Small-Angle X-ray Scattering as a Powerful Tool for Phase and Crystallinity Assessment of Monoclonal Antibody Crystallites in Support of Batch Crystallization.

Crystalline suspensions of monoclonal antibodies (mAbs) have great potential to improve drug substance isolation and purification on a large scale and to be used for drug delivery via high-concentration formulations. Crystalline mAb suspensions are expected to have enhanced chemical and physical properties relative to mAb solutions delivered intravenously, making them attractive candidates for subcutaneous delivery. In contrast to small molecules, the development of protein crystalline suspensions is not a widely used approach in the pharmaceutical industry. This is mainly due to the challenges in finding crystalline hits and the suboptimal physical properties of the resulting crystallites when hits are found. Modern advances in instrumentation and increased knowledge of mAb crystallization have, however, resulted in higher probabilities of discovering crystal forms and improving their particle properties and characterization. In this regard, physical, analytical characterization plays a central role in the initial steps of understanding and later optimizing the crystallization of mAbs and requires careful selection of the appropriate tools. This contribution describes a novel crystal structure of the antibody pembrolizumab and demonstrates the usefulness of small-angle X-ray scattering (SAXS) for characterizing its crystalline suspensions. It illustrates the advantages of SAXS when used to (i) confirm crystallinity and crystal phase of crystallites produced in batch mode; (ii) confirm crystallinity under various conditions and detect variations in crystal phases, enabling fine-tuning of the crystallizations for phase control across multiple batches; (iii) monitor the physical response and stability of the crystallites in suspension with regard to filtration and washing; and (iv) monitor the physical stability of the crystallites upon drying. Overall, this work highlights how SAXS is an essential tool for mAb crystallization characterization.

Identification of Surfactant Impact on a Monoclonal Antibody Characterization via HPLC-Separation Based and Biophysical Methods.

PURPOSE OR OBJECTIVE: Surfactants, including polysorbates and poloxamers, play a crucial role in the formulation of therapeutic proteins by acting as solubilizing and stabilizing agents. They help prevent protein aggregation and adsorption, thereby enhancing the stability of drug substance and products., However, it is important to note that utilizing high concentrations of surfactants in protein formulations can present significant analytical challenges, which can ultimately affect the product characterization. METHODS: In our study, we specifically investigated the impact of elevated surfactant concentrations on the characterization of monoclonal antibodies. We employed various analytical techniques including size-exclusion chromatography (SEC), capillary electrophoresis (CE-SDS), a cell based functional assay, and biophysical characterization. RESULTS: The findings of our study indicate that higher levels of Polysorbate 80 (PS-80) have adverse effects on the measured purity, biological activity, and biophysical characterization of biologic samples. Specifically, the elevated levels of PS-80 cause analytical interferences, which can significantly impact the accuracy and reliability of analytical studies. CONCLUSIONS: Our study results highlight a significant risk in analytical investigations, especially in studies involving the isolation and characterization of impurities. It is important to be cautious of surfactant concentrations, as they can become more concentrated during common sample manipulations like buffer exchange. Indeed, the research presented in this work emphasizes the necessity to evaluate the impact on analytical assays when there are substantial alternations in the matrix composition. By doing so, valuable insights can be gained regarding potential challenges associated with assay development and characterization of biologics with complex formulations.

Learning Relationships Between Chemical and Physical Stability for Peptide Drug Development.

PURPOSE OR OBJECTIVE: Chemical and physical stabilities are two key features considered in pharmaceutical development. Chemical stability is typically reported as a combination of potency and degradation product. Moreover, fluorescent reporter Thioflavin-T is commonly used to measure physical stability. Executing stability studies is a lengthy process and requires extensive resources. To reduce the resources and shorten the process for stability studies during the development of a drug product, we introduce a machine learning-based model for predicting the chemical stability over time using both formulation conditions as well as aggregation curves. METHODS: In this work, we develop the relationships between the formulation, stability timepoint, and the chemical stability measurements and evaluated the performance on a random test set. We have developed a multilayer perceptron (MLP) for total degradation prediction and a random forest (RF) model for potency. RESULTS: The coefficient of determination (R2) of 0.945 and a mean absolute error (MAE) of 0.421 were achieved on the test set when using MLP for total degradation. Similarly, we achieved a R2 of 0.908 and MAE of 1.435 when predicting potency using the RF model. When physical stability measurements are included into the MLP model, the MAE of predicting TD decreases to 0.148. Using a similar strategy for potency prediction, the MAE decreases to 0.705 for the RF model. CONCLUSIONS: We conclude two important points: first, chemical stability can be modeled using machine learning techniques and second there is a relationship between the physical stability of a peptide and its chemical stability.

