QuantiSpec — Quantitative mass spectrometry data analysis of N-metabolically labeled proteins

For relative protein quantitation by mass spectrometry we metabolically labeled E. coli bacteria with ¹⁵N-enriched diets. Proteins extracted from ¹⁵N-labeled and unlabeled E. coli bacteria were mixed, separated by two-dimensional gel electrophoresis and enzymatically digested. The resulting tryptic peptides were analyzed by MALDI mass spectrometry. For the relative protein quantitation we developed fully automated software, QuantiSpec (Quantitative Mass Spectrometry Analysis Software), which uses data from MALDI TOF mass spectrometry and the Mascot database search engine. QuantiSpec detects natural as well as partially or fully labeled peptide isotope distributions. For each identified peptide the ¹⁵N incorporation rate is determined by comparing the experimental to a set of theoretical isotope patterns based on the peptide sequence. Relative quantitation is accomplished by calculating the signal intensity ratios for each ¹⁴N/¹⁵N peptide pair.     

Enhancement of antitumor effect by peptide vaccine therapy in combination with anti-CD4 antibody: Study in a murine model

PurposeThe clinical efficacy of cancer peptide vaccine therapy is insufficient. To enhance the anti-tumor effect of peptide vaccine therapy, we combined this therapy with an anti-CD4 mAb (GK1.5), which is known to deplete CD4⁺ cells, including regulatory T cells (Tregs).MethodsTo determine the treatment schedule, the number of lymphocyte subsets in the peripheral blood of mice was traced by flow cytometry after administration of anti-CD4 mAb. The ovalbumin (OVA)_257–264 peptide vaccine was injected intradermally and anti-CD4 mAb was administered intraperitoneally into C57BL/6 mice at different schedules. We evaluated the enhancement of OVA peptide-specific cytotoxic T lymphocyte (CTL) induction in the combination therapy using the ELISPOT assay, CD107a assay, and cytokine assay. We then examined the in vivo metastasis inhibitory effect by OVA peptide vaccine therapy in combination with anti-CD4 mAb against OVA-expressing thymoma (EG7) in a murine liver metastatic model.ResultsWe showed that peptide-specific CTL induction was enhanced by the peptide vaccine in combination with anti-CD4 mAb and that the optimized treatment schedule had the strongest induction effect of peptide-specific CTLs using an IFN-γ ELISPOT assay. We also confirmed that the CD107a⁺ cells secreted perforin and granzyme B and the amount of IL-2 and TNF produced by these CTLs increased when the peptide vaccine was combined with anti-CD4 mAb. Furthermore, metastasis was inhibited by peptide vaccines in combination with anti-CD4 mAb compared to peptide vaccine alone in a murine liver metastatic model.ConclusionThe use of anti-CD4 mAb in combination with the OVA peptide vaccine therapy increased the number of peptide-specific CTLs and showed a higher therapeutic effect against OVA-expressing tumors. The combination with anti-CD4 mAb may provide a new cancer vaccine strategy.     

Analysis of monoclonal antibody product heterogeneity resulting from alternate cleavage sites of signal peptide

Signal peptides used in biosynthesis of proteins are cleaved at a very specific site by signal peptidase during posttranslational translocation of cytoplasmic proteins across the membrane. In some cases, however, there can be cleavage at nonspecific sites, giving rise to heterogeneity in the mature protein, which manifests itself as either elongation or truncation of the N terminus of the mature protein. When used as biopharmaceutical therapeutics, such heterogeneities may be a cause for concern, depending on the nature of the heterogeneity. This article describes the determination of such heterogeneity by peptide mapping in both the heavy chain and the light chain (LC) of a Chinese hamster ovary (CHO) cell-expressed monoclonal antibody (mAb). The peptide map method described here was capable of detecting the extended N-terminal peptides at levels as low as 1% relative to the peak area of the intact N-terminal peptide. The LC of a mAb product was truncated at its N termini by two amino acid residues at approximately 3-4% levels, resulting from alternate signal peptide cleavage. This article describes the quantitation of this truncation by liquid chromatography-mass spectrometry (LC-MS) peptide mapping. Also described is analysis and characterization of LC truncation by reduced and denatured capillary electrophoresis in sodium dodecyl sulfate (CE-SDS). The truncated mAb, which was devoid of the two N-terminal amino acids, was engineered and shown to migrate as the “pre-LC” peak in reduced CE-SDS assay. The amount of the pre-LC peak recovered from the CE-SDS assay was shown to correlate with the amount of truncated peptide observed from the reduced and alkylated peptide map of the engineered mAb.     

