Hydrophobic Interaction Chromatography Method Research Articles

In-Depth Characterization for Methionine Oxidization in Complementary Domain Region by Hydrophobic Interaction Chromatography.

The oxidation of the complementarity-determining region (CDR) in monoclonal antibodies (mAbs) is a critical quality attribute that can affect the clinical efficacy and safety of recombinant mAb therapeutics. In this study, a robust hydrophobic interaction chromatography (HIC) method was developed to quantify and characterize CDR oxidation variants in mAb-A by using a Proteomix Butyl-NP5 column. The HIC analysis revealed oxidation variants that eluted earlier than the main species with weaker hydrophobicity. It was found that Met105 in the CDR was more susceptible to oxidation. Additionally, it was noted that the oxidation of Met105 on a single heavy chain resulted in elution at a distinct position compared to the oxidation on two heavy chains. This observation led to the fractionation and enrichment of the oxidized variants for further evaluation of their biofunction. The study also demonstrated that the oxidation of Met105 did not impact the antigen-binding capacity but significantly reduced the PD-1/PD-L1 blockade activity of mAb-A. The HIC method, which was employed to quantify CDR oxidation, underwent validation and was subsequently utilized for stability studies as well as for assessing the similarity between mAb-A and its reference product.

Possibility of using the imaged capillary isoelectric method as a multi-attribute method for bispecific antibodies.

An imaged capillary isoelectric focusing (icIEF)-based method was developed and validated as a multi-attribute method for a bispecific antibody (BsAb). First, as the traditional application of the icIEF method, it serves as an identity assay and purity assay for the BsAb. Second, the method can also be used as an impurity assay for the homodimer monoclonal antibodies generated during BsAb assembly. The homodimer impurity analysis for BsAb is usually done by hydrophobic interaction chromatography methods in the industry. The icIEF method has good sensitivity (down to 4µg/mL in a limit of quantitation) when UV fluorescence detection is used, which detects the native fluorescence of proteins. This is the first report that an icIEF method has been applied as impurity assay.

Structure-function relationship study for sulfated protein therapeutics using hydrophobic interaction chromatography and mass spectrometry

Protein tyrosine sulfation is a post-translational modification (PTM) that is rarely reported in recombinant therapeutic proteins. However, when sulfation does occur, the additional negative charge from the modification can influence intermolecular interactions and antigen-binding activity, making it a critical quality attribute that necessitates stringent control. In this study, we developed a unique hydrophobic interaction chromatography (HIC) method for the separation and quantification of a therapeutic bispecific antibody with varying degrees of sulfation. Despite the increased surface hydrophilicity of sulfated species, the HIC method provides enhanced retention. Baseline resolution was attained based on the degree of sulfation, independent of other PTMs such as C-terminal amidation and forced deamidation. Further structure-function relationship studies of enriched sulfated bispecific antibody species were conducted using mass spectrometry and fluorescence-linked immunosorbent assay (FLISA). These studies revealed that the tyrosine sulfation modification, which occurs in the complementarity-determining region (CDR), is a critical quality attribute and can adversely impact the antibody’s binding to its cognate antigen. The evaluation of sulfation assay using HIC method confirmed it is an effective means for controlling this critical quality attribute.

Characterization and validation of a middle-down hydrophobic interaction chromatography method to monitor methionine oxidation in IgG1

Post-translational modifications (PTMs) of therapeutic monoclonal antibodies (mAbs) can impact the efficacy of a drug. Methionine oxidation can alter the overall hydrophobicity of an antibody, thereby inducing conformational changes and affecting its biological activity. To ensure high quality, safety, and efficacy of mAbs, routine monitoring of PTMs such as methionine (Met) oxidation is essential. Met oxidation in the fragment crystallizable (Fc) region of immunoglobulin-G1 (IgG1) is a potential critical quality attribute because it impacts not only the interaction with the neonatal Fc receptor and protein A but also the half-life of mAbs in serum circulation. Although bottom-up mass spectrometry provides high site specificity, it may have limited application in quality control workflows, and its complicated sample preparation could result in procedure-induced oxidation. In this study, we describe the development and characterization of a rapid and robust middle-down hydrophobic interaction chromatography method for monitoring Met oxidation in the Fc region of IgG1. Additionally, we assessed a comprehensive method validation package and demonstrated the specificity, linearity, precision, and accuracy of the new method within a range of 3.8–37.7%. The relative quantitative data provided by this method may be used in a regulated workflow to support process and formulation development as well as in the later stages of drug development and batch release and stability studies.

