Points to Consider: Best Practices to Identify Particle Entry Routes along the Manufacturing Process for Parenteral Formulations.

During the processes involved in pharmaceutical manufacturing, particulate matter may be introduced into a product from a variety of sources and at different points in the manufacturing process. Companies design quality at the beginning of the process to ensure against defects and strive to manufacture products that meet the pharmacopeial standard of being "practically/essentially free" of particles, which can be challenging, though necessary. As particulate matter recalls are predominantly associated with parenteral products, most companies employ a quality risk management program to identify critical parameters or conditions that could affect product quality or patient safety and incorporate systemic and procedural controls to mitigate or reduce the probability of their occurrence. Yet, determining where particulates are most likely to enter the process, what types of materials are most vulnerable, and how the size and number of particles might affect product quality can be very complex. Visual inspection and sampling of the manufactured drug product are designed to control the risk of particulate contamination; building prevention controls will ensure sustainability. This concept paper highlights the necessity of a more thorough understanding of the failure mechanisms that result in particle contamination across a range of products, such as elastomeric components and glass, and processes, such as the formulation and filling of injectables. The goal is to identify process steps within the end-to-end manufacturing process that are most critical to particle generation and entering of visible particles into the final drug product.LAY ABSTRACT: This concept paper highlights the necessity of a more thorough understanding of the failure mechanisms that result in particle contamination across a range of products, such as elastomeric components and glass, and processes, such as the formulation and filling of injectables. The goal is to identify process steps within the end-to-end manufacturing process that are most critical to particle generation and entering of visible particles into the final drug product.

Forces and dynamics of glucose and inhibitor binding to sodium glucose co-transporter SGLT1 studied by single molecule force spectroscopy.

Single molecule force spectroscopy was employed to investigate the dynamics of the sodium glucose co-transporter (SGLT1) upon substrate and inhibitor binding on the single molecule level. CHO cells stably expressing rbSGLT1 were probed by using atomic force microscopy tips carrying either thioglucose, 2'-aminoethyl ß-d-glucopyranoside, or aminophlorizin. Poly(ethylene glycol) (PEG) chains of different length and varying end groups were used as tether. Experiments were performed at 10, 25 and 37 °C to address different conformational states of SGLT1. Unbinding forces between ligands and SGLT1 were recorded at different loading rates by changing the retraction velocity, yielding binding probability, width of energy barrier of the binding pocket, and the kinetic off rate constant of the binding reaction. With increasing temperature, width of energy barrier and average life time increased for the interaction of SGLT1 with thioglucose (coupled via acrylamide to a long PEG) but decreased for aminophlorizin binding. The former indicates that in the membrane-bound SGLT1 the pathway to sugar translocation involves several steps with different temperature sensitivity. The latter suggests that also the aglucon binding sites for transport inhibitors have specific, temperature-sensitive conformations.

Probing binding pocket of serotonin transporter by single molecular force spectroscopy on living cells.

The serotonin transporter (SERT) terminates neurotransmission by removing serotonin from the synaptic cleft. In addition, it is the site of action of antidepressants (which block the transporter) and of amphetamines (which induce substrate efflux). The interaction energies involved in binding of such compounds to the transporter are unknown. Here, we used atomic force microscopy (AFM) to probe single molecular interactions between the serotonin transporter and MFZ2-12 (a potent cocaine analog) in living CHOK1 cells. For the AFM measurements, MFZ2-12 was immobilized on AFM tips by using a heterobifunctional cross-linker. By varying the pulling velocity in force distance cycles drug-transporter complexes were ruptured at different force loadings allowing for mapping of the interaction energy landscape. We derived chemical rate constants from these recordings and compared them with those inferred from inhibition of transport and ligand binding: koff values were in good agreement with those derived from uptake experiments; in contrast, the kon values were scaled down when determined by AFM. Our observations generated new insights into the energy landscape of the interaction between SERT and inhibitors. They thus provide a useful framework for molecular dynamics simulations by exploring the range of forces and energies that operate during the binding reaction.

Determination of the kinetic on- and off-rate of single virus-cell interactions.

Human rhinoviruses are the causative agents of the common cold. The serotypes belonging to the minor receptor group attach to members of the low-density lipoprotein receptor family and enter the host cell via receptor-mediated endocytosis. Receptor binding, the very first step in infection, was characterized by force spectroscopy measurements at the single molecule level. We demonstrate how kinetic on- and off-rate constants can be derived from such experiments carried out with the atomic force microscope.

