to bridge glycobiology with different fields and integrate the heterogeneous glyco-related information are presented. CONCLUSIONS: Despite the initial stage of our integrative efforts, this paper highlights the rapid expansion of glycomics, the validity of existing resources and the bright future of glycobioinformatics.

Resin structure impacts two-component protein adsorption and separation in anion exchange chromatography.

The influence of the resin structure, on the competitive binding and separation of a two-component protein mixture with anion exchange resins is evaluated using conalbumin and green fluorescent protein as a model system. Two macroporous resins, one with large open pores and one with smaller pores, are compared to a resin with grafted polymers. Investigations include measurements of single and two-component isotherms, batch uptake kinetics and two-component column breakthrough. On both macroporous resins, the weaker binding protein, conalbumin, is displaced by the stronger binding green fluorescent protein. For the large pore resin, this results in a pronounced overshoot and efficient separation by frontal chromatography. The polymer-grafted resin exhibits superior capacity and kinetics for one-component adsorption, but is unable to achieve separation due to strongly hindered counter-diffusion. Intermediate separation efficiency is obtained with the smaller pore resin. Confocal laser scanning microscopy provides a mechanistic explanation of the underlying intra-particle diffusional phenomena revealing whether unhindered counter-diffusion of the displaced protein can occur or not. This study demonstrates that the resin's intra-particle structure and its effects on diffusional transport are crucial for an efficient separation process. The novelty of this work lies in its comprehensive nature which includes examples of the three most commonly used resin structures: a small pore agarose matrix, a large-pore polymeric matrix, and a polymer grafted resin. Comparison of the protein adsorption properties of these materials provides valuable clues about advantages and disadvantages of each for anion exchange chromatography applications.

Protein adsorption on core-shell resins for flow-through purifications: Effect of protein molecular size, shape, and salt concentration.

This work addresses the functional properties of the core-shell resins Capto Core 400 and 700 for a broad range of proteins spanning 66.5 to 660 kDa in molecular mass, including bovine serum albumin (BSA) in monomer and dimer form, fibronectin, thyroglobulin, and BSA conjugates with 10 and 30 kDa poly(ethylene glycol) chains. Negatively charged latex nanoparticles (NPs) with nominal diameters of 20, 40, and 100 nm are also studied as surrogates for bioparticles. Protein binding and its trends with respect to salt concentration depend on the protein size and are different for the two agarose-based multimodal resins. For the smaller proteins, the amount of protein bound over practical time scales is limited by the resin surface area and is larger for Capto Core 400 compared with Capto Core 700. For the larger proteins, diffusion is severely restricted in Capto Core 400, resulting in lower binding capacities than those observed for Capto Core 700 despite the larger surface area. Adding 500 mM NaCl reduces the local bound protein concentration and diffusional hindrance resulting in higher binding capacities for the large proteins in Capto Core 400 compared with low ionic strength conditions. The NPs are essentially completely excluded from the Capto Core 400 pores. However, 20 and 40 nm NPs bind significantly to Capto Core 700, further hindering protein diffusion. A model is provided to predict the dynamic binding capacities as a function of residence time.

Identification of plasmepsin inhibitors as selective anti-malarial agents using ligand based drug design.

We describe the application of ligand based virtual screening technologies towards the discovery of novel plasmepsin (PM) inhibitors, a family of malarial parasitic aspartyl proteases. Pharmacophore queries were used to screen vendor libraries in search of active and selective compounds. The virtual hits were biologically assessed for activity and selectivity using whole cell Plasmodium falciparum parasites and on target in PM II, PM IV and the closely related human homologue, Cathepsin D assays. Here we report the virtual screening highlights, structures of the hits and their demonstrated biological activity.

Relationship between HETP measurements and breakthrough curves in short chromatography columns.

An analysis of the relationship between the number of plates measured with a small molecule tracer and the breakthrough curve of a strongly bound protein in short laboratory chromatography columns (1-5 cm) considering flow nonuniformity is presented. For practical conditions, while axial dispersion has only a small impact on the breakthrough curve, radial flow nonuniformity has a profound effect. Radial parabolic velocity profiles lead to tailing tracer peaks and broader breakthrough curves. Profiles where the velocity varies radially only in a thin region near the column wall lead to fronting tracer peaks and early breakthrough when the velocity at the wall is higher than the average and to tailing peaks and tailing breakthrough curves when the velocity at the wall is lower than the average. Experiments conducted in laboratory minicolumns (0.5-1 cm diameter, 0.5-1 ml volume) show tracer peaks and protein breakthrough curves that are consistent with higher velocities at the wall. The model presented in this work provides a tool to model experimental breakthrough data and to assess the degree of flow uniformity required to obtain meaningful dynamic binding capacity measurements using minicolumns in a high-throughput lab setting.