Automated Hydrophobic Interaction Chromatography Screening Combined with In Silico Optimization as a Framework for Nondenaturing Analysis and Purification of Biopharmaceuticals.

The mounting complexity of new modalities in the biopharmaceutical industry entails a commensurate level of analytical innovations to enable the rapid discovery and development of novel therapeutics and vaccines. Hydrophobic interaction chromatography (HIC) has become one of the widely preferred separation techniques for the analysis and purification of biopharmaceuticals under nondenaturing conditions. Inarguably, HIC method development remains very challenging and labor-intensive owing to the numerous factors that are typically optimized by a "hit-or-miss" strategy (e.g., the nature of the salt, stationary phase chemistry, temperature, mobile phase additive, and ionic strength). Herein, we introduce a new HIC method development framework composed of a fully automated multicolumn and multieluent platform coupled with in silico multifactorial simulation and integrated fraction collection for streamlined method screening, optimization, and analytical-scale purification of biopharmaceutical targets. The power and versatility of this workflow are showcased by a wide range of applications including trivial proteins, monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), oxidation variants, and denatured proteins. We also illustrate convenient and rapid HIC method development outcomes from the effective combination of this screening setup with computer-assisted simulations. HIC retention models were built using readily available LC simulator software outlining less than a 5% difference between experimental and simulated retention times with a correlation coefficient of >0.99 for pharmaceutically relevant multicomponent mixtures. In addition, we demonstrate how this approach paves the path for a straightforward identification of first-dimension HIC conditions that are combined with mass spectrometry (MS)-friendly reversed-phase liquid chromatography (RPLC) detection in the second dimension (heart-cutting two-dimensional (2D)-HIC-RPLC-diode array detector (DAD)-MS), enabling the analysis and purification of biopharmaceutical targets.

Evaluating Nanoparticle Hydrophobicity Using Analytical Membrane Hydrophobic Interaction Chromatography.

Nanoparticle hydrophobicity is a key factor controlling the stability, adhesion, and transport of nanoparticle suspensions. Although a number of approaches have been presented for evaluating nanoparticle hydrophobicity, these methods are difficult to apply to larger nanoparticles and viruses (>100 nm in size) that are of increasing importance in drug delivery and gene therapy. This study investigated the use of a new analytical hydrophobic interaction chromatography method employing a 5.0 μm pore size polyvinylidene fluoride membrane as the stationary-phase in membrane hydrophobic interaction chromatography (MHIC). Experimental data obtained using a series of model proteins were in good agreement with literature values for the hydrophobicity (both experimental and computational). MHIC was then used to evaluate the hydrophobicity of a variety of nanoparticles, including a live attenuated viral vaccine, both in water and in the presence of different surfactants. This new method can be implemented on any liquid chromatography system, run times are typically <20 min, and the experiments avoid the use of organic solvents that could alter the structure of many biological nanoparticles.

Comparison of RNA content from hydrophobic interaction chromatography-isolated seminal plasma exosomes from intrauterine insemination (IUI) pregnancies.

Male factors account for roughly half of infertility cases, with most male infertility diagnosed as idiopathic. Researchers predicting intrauterine insemination success rates have identified multiple prognostic factors, including semen parameters and seminal fluid composition. Seminal plasma contains extracellular exosomes, which contain RNAs and proteins involved in spermatogenesis. The contents of seminal plasma exosomes may be an indicator of overall sperm quality or fertility potential; therefore, analysis of exosomes may provide a measure for sperm viability and fertilization potential. In our study, exosomes were isolated and purified from seminal plasma obtained from IUI treatments with known pregnancy outcomes. We used a unique method to isolate the exosomes which made use of the hydrophobic interaction chromatography method. RNASeq was performed on RNAs from the purified exosomes. This analysis revealed holistic trends, including increased expression associated with RNA originating from chromosomes 1, 10, 12, 16 and 21. Overall, total RNA was significantly decreased and rRNA was significantly increased in successful IUI attempts. Furthermore, we found specific mRNAs and lincRNAs associated with positive versus negative pregnancy outcomes. Our study isolated and purified seminal plasma exosomes without ultracentrifugation, and it provides further evidence for differences in seminal plasma exosome molecular contents associated with pregnancy status.