Linking of sensor molecules with amino groups to amino-functionalized AFM tips.

The measuring tip of an atomic force microscope (AFM) can be upgraded to a specific biosensor by attaching one or a few biomolecules to the apex of the tip. The biofunctionalized tip is then used to map cognate target molecules on a sample surface or to study biophysical parameters of interaction with the target molecules. The functionality of tip-bound sensor molecules is greatly enhanced if they are linked via a thin, flexible polymer chain. In a typical scheme of tip functionalization, reactive groups are first generated on the tip surface, a bifunctional cross-linker is then attached with one of its two reactive ends, and finally the probe molecule of interest is coupled to the free end of the cross-linker. Unfortunately, the most popular functional group generated on the tip surface is the amino group, while at the same time, the only useful coupling functions of many biomolecules (such as antibodies) are also NH(2) groups. In the past, various tricks or detours were applied to minimize the undesired bivalent reaction of bifunctional linkers with adjacent NH(2) groups on the tip surface. In the present study, an uncompromising solution to this problem was found with the help of a new cross-linker ("acetal-PEG-NHS") which possesses one activated carboxyl group and one acetal-protected benzaldehyde function. The activated carboxyl ensures rapid unilateral attachment to the amino-functionalized tip, and only then is the terminal acetal group converted into the amino-reactive benzaldehyde function by mild treatment (1% citric acid, 1-10 min) which does not harm the AFM tip. As an exception, AFM tips with magnetic coating become demagnetized in 1% citric acid. This problem was solved by deprotecting the acetal group before coupling the PEG linker to the AFM tip. Bivalent binding of the corresponding linker ("aldehyde-PEG-NHS") to adjacent NH(2) groups on the tip was largely suppressed by high linker concentrations. In this way, magnetic AFM tips could be functionalized with an ethylene diamine derivative of ATP which showed specific interaction with mitochondrial uncoupling protein 1 (UCP1) that had been purified and reconstituted in a mica-supported planar lipid bilayer.

AFM functional imaging on vascular endothelial cells.

Vascular endothelial (VE)-cadherin is predominantly responsible for the mechanical linkage between endothelial cells, where VE-cadherin molecules are clustered and linked through their cytoplasmic domain to the actin-based cytoskeleton. Clustering and linkage of VE-cadherin to actin filaments is a dynamic process and changes according to the functional state of the cells. Here nano-mapping of VE-cadherin was performed using simultaneous topography and recognition imaging (TREC) technique onto microvascular endothelial cells from mouse myocardium (MyEnd). The recognition maps revealed prominent 'dark' spots (domains or clusters) with the sizes from 10 to 250 nm. These spots arose from a decrease of oscillation amplitude during specific binding between VE-cadherin cis-dimers. They were assigned to characteristic structures of the topography images. After treatment with nocodazole so as to depolymerize microtubules, VE-cadherin domains with a typical ellipsoidal form were still found to be collocalized with cytoskeletal filaments supporting the hypothesis that VE-cadherin is linked to actin filaments. Compared to other conventional techniques such as immunochemistry or single molecule optical microscopy, TREC represents an alternative method to quickly obtain the local distribution of receptors on cell surface with an unprecedented lateral resolution of several nanometers.

Antibody recognition force microscopy shows that outer membrane cytochromes OmcA and MtrC are expressed on the exterior surface of Shewanella oneidensis MR-1.

Antibody recognition force microscopy showed that OmcA and MtrC are expressed on the exterior surface of living Shewanella oneidensis MR-1 cells when Fe(III), including solid-phase hematite (Fe(2)O(3)), was the terminal electron acceptor. OmcA was localized to the interface between the cell and mineral. MtrC displayed a more uniform distribution across the cell surface. Both cytochromes were associated with an extracellular polymeric substance.

Multiple receptors involved in human rhinovirus attachment to live cells.

Minor group human rhinoviruses (HRVs) attach to members of the low-density lipoprotein receptor family and are internalized via receptor-mediated endocytosis. The attachment of HRV2 to the cell surface, the first step in infection, was characterized at the single-molecule level by atomic force spectroscopy. Sequential binding of multiple receptors was evident from recordings of characteristic quantized force spectra, which suggests that multiple receptors bound to the virus in a timely manner. Unbinding forces required to detach the virus from the cell membrane increased within a time frame of several hundred milliseconds. The number of receptors involved in virus binding was determined, and estimates for on-rate, off-rate, and equilibrium binding constant of the interaction between HRV2 and plasma membrane-anchored receptors were obtained.