Discovery and Control of Succinimide Formation and Accumulation at Aspartic Acid Residues in The Complementarity-Determining Region of a Therapeutic Monoclonal Antibody.

PURPOSE: Succinimide formation and isomerization alter the chemical and physical properties of aspartic acid residues in a protein. Modification of aspartic acid residues within complementarity-determining regions (CDRs) of therapeutic monoclonal antibodies (mAbs) can be particularly detrimental to the efficacy of the molecule. The goal of this study was to characterize the site of succinimide accumulation in the CDR of a therapeutic mAb and understand its effects on potency. Furthermore, we aimed to mitigate succinimide accumulation through changes in formulation. METHODS: Accumulation of succinimide was identified through intact and reduced LC-MS mass measurements. A low pH peptide mapping method was used for relative quantitation and localization of succinimide formation in the CDR. Statistical modeling was used to correlate levels of succinimide with basic variants and potency measurements. RESULTS: Succinimide accumulation in Formulation A was accelerated when stored at elevated temperatures. A strong correlation between succinimide accumulation in the CDR, an increase in basic charge variants, and a decrease in potency was observed. Statistical modeling suggest that a combination of ion exchange chromatography and potency measurements can be used to predict succinimide levels in a given sample. Reformulation of the mAb to Formulation B mitigates succinimide accumulation even after extended storage at elevated temperatures. CONCLUSION: Succinimide formation in the CDR of a therapeutic mAb can have a strong negative impact on potency of the molecule. We demonstrate that thorough characterization of the molecule by LC-MS, ion exchange chromatography, and potency measurements can facilitate changes in formulation that mitigate succinimide formation and the corresponding detrimental changes in potency.

Coated microneedles for transdermal delivery of a potent pharmaceutical peptide.

Minimally invasive delivery of peptide and protein molecules represents a significant opportunity for product differentiation and value creation versus standard injectable routes of administration. One such technology utilizes microneedle (MN) patches and it has made considerable clinical advances in systemic delivery of potent macromolecules and vaccines. A sub-class of this technology has focused on preparation of solid dense MN arrays followed by precision formulation coating on the tips of the MN. The objective of this study was to develop a drug product using the MN technology that has similar bioperformance when compared to subcutaneous route of delivery and can provide improved stability under storage. Therapeutic peptide (Peptide A, Merck & Co., Inc., Kenilworth, NJ, USA) is being developed as a subcutaneous injection for chronic dosing with a submilligram estimated therapeutic dose. Peptide A has chemical and physical stability challenges in solution and this led to exploration of a viable drug product which could provide therapeutic dosages while overcoming the stability issues seen with the compound. This work focused on developing a coated solid microstructure transdermal system (sMTS) for Peptide A followed by detailed in vitro and preclinical evaluation for two different coating formulations. Based on initial assessment, ~250 µg of Peptide A could be coated with precision on a 1.27cm2 patch which contained 316 MN's. The delivery from these systems was achieved with absolute bioavailability being similar to the subcutaneous delivery (88% and 74% for coated sMTS 1 & 2 and 75% for subcutaneous delivery). Stability of Peptide A was also found to be significantly improved when coated on the sMTS system with minimal degradation recorded at room temperature storage as compared to the subcutaneous liquid formulation. Additionally, skin irritation (on pig skin) was also measured in this study and it was found to be minimal and self-resolving. This evaluation provided a viable option for developing a drug product with improved stability and successful delivery of the investigated molecule. Graphical abstractSchematic showing uncoated sMTS, resulting product with coated peptide, successful skin penetration with high delivery efficiency and bioavailability.

Peptide Oligomerization Memory Effects and Their Impact on the Physical Stability of the GLP-1 Agonist Liraglutide.