Rapid separation of blood plasma exosomes from low-density lipoproteins via a hydrophobic interaction chromatography method on a polyester capillary-channeled polymer fiber phase

Exosomes are membrane-bound, cell-secreted vesicles, with sizes ranging from 30 to 150 nm. Exosomes in blood plasma have become proposed targets as measurable indicators of disease conditions. Current methods for plasma-based exosome isolation are time-consuming, complex, and have high operational costs. One of the most commonly reported shortcomings of current isolation protocols is the co-extraction of lipoproteins (e.g. low-density lipoproteins, LDLs) with the target exosomes. This report describes the use of a rapid, single-operation hydrophobic interaction chromatography (HIC) procedure on a polyester (PET) capillary-channeled polymer (C-CP) fiber column, demonstrating the ability to efficiently purify exosomes. The method has previously been demonstrated for isolation of exosomes from diverse biological matrices, but questions were raised about the potential co-elution of LDLs. In the method described herein, a step-gradient procedure sequentially elutes spiked lipoproteins and blood plasma-originating exosomes in 10 min, with the LDLs excluded from the desired exosome fraction. Mass spectrometry (MS) was used to characterize an impurity in the primary LDL material, identifying the presence of exosomal material. Transmission electron microscopy (TEM) and an enzyme-linked immunosorbent assay (ELISA) were used to identify the various elution components. The method serves both as a rapid means of high purity exosome isolation as well as a screening tool for the purity of LDL samples with respect to extracellular vesicles.

Rapid isolation of lentivirus particles from cell culture media via a hydrophobic interaction chromatography method on a polyester, capillary-channeled polymer fiber stationary phase.

Lentiviruses are increasingly used as gene delivery vehicles for vaccines and immunotherapies. However, the purification of clinical-grade lentivirus vectors for therapeutic use is still troublesome and limits preclinical and clinical experiments. Current purification methods such as ultracentrifugation and ultrafiltration are time consuming and do not remove all of the impurities such as cellular debris, membrane fragments, and denatured proteins from the lentiviruses. The same challenges exist in terms of their analytical characterization. Presented here is the novel demonstration of the chromatographic isolation of virus particles from culture media based on the hydrophobicity characteristics of the vesicles. A method was developed to isolate lentivirus from media using a hydrophobic interaction chromatography (HIC) method performed on a polyester, capillary-channeled polymer (PET C-CP) stationary phase and a standard liquid chromatography apparatus. The method is an extension of the approach developed in this laboratory for the isolation of extracellular vesicles (EVs). Quantitative polymerase chain reaction (qPCR) was used to verify and quantify lentiviruses in elution fractions. Load and elution mobile phase compositions were optimized to affect high efficiency and throughput. The process has been visualized via scanning electron microscopy (SEM) of the fiber surfaces following media injection, the elution of proteinaceous material, and the elution of lentiviruses. This effort has yielded a rapid (<10min), low-cost (< $15 per column, providing multiple separations), and efficient method for the isolation/purification of lentivirus particles from cell culture media at the analytical scale.

Preliminary purification and expression of Asian malaria vaccine candidate recombinant erythrocyte-binding antigen 140

Background: Causing an estimated 219 million cases in 2017, malaria is a life-threatening mosquito-borne disease caused by Plasmodium parasites, with P. falciparum being the most deadly. P. falciparum antigens involved in erythrocyte recognition and invasion during the merozoite stage are likely targets of protective immune responses. Natural antibody responses against the Erythrocyte Binding Antigen 140 (EBA-140) ligand have been found in individuals in malaria-endemic areas. EBA-140, a member of the Pf erythrocyte binding antigen (EBA) family, is one of the prospective candidates for malaria vaccine development. Methods and materials: In the study, the Philippine malaria parasite strain, FCH/4, was identified using its sequences in Genbank. Erythrocyte-binding antigen 140 (EBA-140) was selected by in silico screening of putative vaccine candidates in P. falciparum using the genomic database PlasmoDB and was then synthesized as codon-optimized full-length EBA-140. This 3284 bp gene was cloned into peT30-a(+) vector, transformed and expressed in E. coli BL21(DE3) cells. The EBA-140 gene insert was confirmed via restriction enzyme digestion and its protein 3D conformation was predicted using I-TASSER. Protein expression was optimized at 28 °C induction for 17 h using 0.3 mM IPTG. Recombinant EBA-140 in the native lysate was initially run on a weak anion-exchanger and then on a hydrophobic interaction column for purification. Results: The recombinant EBA-140 was constitutively expressed in the cytosol and highly expressed in the inclusion bodies upon induction at 28 °C for 17 h using 0.3 mM IPTG. The expressed rEBA-140 was then purified by several optimization steps of anion exchange chromatography, producing a 51.9% pure protein. To further purify the protein, hydrophobic interaction chromatography was employed. Conclusion: Recombinant EBA-140 was successfully expressed and purified optimally using anion exchange chromatography. The hydrophobic interaction chromatography method as polishing step is continuously being optimized to purify the 32% hydrophobic rEBA-140. To further confirm the specificity and identity of the purified fractions, Western blot and peptide mass fingerprinting methods are being optimized. Since no vaccine is yet available for malaria, characterization studies of immunoreactive Plasmodium antigens are of significant importance.