Localization of the ergtoxin-1 receptors on the voltage sensing domain of hERG K+ channel by AFM recognition imaging.

The inhibition of the human ether-à-go-go-related (hERG) K+ channels is the major cause of long QT syndromes inducing fatal cardiac arrhythmias. Ergtoxin 1 (ErgTx1) belongs to scorpion-toxins, which are K+ channel-blockers, and binds to hERG channel with 1:1 stoichiometry and high affinity (Kd approximately 10 nM). Nevertheless, patch-clamp recordings recently demonstrated that ErgTx1 does not establish complete blockade of hERG currents, even at high ErgTx1 concentrations. Such phenomenon is supposed to be consistent with highly dynamic conformational changes of the outer pore domain of hERG. In this study, simultaneous topography and recognition imaging (TREC) on hERG HEK 293 cells was used to visualize binding sites on the extracellular part of hERG channel (on S1-S2 region) for Anti-Kv11.1 (hERG-extracellular-antibody). The recognition maps of hERG channels contained recognition spots, haphazardly distributed and organized in clusters. Recognition images after the addition of ErgTx1 at high concentrations ( approximately 1 microM) revealed subsequent partial disappearance of clusters, indicating that ErgTx1 was bound to the S1-S2 region. These results were supported by AFM force spectroscopy data, showing for the first time that voltage sensing domain (S1-S4) of hERG K+ channel might be one of the multiple binding sites of ErgTx1.

Unbinding molecular recognition force maps of localized single receptor molecules by atomic force microscopy.

Atomic force microscopy is a technique capable to study biological recognition processes at the single-molecule level. In this work we operate the AFM in a force-scan based mode, the jumping mode, where simultaneous topographic and tip-sample adhesion maps are acquired. This approach obtains the unbinding force between a well-defined receptor molecule and a ligand attached to the AFM tip. The method is applied to the avidin-biotin system. In contrast with previous data, we obtain laterally resolved adhesion maps of avidin-biotin unbinding forces highly correlated with single avidin molecules in the corresponding topographic map. The scanning rate 250 pixel s(-1) (2 min for a 128 x 128 image) is limited by the hydrodynamic drag force. We are able to build a rupture-force distribution histogram that corresponds to a single defined molecule. Furthermore, we find that due to the motility of the polymer used as spacer to anchor the ligand to the tip, its direction at rupture does not generally coincide with the normal to the tip-sample, this introduces an appreciable error in the measured force.

Recognition imaging and highly ordered molecular templating of bacterial S-layer nanoarrays containing affinity-tags.

Functional nanoarrays were fabricated using the chimeric bacterial cell surface layer (S-layer) protein rSbpA fused with the affinity tag Strep-tagII and characterized using various atomic force microscopy (AFM) techniques in aqueous environment. The accessibility of Strep-tagII was verified by single-molecule force spectroscopy studies employing Strep-Tactin as specific ligand. Simultaneous topography and recognition imaging (TREC) of the nanoarray yielded high resolution maps of the Strep-tagll binding sites with a positional accuracy of 1.5 nm. The nanoarrays were used as template for constructing highly ordered molecular binding blocks.

Functionalization of probe tips and supports for single-molecule recognition force microscopy.

The measuring tip of a force microscope can be converted into a monomolecular sensorif one or few "ligand" molecules are attached to the apex of the tip while maintainingligand function. Functionalized tips are used to study fine details of receptor-ligand interactionby force spectroscopy or to map cognate "receptor" molecules on the sample surface. Thereceptor (or target) molecules can be present on the surface of a biological specimen; alternatively,soluble target molecules must be immobilized on ultraflat supports. This review describes the methodsof tip functionalization, as well as target molecule immobilization. Silicon nitride tips, siliconchips, and mica have usually been functionalized in three steps: (1) aminofunctionalization,(2) crosslinker attachment, and (3) ligand/receptor coupling, whereby numerous crosslinkersare available to couple widely different ligand molecules. Gold-covered tips and/or supports haveusually been coated with a self-assembled monolayer, on top of which the ligand/receptor moleculehas been coupled either directly or via a crosslinker molecule. Apart from these general strategies,many simplified methods have been used for tip and/or support functionalization, even single-stepmethods such as adsorption or chemisorption being very efficient under suitable circumstances. Allmethods are described with the same explicitness and critical parameters are discussed. In conclusion,this review should help to find suitable methods for specific problems of tip and support functionalization.