Peptides and proteins commonly have complex structural landscapes allowing for transformation into a wide array of species including oligomers, aggregates, and fibrils. The formation of undesirable forms including aggregates and fibrils poses serious risks from the perspective of drug development and disease. Liraglutide, a GLP-1 agonist for the treatment of diabetes, is a conjugated peptide that forms oligomers that can be stabilized by pH and organic solvents. We have developed an analytical toolkit to overcome challenges inherent to Liraglutide's conjugated acyl chain and probed the impact its oligomers have on its physical stability. Our studies show that Liraglutide's oligomer states have significant and potentially detrimental impacts on its propensity to aggregate and form fibrils as well as its potency. Liraglutide delivered as a synthetic peptide is able to maintain its oligomerization state in dried lyophilized powders, acting as a memory effect from its synthetic process and purification. Through Liraglutide's oligomer memory effect, we demonstrate the importance and impact the process for synthetic peptides can have on drug development spanning from discovery to formulation development.

The metal chaperone Atox1 regulates the activity of the human copper transporter ATP7B by modulating domain dynamics.

The human transporter ATP7B delivers copper to the biosynthetic pathways and maintains copper homeostasis in the liver. Mutations in ATP7B cause the potentially fatal hepatoneurological disorder Wilson disease. The activity and intracellular localization of ATP7B are regulated by copper, but the molecular mechanism of this regulation is largely unknown. We show that the copper chaperone Atox1, which delivers copper to ATP7B, and the group of the first three metal-binding domains (MBD1-3) are central to the activity regulation of ATP7B. Atox1-Cu binding to ATP7B changes domain dynamics and interactions within the MBD1-3 group and activates ATP hydrolysis. To understand the mechanism linking Atox1-MBD interactions and enzyme activity, we have determined the MBD1-3 conformational space using small angle X-ray scattering and identified changes in MBD dynamics caused by apo-Atox1 and Atox1-Cu by solution NMR. The results show that copper transfer from Atox1 decreases domain interactions within the MBD1-3 group and increases the mobility of the individual domains. The N-terminal segment of MBD1-3 was found to interact with the nucleotide-binding domain of ATP7B, thus physically coupling the domains involved in copper binding and those involved in ATP hydrolysis. Taken together, the data suggest a regulatory mechanism in which Atox1-mediated copper transfer activates ATP7B by releasing inhibitory constraints through increased freedom of MBD1-3 motions.

Tangled web of interactions among proteins involved in iron-sulfur cluster assembly as unraveled by NMR, SAXS, chemical crosslinking, and functional studies.

Proteins containing iron-sulfur (Fe-S) clusters arose early in evolution and are essential to life. Organisms have evolved machinery consisting of specialized proteins that operate together to assemble Fe-S clusters efficiently so as to minimize cellular exposure to their toxic constituents: iron and sulfide ions. To date, the best studied system is the iron-sulfur cluster (isc) operon of Escherichia coli, and the eight ISC proteins it encodes. Our investigations over the past five years have identified two functional conformational states for the scaffold protein (IscU) and have shown that the other ISC proteins that interact with IscU prefer to bind one conformational state or the other. From analyses of the NMR spectroscopy-derived network of interactions of ISC proteins, small-angle X-ray scattering (SAXS) data, chemical crosslinking experiments, and functional assays, we have constructed working models for Fe-S cluster assembly and delivery. Future work is needed to validate and refine what has been learned about the E. coli system and to extend these findings to the homologous Fe-S cluster biosynthetic machinery of yeast and human mitochondria. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.

A canine biorelevant dissolution method for predicting in vivo performance of orally administered sustained release matrix tablets.

Preclinical species are a crucial component of drug development, but critical differences in physiology and anatomy need to be taken into account when attempting to extrapolate to humans or between species. The same is true when trying to develop oral formulations for preclinical species, especially unconventional formulations, such as sustained release tablets. During the evaluation of such specialized dosage forms, dissolution can be a critical in vitro tool used to rank-order formulations and ultimately choose the desired release rate. Here, the development of a canine biorelevant dissolution method for the prediction of the in vivo performance of sustained release matrix tablets in beagle dogs is described. The method accounts for differences in physiology between humans and dogs such as gastrointestinal fluid composition, gastric emptying forces, and gastric residence time. The most critical dissolution method parameters were found to be the paddle speed used to simulate the gastric emptying forces as well as the time spent in simulated gastric fluid. The resulting differences in method conditions are further explored through in silico models of the hydrodynamic forces applied to a dosage form. Two case studies are reported showing that the method was able to obtain excellent in vitro-in vivo relationships (slopes ranging from 1.08-1.01) which are significantly (p < 0.01-0.05) improved compared to human biorelevant dissolution used to predict in vivo performance in humans (slopes ∼1.5-1.75). The quality of the method's predictive ability allows for it to help drive the development of matrix sustained release formulations intended for preclinical studies.