A hydrophobic interaction chromatography method suitable for quantitating individual monoclonal antibodies contained in co-formulated drug products

A co-formulated monoclonal antibody (mAb) product containing two or more antibodies offers several therapeutic advantages. However, quantitating the individual antibodies in a co-formulated product is challenging due to the similar biochemical and biophysical properties of mAbs. To identify a method suitable to support the development of a co-formulated drug product with three mAbs, a hydrophobic interaction chromatography method was developed, utilizing a Dionex ProPac HIC-10 column, 100 mM phosphate buffer (pH 7.0), and an ammonium sulfate gradient. Compared to other methods that were evaluated, the HIC method showed the best separation, as well as accurate quantitation of the three mAbs in the co-formulated drug product. The calibration curves were linear over column loads of 225 μg to 900 μg (R2 > 0.99) and the accuracy was between 91% and 106%. Intra-day and inter-day precisions (RSD) were less than or equal to 0.6 % and 1.7%, respectively. The method was used to quantitate individual mAb concentrations in the co-formulated drug product and to monitor any changes in concentration during stability studies.

Adaptive methodology to determine hydrophobicity of nanomaterials in situ.

The hydrophobicity of nanoparticles (NPs) is a key property determining environmental fate, biological partitioning and toxicity. However, methods to characterize surface hydrophobicity are not uniformly applied to NPs and cannot quantify surface changes in complex environments. Existing methods designed to evaluate the hydrophobicity of bulk solids, chemicals, and proteins have significant limitations when applied to NPs. In this study, we modified and evaluated two methods to determine the hydrophobicity of NPs, hydrophobic interaction chromatography (HIC) and dye adsorption, and compared them to the standard octanol-water partitioning protocol for chemicals. Gold, copper oxide, silica, and amine-functionalized silica NPs were used to evaluate methods based on their applicability to NPs that agglomerate and have surface coatings. The octanol water partitioning and HIC methods both measured Au NPs as hydrophilic, but despite having a small size and stable suspension, NPs could not be fully recovered from the HIC column. For the dye adsorption method, hydrophobic (Rose Bengal) and hydrophilic (Nile Blue) dyes were adsorbed to the NP surface, and linear isotherm parameters were used as a metric for hydrophobicity. CuO was determined to be slightly hydrophilic, while SiO2 was hydrophilic and Ami-SiO2 was hydrophobic. The advantages and limitations of each method are discussed, and the dye adsorption method is recommended as the most suitable for application across broad classes of nanomaterials. The dye assay method was further used to measure changes in the surface hydrophobicity of TiO2 NPs after being suspended in natural water collected from the Alsea Rivers watershed in Oregon. TiO2 NPs adsorbed Rose Bengal when suspended in ultrapure water, but adsorbed Nile Blue after being incubated in natural water samples, demonstrating a shift from hydrophobic to hydrophilic properties on the outer surface. The dye adsorption method can be applied to characterize surface hydrophobicity of NPs and quantify environmental transformations, potentially improving environmental fate models.

Evaluation of exosome loading characteristics in their purification via a glycerol-assisted hydrophobic interaction chromatography method on a polyester, capillary-channeled polymer fiber phase.

Exosomes are membrane-secreted vesicles, with sizes ranging from 30 to 150 nm, which play key roles in intercellular communication. There is intense interest in developing methods to isolate and quantify exosomes toward clinical diagnostics, fundamental studies of intercellular processes, and use of exosomes as delivery vehicles for therapeutic agents. Current methods for exosomes isolation and quantification are time consuming and have operational high costs; few combine isolation and quantification into a singular operation unit. This report describes the use of hydrophobic interaction chromatography on a polyester capillary-channeled polymer fiber column, employing a step gradient for exosome elution, including use of glycerol as a solvent modifier. The entire procedure is completed in 8 min, while maintaining the structural integrity and biological activity of the isolated exosomes. Electron microscopy was used to verify the size and structural fidelity of single exosomes. Absorbance response curves for a commercial exosome sample were used for exosome quantification in the chromatographic separations. In order to determine the dynamic loading capacity for exosomes, different volumes of Dictyostelium discoideum cell culture milieu supernatant were loaded at different column lengths (5-30 cm) and loading flow rates (0.2-0.5 ml/min). A loading capacity of 5.4 × 1012 exosomes derived from D. discoideum milieu was obtained on a 0.8 × 300 mm column; yielding recoveries of over 80%. It is believed that this isolation and purification strategy holds many advantages toward the use of exosomes across a wide breadth of medical and biotechnology applications.