Structure, cell wall elasticity and polysaccharide properties of living yeast cells, as probed by AFM.

Although the chemical composition of yeast cell walls is known, the organization, assembly, and interactions of the various macromolecules remain poorly understood. Here, we used in situ atomic force microscopy (AFM) in three different modes to probe the ultrastructure, cell wall elasticity and polymer properties of two brewing yeast strains, i.e.  Saccharomyces carlsbergensis and S. cerevisiae. Topographic images of the two strains revealed smooth and homogeneous cell surfaces, and the presence of circular bud scars on dividing cells. Nanomechanical measurements demonstrated that the cell wall elasticity of S. carlsbergensis is homogeneous. By contrast, the bud scar of S. cerevisiae was found to be stiffer than the cell wall, presumably due to the accumulation of chitin. Notably, single molecule force spectroscopy with lectin-modified tips revealed major differences in polysaccharide properties of the two strains. Polysaccharides were clearly more extended on S. cerevisiae, suggesting that not only oligosaccharides, but also polypeptide chains of the mannoproteins were stretched. Consistent with earlier cell surface analyses, these findings may explain the very different aggregation properties of the two organisms. This study demonstrates the power of using multiple complementary AFM modalities for probing the organization and interactions of the various macromolecules of microbial cell walls.

Detection, localization, and conformational analysis of single polysaccharide molecules on live bacteria.

The nanoscale exploration of microbes using atomic force microscopy (AFM) is an exciting, rapidly evolving research field. Here, we show that single-molecule force spectroscopy is a valuable tool for the localization and conformational analysis of individual polysaccharides on live bacteria. We focus on the clinically important probiotic bacterium Lactobacillus rhamnosus GG, demonstrating the power of AFM to reveal the coexistence of polysaccharide chains of different nature on the cell surface. Applicable to a wide variety of cells, this single molecule method offers exciting prospects for analyzing the heterogeneity and diversity of macromolecules constituting cell membranes and cell walls.

A new, simple method for linking of antibodies to atomic force microscopy tips.

Functionalization of atomic force microscope (AFM) tips with bioligands converts them into monomolecular biosensors which can detect complementary receptor molecules on the sample surface. Flexible PEG tethers are preferred because the bioligand can freely reorient and locally palpate the sample surface while the AFM tip is moved along. In a well-established coupling scheme [Hinterdorfer et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 3477-3481], a heterobifunctional PEG linker is used to tether thiol-containing bioligands to amino-functionalized AFM tips. Since antibodies contain no free thiol residues, prederivatization with N-succinimidyl 3-(acetylthio)propionate (SATP) is needed which causes a relatively high demand for antibody. The present study offers a convenient alternative with minimal protein consumption (e.g., 5 microg of protein in 50 microL of buffer) and no prederivatization, using a new heterobifunctional cross-linker that has two different amino-reactive functions. One end is an activated carboxyl (N-hydroxysuccinimide ester) which is much faster to react with the amino groups of the tips than the benzaldehyde function on its other end. The reactivity of the latter is sufficient, however, to covalently bind lysine residues of proteins via Schiff base formation. The method has been critically examined, using biotinylated IgG as bioligand on the tip and mica-bound avidin as complementary receptor. These experiments were well reproduced on amino-functionalized silicon nitride chips where the number of specifically bound IgG molecules (approximately 2000 per microm2) was estimated from the amount of specifically bound ExtrAvidin-peroxidase conjugate. For a bioscientific application, human rhinovirus particles were tethered to the tip, very-low-density lipoprotein receptor fragments were tethered to mica, and the specific interaction was studied by force microscopy.

Nano-scale dynamic recognition imaging on vascular endothelial cells.