The Complex Energy Landscape of the Protein IscU.

IscU, the scaffold protein for iron-sulfur (Fe-S) cluster biosynthesis in Escherichia coli, traverses a complex energy landscape during Fe-S cluster synthesis and transfer. Our previous studies showed that IscU populates two interconverting conformational states: one structured (S) and one largely disordered (D). Both states appear to be functionally important because proteins involved in the assembly or transfer of Fe-S clusters have been shown to interact preferentially with either the S or D state of IscU. To characterize the complex structure-energy landscape of IscU, we employed NMR spectroscopy, small-angle x-ray scattering (SAXS), and differential scanning calorimetry. Results obtained for IscU at pH 8.0 show that its S state is maximally populated at 25°C and that heating or cooling converts the protein toward the D state. Results from NMR and DSC indicate that both the heat- and cold-induced S→D transitions are cooperative and two-state. Low-resolution structural information from NMR and SAXS suggests that the structures of the cold-induced and heat-induced D states are similar. Both states exhibit similar (1)H-(15)N HSQC spectra and the same pattern of peptidyl-prolyl peptide bond configurations by NMR, and both appear to be similarly expanded compared with the S state based on analysis of SAXS data. Whereas in other proteins the cold-denatured states have been found to be slightly more compact than the heat-denatured states, these two states occupy similar volumes in IscU.

Role of IscX in iron-sulfur cluster biogenesis in Escherichia coli.

The Escherichia coli isc operon encodes key proteins involved in the biosynthesis of iron-sulfur (Fe-S) clusters. Whereas extensive studies of most ISC proteins have revealed their functional properties, the role of IscX (also dubbed YfhJ), a small acidic protein encoded by the last gene in the operon, has remained in question. Previous studies showed that IscX binds iron ions and interacts with the cysteine desulfurase (IscS) and the scaffold protein for cluster assembly (IscU), and it has been proposed that IscX functions either as an iron supplier or a regulator of Fe-S cluster biogenesis. We have used a combination of NMR spectroscopy, small-angle X-ray scattering (SAXS), chemical cross-linking, and enzymatic assays to enlarge our understanding of the interactions of IscX with iron ions, IscU, and IscS. We used chemical shift perturbation to identify the binding interfaces of IscX and IscU in their complex. NMR studies showed that Fe(2+) from added ferrous ammonium sulfate binds IscX much more avidly than does Fe(3+) from added ferric ammonium citrate and that Fe(2+) strengthens the interaction between IscX and IscU. We found that the addition of IscX to the IscU-IscS binary complex led to the formation of a ternary complex with reduced cysteine desulfurase activity, and we determined a low-resolution model for that complex from a combination of NMR and SAXS data. We postulate that the inhibition of cysteine desulfurase activity by IscX serves to reduce unproductive conversion of cysteine to alanine. By incorporating these new findings with results from prior studies, we propose a detailed mechanism for Fe-S cluster assembly in which IscX serves both as a donor of Fe(2+) and as a regulator of cysteine desulfurase activity.

Characterizing RNA dynamics at atomic resolution using solution-state NMR spectroscopy.

Many recently discovered noncoding RNAs do not fold into a single native conformation but sample many different conformations along their free-energy landscape to carry out their biological function. Here we review solution-state NMR techniques that measure the structural, kinetic and thermodynamic characteristics of RNA motions spanning picosecond to second timescales at atomic resolution, allowing unprecedented insights into the RNA dynamic structure landscape. From these studies a basic description of the RNA dynamic structure landscape is emerging, bringing new insights into how RNA structures change to carry out their function as well as applications in RNA-targeted drug discovery and RNA bioengineering.

Incorporation of CC steps into Z-DNA: interplay between B-Z junction and Z-DNA helical formation.