Native Hydrophobic Interaction Chromatography Hyphenated to Mass Spectrometry for Characterization of Monoclonal Antibody Minor Variants.

Conventionally, hydrophobic interaction chromatography (HIC) uses mobile phases with high salt concentration that are not compatible with mass spectrometry (MS). Here we describe development of an HIC method coupled with MS detection (HIC-MS) utilizing an aqueous mobile phase with a low concentration of a volatile salt for characterizing recombinant monoclonal antibody (mAb) post-translational modifications (PTMs). The ability of HIC to separate the oxidation and free thiol variants of the mAbs enables their isolation and rapid characterization of these attributes under native conditions, an important step toward understanding the role they play.

ADC Analysis by Hydrophobic Interaction Chromatography.

Hydrophobic interaction chromatography (HIC) is a traditional technique used for the separation, purification, and characterization of proteins. As the number of antibody-drug conjugates (ADCs) continues to increase in clinical trials, HIC and other orthogonal methods utilizing changes in hydrophobicity are being used for ADC characterization and analysis. Unlike other techniques, HIC uniquely allows for protein analysis under mild nondenaturing conditions that preserve the native structure and activity of the molecules. Analysis of the ADC in its native form is advantageous. Herein, we describe a generic HIC protocol for the screening, analysis, and characterization of ADCs using an ammonium sulfate buffer and a high-pressure liquid chromatography system. Parameters affecting data quality and interpretation are addressed. In addition, several recommendations are included for method optimization and troubleshooting.

Protocols for the analytical characterization of therapeutic monoclonal antibodies. I - Non-denaturing chromatographic techniques.

Size-, charge- and hydrophobicity-related variants of a biopharmaceutical product have to be deeply characterized for batch consistency and for the assessment of immunogenicity and safety effects. Size exclusion chromatography (SEC) and ion exchange chromatography (IEX) are considered as the gold standard for the analysis of high molecular weight species (HMWS) and charge-related variants, respectively. Hydrophobic interaction chromatography (HIC) has drawn renewed attention to monitor the small drug payload distribution in the cysteine-linked antibody-drug conjugates (ADC). These three chromatographic techniques, namely SEC, HIC and IEX, are historical, non-denaturing and robust approaches widely used for the characterization of biopharmaceutical proteins. Despite the broad spectrum of monoclonal antibodies (mAbs) structures, isoelectric points (pIs) and hydrophobicities, generic protocols can be applied to separate their size-, charge- and hydrophobicity-related variants, using the last generation of chromatographic columns and appropriate mobile phase conditions. Straightforward protocols are described in this manuscript with representative chromatograms of ten distinct Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved therapeutic mAb products to illustrate the performance of the SEC, IEX and HIC methods.

A fucose specific lectin from Aspergillus flavus induced interleukin-8 expression is mediated by mitogen activated protein kinase p38.

Aspergillus flavus is an ubiquitous, opportunistic fungus responsible to cause invasive fungal allergic diseases, including bronchopulmonary invasive aspergillosis in persons with altered immune function. Lectins have been implicated as interaction mediators between the pathogenic fungi and human host. We isolated L-fucose specific lectin from A. flavus (FFL) and purified it to homogeneity with a combination of ion exchange and hydrophobic interaction chromatography methods. Its hemagglutination activity was significantly inhibited by 125 μM L-fucose as compared to other sugars and sugar derivatives. We, then used human cell line L-132, and U937 cell line to explore the possible cytotoxicity and proinflammatory effect of this fucose-specific lectin. The lectin induced the expression of proinflammatory cytokine interleukin-8 (IL-8) in a dose-dependent manner, and it was found to be associated with the p38 mitogen activated protein kinase (MAPK). The p38MAPK signalling pathway regulates the transcription factor activator protein-1 (AP-1) activity, which is the integration point of many signals that can differentially affect the expression and transcriptional activity of a cell. We observed activation of c-Jun, a critical component of the AP-1 complex, mediated by p38MAPK upon the FFL treatment in L-132 cells. Finally, inhibition of p38MAPK by a specific inhibitor attenuates the c-Jun, suggesting the p38MAPK involvement in the c-Jun activation, which in turn transcriptionally activates the induction of IL-8 in response to the lectin. Thus, this study showed a potential lectin-mediated mechanism to modulate the immune response during host-fungus interactions.