Role of configurational flexibility on the adsorption kinetics of bivalent bispecific antibodies on porous cation exchange resins.

The adsorption kinetics of a monoclonal antibody (mAb) used as a reference and of bivalent bispecific antibodies (BiSAb) on a macroporous cation exchanger is studied experimentally by examining the transient patterns of bound protein within the particles using confocal microscopy for a range of protein concentrations, buffer concentrations and pH, and temperatures. The mAb adsorption kinetics is controlled by pore diffusion and conforms to the classical shrinking core model. While the mAb adsorption rate increases with temperature, the ratio of effective and free solution diffusivity, De /D0, remains constant and has a value of 0.20. The BiSAb's structure is comprised of scFv domains that are genetically fused to a framework IgG through flexible peptide linkers which results in conformational flexibility leading to multiple binding forms with varying affinity for the adsorbent surface. As a result, adsorption of the BiSAbs shows complex patterns of total bound protein within the particles. These BiSAb adsorption patterns are influenced by buffer ionic strength, pH, and temperature in unique ways. Sharper intraparticle profiles are observed for conditions where the binding strength is greater (lower buffer concentration and/or pH) or when the protein is chemically crosslinked to restrict configurational flexibility. Temperature affects the BiSAb pore diffusivity as well as the interconversion kinetics. While the effects of temperature on BiSAb transport are also described by a constant De /D0 = 0.15, the temperature also affects the rate of interconversion between binding forms leading to faster equilibration at higher temperatures. A phenomenological model indicates that the interplay of pore diffusion and adsorption with the kinetically limited interconversion between binding forms is responsible for the experimental trends.

Chromatographic and adsorptive behavior of a bivalent bispecific antibody and associated fragments.

The elution and adsorptive behavior of a bivalent bispecific antibody (BiSAb), comprising an IgG1 framework with a scFv domain genetically fused to each heavy chain C-terminus via flexible linkers, and of two associated fragments were studied on two cation exchange chromatography media - ProPac WCX-10, which is pellicular and suitable for analytical use, and Nuvia HR-S, which is macroporous and suitable for preparative and process scale uses. Both fragments were identified by MS as missing one of the two scFv domains and its flexible linker, but one of them also contains an additional C-terminal lysine. The separation of these fragments on both resins occurs as a result of differences in non-specific ligand-protein interactions that are modulated by the salt concentration. For the ProPac WCX-10 column, complex, multipeak elution behaviors are observed, since, as a result of the linker flexibility, both the intact molecule and the fragments appear to exist in multiple binding configurations with each scFv domains either collapsed onto the IgG framework or extended away from it. With a residence time of 2.5 min and at 21 °C, two peak elution is observed for the fragments which contain a single linked scFv and three peak elution for the intact molecule which contains two linked scFvs. This behavior is affected by residence time, temperature, and hold time. Increasing the residence time to 25 min or increasing temperature to 40°C results in elution of a single, merged peak for each of the protein species. For Nuvia HR-S, the broader peaks, obtained as a result of mass transfer limitations, tend to obscure the multipeak elution behavior. Nevertheless, even for this resin, the effects of configurational flexibility are still manifested at the single-particle scale and affect the evolution of the patterns of protein binding within individual resin particles as evident from confocal microscopy observations.

Protein Adsorption on Core-shell Particles: Comparison of Capto™ Core 400 and 700 Resins.

Structural and functional characteristics of the two core-shell resins Capto™ Core 400 and 700, which are useful for the flow-through purification of bioparticles such as viruses, viral vectors, and vaccines, are compared using bovine serum albumin (BSA) and thyroglobulin (Tg) as models for small and large protein contaminants. Both resins are agarose-based and contain an adsorbing core surrounded by an inert shell. Although shell thicknesses are comparable (3.6 and 4.2 µm for Capto Core 400 and 700, respectively), the two resins differ substantially in pore size (pore radii of 19 and 50 nm, respectively). Because of the smaller pores and higher surface area, the BSA binding capacity of Capto Core 400 is approximately double that of Capto Core 700. However, for the much larger Tg, the attainable capacity is substantially larger for Capto Core 700. Mass transfer in both resins is affected by diffusional resistances through the shell and within the adsorbing core. For BSA, core and shell effective pore diffusivities are about 0.25 × 10-7 and 0.6 × 10-7 cm2/s, respectively, for Capto Core 400, and about 1.6 × 10-7 and 2.6 × 10-7 cm2/s, respectively, for Capto Core 700. These values decrease dramatically for Tg to 0.022 × 10-7 and 0.088 × 10-7 cm2/s and to 0.13 × 10-7 and 0.59 × 10-7 cm2/s for Capto Core 400 and 700, respectively. Adsorbed Tg further hinders diffusion of BSA in both resins. Column measurements show that, despite the higher static capacity of Capto Core 400 for BSA, the dynamic binding capacity is greater for Capto Core 700 as a result of its faster kinetics. However, some of this advantage is lost if the feed is a mixture of BSA and Tg since, in this case, Tg binding leads to greater diffusional hindrance for BSA.