The left-handed DNA structure, Z-DNA, is believed to play important roles in gene expression and regulation. Z-DNA forms sequence-specifically with a preference for sequences rich in pyrimidine/purine dinucleotide steps. In vivo, Z-DNA is generated in the presence of negative supercoiling or upon binding proteins that absorb the high energetic cost of the B-to-Z transition, including the creation of distorted junctions between B-DNA and Z-DNA. To date, the sequence preferences for the B-to-Z transition have primarily been studied in the context of sequence repeats lacking B-Z junctions. Here, we develop a method for characterizing sequence-specific preferences for Z-DNA formation and B-Z junction localization within heterogeneous DNA duplexes that is based on combining 2-aminopurine fluorescence measurements with a new quantitative application of circular dichroism spectroscopy for determining the fraction of B- versus Z-DNA. Using this approach, we show that at least three consecutive CC dinucleotide steps, traditionally thought to disfavor Z-DNA, can be incorporated within heterogeneous Z-DNA containing B-Z junctions upon binding to the Zα domain of the RNA adenosine deaminase protein. Our results indicate that the incorporation of CC steps into Z-DNA is driven by favorable sequence-specific Z-Z and B-Z stacking interactions as well as by sequence-specific energetics that localize the distorted B-Z junction at flexible sites. Together, our results expose higher-order complexities in the Z-DNA code within heterogeneous sequences and suggest that Z-DNA can in principle propagate into a wider range of genomic sequence elements than previously thought.

Sequence-specific B-DNA flexibility modulates Z-DNA formation.

Conversion of right-handed B-DNA into left-handed Z-DNA is one of the largest structural transitions in biology that plays fundamental roles in gene expression and regulation. Z-DNA segments must form within genomes surrounded by a sea of B-DNA and require creation of energetically costly B/Z junctions. Here, we show using a combination of natural abundance NMR R(1ρ) carbon relaxation measurements and CD spectroscopy that sequence-specific B-DNA flexibility modulates the thermodynamic propensity to form Z-DNA and the location of B/Z junctions. We observe sequence-specific flexibility in B-DNA spanning fast (ps-ns) and slow (µs-ms) time scales localized at the site of B/Z junction formation. Further, our studies show that CG-repeats play an active role tuning this intrinsic B-DNA flexibility. Taken together, our results suggest that sequence-specific B-DNA flexibility may provide a mechanism for defining the length and location of Z-DNA in genomes.

Development of a Robust Manufacturing Route for Molnupiravir, an Antiviral for the Treatment of COVID-19.

Herein is described the development of a large-scale manufacturing process for molnupiravir, an orally dosed antiviral that was recently demonstrated to be efficacious for the treatment of patients with COVID-19. The yield, robustness, and efficiency of each of the five steps were improved, ultimately culminating in a 1.6-fold improvement in overall yield and a dramatic increase in the overall throughput compared to the baseline process.

Phase diagrams and water activities of aqueous dicarboxylic acid systems of atmospheric importance.

We have studied liquid/solid phase diagrams and water activities of the dicarboxylic acid/water binary systems for maleic, dl-malic, glutaric, and succinc acids using differential scanning calorimetry, infrared (IR) spectroscopy of thin films, and conductivity analysis of saturated solutions. For each binary system we report the measurements of the ice melting envelope, the acid dissolution envelope, and the ice/acid eutectic temperature and composition. Water activities have been determined by using the freezing point depression of ice. Additionally, an irreversible solid/solid phase transition for maleic acid was observed in both DSC and IR studies likely due to the conversion of a meta-stable crystal form of maleic acid to its most stable crystal form. In general we find good agreement with literature values for temperature-dependent acid solubilities.

Development and Application of a High Throughput Protein Unfolding Kinetic Assay.

The kinetics of folding and unfolding underlie protein stability and quantification of these rates provides important insights into the folding process. Here, we present a simple high throughput protein unfolding kinetic assay using a plate reader that is applicable to the studies of the majority of 2-state folding proteins. We validate the assay by measuring kinetic unfolding data for the SH3 (Src Homology 3) domain from Actin Binding Protein 1 (AbpSH3) and its stabilized mutants. The results of our approach are in excellent agreement with published values. We further combine our kinetic assay with a plate reader equilibrium assay, to obtain indirect estimates of folding rates and use these approaches to characterize an AbpSH3-peptide hybrid. Our high throughput protein unfolding kinetic assays allow accurate screening of libraries of mutants by providing both kinetic and equilibrium measurements and provide a means for in-depth ϕ-value analyses.

New and Evolving Techniques for the Characterization of Peptide Therapeutics.