Why working with porcine circulating serum amyloid A is a pig of a job

Serum amyloid A (SAA) is a major acute phase protein in most species, and is widely employed as a health marker. Systemic SAA isoforms (SAA1, and SAA2) are apolipoproteins synthesized by the liver which associate with high density lipoproteins (HDL). Local SAA (SAA3) isoforms are synthesized in other tissues and are present in colostrums, mastitic milk and mammary dry secretions. Of systemic SAA the bulk is monomeric and bound to HDL, and a small proportion is found in serum in a multimeric form with a buried HDL binding site. In most species, systemic SAA could easily be studied by purifying it from serum of diseased individuals by hydrophobic interaction chromatography methods. For years, we were not able to isolate systemic pig SAA using the latter methods, and found that the bulk of pig SAA did not reside in the HDL-rich serum fractions but in the soluble protein fraction mainly as a multimeric protein.Based on these surprising results, we analysed in silico the theoretical properties and predicted the secondary structure of pig SAA by using the published pig primary SAA amino acid sequence. Results of the analysis confirmed that systemic pig SAA had the highest homology with local SAA3 which in other species is the isoform associated with non-hepatic production in tissues such as mammary gland and intestinal epithelium. Furthermore, the primary sequence of the pig SAA N-terminal HDL binding site did differ considerably from SAA1/2. Secondary structure analysis of the predicted alpha–helical structure of this HDL binding site showed a considerable reduction in hydrophobicity compared to SAA1/2. Based on these results, it is argued that systemic acute phase SAA in the pig has the structural properties of locally produced SAA (SAA3). It is proposed that in pig SAA multimers the charged N-terminal sequence is buried, which would explain their different properties.It is concluded that pig systemic SAA is unique compared to other species, which raises questions about the proposed importance of acute phase SAA in HDL metabolism during inflammation in this species.

HIC resolution of an IgG1 with an oxidized Trp in a complementarity determining region

Susceptibility of tryptophan (Trp) in a complementarity-determining region (CDR) to oxidation is a significant issue for recombinant monoclonal antibody (mAb) therapeutics due to the clinical efficacy and stability concerns. Here we present a case study using hydrophobic interaction chromatography (HIC) to separate an oxidized Trp containing population of an IgG1. The best separation was achieved using dual Dionex ProPac HIC-10 columns, and the oxidized Trp population was isolated as a separated pre-peak. Peptide map analysis revealed that the oxidized Trp is located in a heavy chain CDR. In addition, the HIC method was capable of monitoring the oxidation status of the CDR Trp, as the oxidation rate of the CDR Trp measured by HIC directly correlated with the results of the peptide maps. The same method conditions were also capable of separating oxidized methionine (Met) and isomerization/deamidation products, which co-elute as another pre-peak at a different retention time from the oxidized Trp species. These observations indicate that the HIC procedure can be utilized to monitor the oxidative status of the CDR Trp in the IgG1.

ADP Competes with FAD Binding in Putrescine Oxidase

Putrescine oxidase from Rhodococcus erythropolis NCIMB 11540 (PuO(Rh)) is a soluble homodimeric flavoprotein of 100 kDa, which catalyzes the oxidative deamination of putrescine and some other aliphatic amines. The initial characterization of PuO(Rh) uncovered an intriguing feature: the enzyme appeared to contain only one noncovalently bound FAD cofactor per dimer. Here we show that this low FAD/protein ratio is the result of tight binding of ADP, thereby competing with FAD binding. MS analysis revealed that the enzyme is isolated as a mixture of dimers containing two molecules of FAD, two molecules ADP, or one FAD and one ADP molecule. In addition, based on a structural model of PuO(Rh) that was built using the crystal structure of human monoamine oxidase B (MAO-B), we constructed an active mutant enzyme, PuO(Rh) A394C, that contains covalently bound FAD. These findings show that the covalent FAD-protein linkage can be formed autocatalytically and hint to a new-found rationale for covalent flavinylation: covalent flavinylation may have evolved to prevent binding of ADP or related cellular compounds, which would prohibit formation of flavinylated and functional enzyme.