Patterns of protein adsorption in ion-exchange particles and columns: Evolution of protein concentration profiles during load, hold, and wash steps predicted for pore and solid diffusion mechanisms.

Elucidation of protein transport mechanism in ion exchanges is essential to model separation performance. In this work we simulate intraparticle adsorption profiles during batch adsorption assuming typical process conditions for pore, solid and parallel diffusion. Artificial confocal laser scanning microscopy images are created to identify apparent differences between the different transport mechanisms. Typical sharp fronts for pore diffusion are characteristic for Langmuir equilibrium constants of KL ≥1. Only at KL = 0.1 and lower, the profiles are smooth and practically indistinguishable from a solid diffusion mechanism. During hold and wash steps, at which the interstitial buffer is removed or exchanged, continuation of diffusion of protein molecules is significant for solid diffusion due to the adsorbed phase concentration driving force. For pore diffusion, protein mobility is considerable at low and moderate binding strength. Only when pore diffusion if completely dominant, and the binding strength is very high, protein mobility is low enough to restrict diffusion out of the particles. Simulation of column operation reveals substantial protein loss when operating conditions are not adjusted appropriately.

Separation of monoclonal antibody monomer-dimer mixtures by gradient elution with ceramic hydroxyapatite.

Modeling the chromatographic separations of proteins at manufacturing scale is important since downstream processing costs are often dominant. At such scales, the columns are highly overloaded heightening the challenge of predicting performance. In this work, the separation of a monoclonal antibody monomer-dimer mixture is conducted by gradient elution chromatography with ceramic hydroxyapatite (CHT) columns Type I and Type II under overloaded conditions. Phosphate gradients are shown to be preferable over sodium chloride gradients since the latter result in undesirable pH transitions generated within the column itself. Using sodium phosphate gradients separation is obtained with both CHT types, achieving approximately 90% recovery at 99% monomer purity starting with a mixture containing 30% dimer at total protein loads up to 30 mg/mL. Because of its higher binding capacity, even higher loadings can be obtained with CHT Type I without monomer breakthrough. A hybrid model is developed to describe the separation. The model, based on an empirical description of two-component, competitive isotherms at low sodium phosphate concentration coupled with the stoichiometric displacement model at higher sodium phosphate concentrations, is in good agreement with the experiments using the linear driving force (LDF) approximation to describe adsorption/desorption kinetics. The same LDF rate coefficient predicts the separation at loadings between 0.8 and 30 mg/mL. The model developed in this work can be used as a general tool to optimize operating conditions, understand what factors limit performance, and compare different operating modes.

Dynamics of competitive binding and separation of monoclonal antibody monomer-dimer mixtures in ceramic hydroxyapatite columns.

This work examines the separation dynamics of monoclonal antibody monomer/dimer mixtures by frontal analysis using ceramic hydroxyapatite CHT Type I and Type II columns. The binding capacity and selectivity are dependent on the CHT type and salt concentration. While the rate of protein adsorption on CHT Type I is slow and controlled largely by pore diffusion resulting in relatively poor separation, adsorption on CHT Type II is much faster and better separation is obtained than with Type I. However, comparison with predictions based on pore diffusion alone, reveals the presence of additional resistances associated with adsorption and displacement kinetics. A spreading kinetics model assuming multiple binding configurations coupled with pore diffusion was developed to describe these effects and found to be in quantitative agreement with the frontal analysis results and able to predict the separation achieved for conditions outside the range of the experiments. To help validate the assumed mechanism, isocratic elution experiments were also conducted at low protein loads. The chromatograms could be described by the solution of the spreading model coupled with pore diffusion in the linear region of the isotherm with parameters determined from the analytical expressions for the peak moments. This confirms that there is an increasing tendency to spread with slower kinetics as the salt concentration is decreased and binding strength is increased.