Platform development for expression and purification of stable isotope labeled monoclonal antibodies in Escherichia coli

The widespread use of monoclonal antibodies (mAbs) as a platform for therapeutic drug development in the pharmaceutical industry has led to an increased interest in robust experimental approaches for assessment of mAb structure, stability and dynamics. The ability to enrich proteins with stable isotopes is a prerequisite for the in-depth application of many structural and biophysical methods, including nuclear magnetic resonance (NMR), small angle neutron scattering, neutron reflectometry, and quantitative mass spectrometry. While mAbs can typically be produced with very high yields using mammalian cell expression, stable isotope labeling using cell culture is expensive and often impractical. The most common and cost-efficient approach to label proteins is to express proteins in Escherichia coli grown in minimal media; however, such methods for mAbs have not been reported to date. Here we present, for the first time, the expression and purification of a stable isotope labeled mAb from a genetically engineered E. coli strain capable of forming disulfide bonds in its cytoplasm. It is shown using two-dimensional NMR spectral fingerprinting that the unlabeled mAb and the mAb singly or triply labeled with ¹³C, ¹⁵N, ²H are well folded, with only minor structural differences relative to the mammalian cell-produced mAb that are attributed to the lack of glycosylation in the Fc domain. This advancement of an E. coli-based mAb expression platform will facilitate the production of mAbs for in-depth structural characterization, including the high resolution investigation of mechanisms of action.     

QuantiSpec — Quantitative mass spectrometry data analysis of 15N-metabolically labeled proteins

For relative protein quantitation by mass spectrometry we metabolically labeled E. coli bacteria with ¹⁵N-enriched diets. Proteins extracted from ¹⁵N-labeled and unlabeled E. coli bacteria were mixed, separated by two-dimensional gel electrophoresis and enzymatically digested. The resulting tryptic peptides were analyzed by MALDI mass spectrometry. For the relative protein quantitation we developed fully automated software, QuantiSpec (Quantitative Mass Spectrometry Analysis Software), which uses data from MALDI TOF mass spectrometry and the Mascot database search engine. QuantiSpec detects natural as well as partially or fully labeled peptide isotope distributions. For each identified peptide the ¹⁵N incorporation rate is determined by comparing the experimental to a set of theoretical isotope patterns based on the peptide sequence. Relative quantitation is accomplished by calculating the signal intensity ratios for each ¹⁴N/¹⁵N peptide pair.     

Application of Absorption Modeling to Predict Bioequivalence Outcome of Two Batches of Etoricoxib Tablets

As part of the overall product development and manufacturing strategy, pharmaceutical companies routinely change formulation and manufacturing site. Depending on the type and level of change and the BCS class of the molecule, dissolution data and/or bioequivalence (BE) may be needed to support the change for immediate release dosage forms. In this report, we demonstrate that for certain weakly basic low-solubility molecules which rapidly dissolve in the stomach, absorption modeling could be used to justify a BE study waiver even when there is failure to show dissolution similarity under some conditions. The development of an absorption model for etoricoxib is described here, which was then used to a priori predict the BE outcome of tablet batches manufactured at two sites. Dissolution studies in 0.01 N HCl media (pH 2.0) had demonstrated similarity of etoricoxib tablets manufactured at two different sites. However, dissolution testing at pH 4.5 and pH 6.8 media failed to show comparability of the tablets manufactured at the two sites. Single simulations and virtual trials conducted using the 0.01 N HCl dissolution showed similarity in AUC and C_max for all tablet strengths for batches manufactured at the two manufacturing sites. These predicted results were verified in a definitive bioequivalence study, which showed that both tablet batches were bioequivalent. Since the development of traditional in vitro–in vivo correlations (IVIVC) for immediate release (IR) products is challenging, in cases such as etoricoxib, absorption modeling could be used as an alternative to support waiver of a BE study.KEY WORDS: bioequivalence, dissolution, modeling, pharmacokinetics, SUPAC     