Combination of high-resolution atomic force microscope topography imaging with single molecule force spectroscopy provides a unique possibility for the detection of specific molecular recognition events. The identification and localization of specific receptor binding sites on complex heterogeneous biosurfaces such as cells and membranes are of particular interest in this context. Here simultaneous topography and recognition imaging (TREC) was applied to gently fixed microvascular endothelial cells from mouse myocardium (MyEnd) to identify binding sites of vascular endothelial (VE)-cadherin, known to play a crucial role in calcium-dependent, homophilic cell-to-cell adhesion. TREC images were acquired with magnetically oscillating atomic-force microscope tips functionalized with a recombinant VE-cadherin-Fc cis-dimer. The recognition images revealed single molecular binding sites and prominent, irregularly shaped dark spots (domains) with sizes ranging from 10 to 100 nm. These domains arose from a decrease of the oscillation amplitude during specific binding between active VE-cadherin cis-dimers. The VE-cadherin clusters were subsequently assigned to topography features. TREC represents an exquisite method to quickly obtain the local distribution of receptors on cellular surface with an unprecedented lateral resolution of 5 nm.

Antibody linking to atomic force microscope tips via disulfide bond formation.

Covalent binding of bioligands to atomic force microscope (AFM) tips converts them into monomolecular biosensors by which cognate receptors can be localized on the sample surface and fine details of ligand-receptor interaction can be studied. Tethering of the bioligand to the AFM tip via a approximately 6 nm long, flexible poly(ethylene glycol) linker (PEG) allows the bioligand to freely reorient and to rapidly "scan" a large surface area while the tip is at or near the sample surface. In the standard coupling scheme, amino groups are first generated on the AFM tip. In the second step, these amino groups react with the amino-reactive ends of heterobifunctional PEG linkers. In the third step, the 2-pyridyl-S-S groups on the free ends of the PEG chains react with protein thiol groups to give stable disulfide bonds. In the present study, this standard coupling scheme has been critically examined, using biotinylated IgG with free thiols as the bioligand. AFM tips with PEG-tethered biotin-IgG were specifically recognized by avidin molecules that had been adsorbed to mica surfaces. The unbinding force distribution showed three maxima that reflected simultaneous unbinding of 1, 2, or 3 IgG-linked biotin residues from the avidin monolayer. The coupling scheme was well-reproduced on amino-functionalized silicon nitride chips, and the number of covalently bound biotin-IgG per microm2 was estimated by the amount of specifically bound ExtrAvidin-peroxidase conjugate. Coupling was evidently via disulfide bonds, since only biotin-IgG with free thiol groups was bound to the chips. The mechanism of protein thiol coupling to 2-pyridyl-S-S-PEG linkers on AFM tips was further examined by staging the coupling step in bulk solution and monitoring turnover by release of 2-pyridyl-SH which tautomerizes to 2-thiopyridone and absorbs light at 343 nm. These experiments predicted 10(3)-fold slower rates for the disulfide coupling step than actually observed on AFM tips and silicon nitride chips. The discrepancy was reconciled by assuming 10(3)-fold enrichment of protein on AFM tips via preadsorption, as is known to occur on comparable inorganic surfaces.

Ligands on the string: single-molecule AFM studies on the interaction of antibodies and substrates with the Na+-glucose co-transporter SGLT1 in living cells.

Atomic force microscopy (AFM) was used to probe topology, conformational changes and initial substrate-carrier interactions of Na+-glucose co-transporter (SGLT1) in living cells on a single-molecule level. By scanning SGLT1-transfected Chinese hamster ovary (CHO) cells with AFM tips carrying an epitope-specific antibody directed against the extramembranous C-terminal loop 13, significant recognition events could be detected. Specificity was confirmed by the absence of events in nontransfected CHO cells and by the use of free antigen and free antibody superfusion. Thus, contrary to computer predictions on SGLT1 topology, loop 13 seems to be part of the extracellular surface of the transporter. Binding probability of the antibody decreased upon addition of phlorizin, a specific inhibitor of SGLT1, suggesting a considerable conformational change of loop 13 when the inhibitor occludes the sugar translocation pathway. Using an AFM tip carrying 1-thio-D-glucose, direct evidence could be obtained that in the presence of Na+ a sugar-binding site appears on the transporter surface. The binding site accepts the sugar residue of the glucoside phlorizin, free D-glucose, and D-galactose, but not free L-glucose and probably represents the first of several selectivity filters of the transporter. This work demonstrates the potential of AFM to study the presence and dynamics of plasma membrane transporters in intact cells on the single molecule level.