Effects of molecule size and resin structure on protein adsorption on multimodal anion exchange chromatography media.

The effect of bead and ligand structure on protein adsorption was investigated for multimodal anion exchangers combining a quaternary ammonium ion group with hydrophobic moieties: Nuvia aPrime 1 and aPrime 2, based on a 54 µm diameter polymeric bead, and Capto Adhere ImpRes and Capto Adhere, based on agarose beads 51 and 78 µm diameter, respectively. Bovine serum albumin (BSA) monomer, BSA dimer, and thyroglobulin (Tg) were used as model proteins. Based on TEM imaging and iSEC, the Nuvia resins have a microgranular structure and large pores (110 nm radius), while the Capto resins have a fibrous structure and smaller pores (32-36 nm radius). Comparable binding capacities (80-110 mg/mL), decreasing as salt is added, are observed for all three proteins on the Nuvia resins. Higher capacities (110-130 mg/mL), also decreasing as salt is added, are observed for BSA monomer and dimer on the Capto resins. However, the Tg binding capacity is very low in this case and increases as salt is added. Confocal laser scanning microscopy show that the kinetics are controlled by pore diffusion for all four resins, but with diffusivities that decrease as the protein size increases especially for the Capto resins. For Tg at low salt, binding is restricted to a thin shell close to the bead surface for both Capto resins. The ratio of effective and free diffusivity is about 0.30, 0.18, and 0.08 for BSA monomer, BSA dimer, and Tg, respectively, on the Nuvia resin. These values decrease to about 0.11, 0.04, and 0.01, respectively, for the Capto resins as a result of diffusional hindrance. Dynamic binding capacities are consistent with the equilibrium and rate behaviors.

Effects of negative and positive cooperative adsorption of proteins on hydrophobic interaction chromatography media.

The adsorption behavior of the model proteins: alpha-Lactalbumin, Bovine Serum Albumin, Lysozyme, and a monoclonal antibody, in single component and in binary mixtures, was investigated on two different hydrophobic interaction chromatography resins using both static and dynamic methods. A kinetic model of the adsorption process was developed, which accounted for protein unfolding and intermolecular interactions in the adsorbed phase. The latter incorporated positive cooperative interactions, resulting from preferred and multilayer adsorption on the adsorbent surface, as well as negative cooperative interactions attributed to exclusion effects due to size exclusion and repulsion. Cooperative adsorption resulted in negative or positive deviations from the Langmuir model for both single and multicomponent isotherms. The model was used to assess possible contributions of different adsorption mechanisms of proteins and their structurally different forms to the overall adsorption pattern, as well as to simulate chromatographic band profiles under different loading conditions. For proteins with unstable structure, the overall adsorption isotherm was dominated by binding of unfolded species at low surface coverage and by positive cooperative adsorption at high surface coverage. Furthermore, regardless of structural stability, exclusion effects influenced strongly adsorption equilibrium, particularly at low surface coverages. In case of chromatographic elution, i.e. under dynamic conditions, unfolding, negative cooperative adsorption, and kinetic effects governed the retention behavior and determined peak shapes, whereas the effect of positive cooperative adsorption was negligible.

Chromatographic behavior of bivalent bispecific antibodies on hydrophobic interaction chromatography columns.

The elution behavior of bivalent bispecific antibodies (BiSAb) comprising an immunoglobulin G framework genetically fused to a pair of single chain variable fragments (scFvs) was studied on hydrophobic interaction chromatography (HIC) columns using ammonium sulfate gradients. Each of the BiSAb molecules studied exhibited a three-peak elution behavior regardless of the location of scFv attachment to the framework IgG. Collecting and re-injecting each of the isolated peaks and eluting with the same gradient resulted in the same three-peak profile indicating that the behavior is reversible. Analogous behavior was observed for HIC resins with different functional ligands, matrix structures, and particle sizes. Residence time, operating temperature, and hold time were shown to affect the elution behavior. While three peaks were obtained at short residence times and room temperature, residence times longer than about 27 min or operating at 45 °C resulted in a single merged peak indicating that the underlying mechanism occurs on time scales comparable to that of chromatographic separation. Holding the protein on the resins prior to elution enriched the late eluting peak indicating that multiple binding states formed on the chromatographic surface are responsible for this behavior. Tryptophan auto-fluorescence measurements show that stronger binding forms have increased solvent exposure indicating that surface-catalyzed conformational changes play a role. A model was developed to describe the interplay of chromatographic separation and slow conformational changes.