Advances in technologies related to the design and manufacture of therapeutic peptides have enabled researchers to overcome the biological and technological challenges that have limited their application in the past. As a result, peptides of increasing complexity have become progressively important against a variety of disease targets. Developing peptide drug products brings with it unique scientific challenges consistent with the unique physicochemical properties of peptide molecules. The identification of the proper characterization tools is required in order to develop peptide formulations with the appropriate stability, manufacturability, and bioperformance characteristics. This knowledge supports the build of critical quality attributes and, ultimately, regulatory specifications. The purpose of this review article is to provide an overview of the techniques that are employed for analytical characterization of peptide drug products. The techniques covered are highlighted in the context of peptide drug product understanding and include chemical and biophysical approaches. Emphasis is placed on summarizing the recent literature experience in the field. Finally, the authors provide regulatory perspective on these characterization approaches and discuss some potential areas for further research in the field.

Evaluating the uncertainty in exchange parameters determined from off-resonance R1ρ relaxation dispersion for systems in fast exchange.

Spin relaxation in the rotating frame (R1ρ) is a powerful NMR technique for characterizing fast microsecond timescale exchange processes directed toward short-lived excited states in biomolecules. At the limit of fast exchange, only k(ex)=k(1)+k(-1) and Φex=p(G)p(E)(Δω)(2) can be determined from R1ρ data limiting the ability to characterize the structure and energetics of the excited state conformation. Here, we use simulations to examine the uncertainty with which exchange parameters can be determined for two state systems in intermediate-to-fast exchange using off-resonance R1ρ relaxation dispersion. R1ρ data computed by solving the Bloch-McConnell equations reveals small but significant asymmetry with respect to offset (R1ρ (ΔΩ)≠R1ρ (-ΔΩ)), which is a hallmark of slow-to-intermediate exchange, even under conditions of fast exchange for free precession chemical exchange line broadening (k(ex)/Δω>10). A grid search analysis combined with bootstrap and Monte-Carlo based statistical approaches for estimating uncertainty in exchange parameters reveals that both the sign and magnitude of Δω can be determined at a useful level of uncertainty for systems in fast exchange (k(ex)/Δω<10) but that this depends on the uncertainty in the R1ρ data and requires a thorough examination of the multidimensional variation of χ(2) as a function of exchange parameters. Results from simulations are complemented by analysis of experimental R1ρ data measured in three nucleic acid systems with exchange processes occurring on the slow (k(ex)/Δω=0.2; pE=∼0.7%), fast (k(ex)/Δω=∼10-16; p(E)=∼13%) and very fast (k(ex)=39,000 s(-1)) chemical shift timescales.

Novel entries in a fungal biofilm matrix encyclopedia.

Virulence of Candida is linked with its ability to form biofilms. Once established, biofilm infections are nearly impossible to eradicate. Biofilm cells live immersed in a self-produced matrix, a blend of extracellular biopolymers, many of which are uncharacterized. In this study, we provide a comprehensive analysis of the matrix manufactured by Candida albicans both in vitro and in a clinical niche animal model. We further explore the function of matrix components, including the impact on drug resistance. We uncovered components from each of the macromolecular classes (55% protein, 25% carbohydrate, 15% lipid, and 5% nucleic acid) in the C. albicans biofilm matrix. Three individual polysaccharides were identified and were suggested to interact physically. Surprisingly, a previously identified polysaccharide of functional importance, ß-1,3-glucan, comprised only a small portion of the total matrix carbohydrate. Newly described, more abundant polysaccharides included α-1,2 branched α-1,6-mannans (87%) associated with unbranched ß-1,6-glucans (13%) in an apparent mannan-glucan complex (MGCx). Functional matrix proteomic analysis revealed 458 distinct activities. The matrix lipids consisted of neutral glycerolipids (89.1%), polar glycerolipids (10.4%), and sphingolipids (0.5%). Examination of matrix nucleic acid identified DNA, primarily noncoding sequences. Several of the in vitro matrix components, including proteins and each of the polysaccharides, were also present in the matrix of a clinically relevant in vivo biofilm. Nuclear magnetic resonance (NMR) analysis demonstrated interaction of aggregate matrix with the antifungal fluconazole, consistent with a role in drug impedance and contribution of multiple matrix components. Importance: This report is the first to decipher the complex and unique macromolecular composition of the Candida biofilm matrix, demonstrate the clinical relevance of matrix components, and show that multiple matrix components are needed for protection from antifungal drugs. The availability of these biochemical analyses provides a unique resource for further functional investigation of the biofilm matrix, a defining trait of this lifestyle.