P23-M Limit of Quantitation for Low-Abundance Proteins in Plasma by Targeted MS

Biomarker discovery results in the creation of candidate lists of potential markers that must be subsequently verified in plasma.¹ The most mature methods at present require abundant protein depletion and fractionation at the protein/peptide levels in order to detect and quantitate low ng/mL concentrations of plasma proteins by stable isotope dilution mass spectrometry. Sample-processing methods with sufficient throughput, recovery, and reproducibility to enable robust detection and quantitation of candidate bio-marker proteins were evaluated by adding five non-native proteins to immunoaffinity-depleted female plasma at varying concentrations (1000, 100, 50, 25, and 10 ng/mL). Each protein was monitored by one or more representative synthetic tryptic peptides labeled with [¹³C₆]leucine or [¹³C₅] valine. Following reduction, carbamidomethylation, and enzymatic digestion, two separate processing paths were compared. In path 1, digested plasma was diluted 1:10 and [¹³C] internal standards were added just prior to direct analysis by multiple reaction monitoring with LC-MS/MS (MRM LC-MS/MS). In path 2, peptides were separated by strong cation exchange, and [¹³C] internal standards were added to corresponding SCX fractions prior to analysis by MRM LC-MS/MS. Detection and quantitation by MRM used the response of at least two product ions from each of the signature peptides. Using processing path 1, we achieved detection and quantitation down to 50 ng/mL in depleted plasma. However, using processing path 2, we achieved detection and quantitation of all spiked proteins, including the non-native protein at 10 ng/mL. While analysis of non-fractionated plasma achieved higher recovery of those proteins detected in both processes, SCX fractionation at the peptide level clearly increases detection and LOQs for potential biomarker proteins in plasma.     

In vivo D2O labeling to quantify static and dynamic changes in cholesterol and cholesterol esters by high resolution LC/MS

High resolution LC/MS-MS and LC/APPI-MS methods have been established for the quantitation of flux in the turnover of cholesterol and cholesterol ester. Attention was directed toward quantifying the monoisotopic mass (M0) and that of the singly deuterated labeled (M+1) isotope. A good degree of isotopic dynamic range has been achieved by LC/MS-MS ranging from 3-4 orders of magnitude. Correlation between the linearity of GC/MS and LC atmospheric pressure photoionization (APPI)-MS are complimentary (r² = 0.9409). To prove the viability of this particular approach, male C57Bl/6 mice on either a high carbohydrate (HC) or a high fat (HF) diet were treated with ²H₂O for 96 h. Gene expression analysis showed an increase in the activity of stearoyl-CoA desaturase (Scd1) in the HC diet up to 69-fold (P < 0.0008) compared with the HF diet. This result was supported by the quantitative flux measurement of the isotopic incorporation of ²H into the respective cholesterol and cholesterol ester (CE) pools. We concluded that it is possible to readily obtain static and dynamic measurement of cholesterol and CEs in vivo by coupling novel LC/MS methods with stable isotope-based protocols.     

Fucosebeta-1-P-Ser is a new type of glycosylation: using antibodies to identify a novel structure in Dictyostelium discoideum and study multiple types of fucosylation during growth and development

Three antibodies that recognize distinct fucose epitopes were used to study fucosylation during growth and development of Dictyostelium discoideum. mAb83.5 is known to recognize an undefined "fucose epitope" on several proteins with serine-rich domains, while mAb CAB4, and a component of anti-horse-radish peroxidase, specifically recognize Fucalpha1,6GlcNAc and Fucalpha1,3GlcNAc residues respectively in the core of N-linked oligosaccharides. We show that mAb 83.5 defines a new type of O-glycosylation. Serine-containing peptides incubated with GDPbeta[3H]Fuc and microsomes formed two fucosylated products. A neutral product accounting for 30% of the label did not react with the antibody, while the rest of the label was incorporated into a charged product which contained all the mAb83.5 reactive material. beta- Elimination of the labeled peptide or endogenous products produced [3H]Fuc-1-P, indicating phosphodiester linkage to serine. Fucbeta-1-P and GDP-betaFuc at 100 microM blocked mAb83.5 binding to endogenous and peptide products, but their alpha-linked anomers did not. Electrospray ionization mass spectra of the neutral and anionic labeled products showed major peaks of mass units corresponding to O-Fuc-Ser peptide and O-Fuc-phospho-Ser peptide, respectively. The activity of Fuc- phosphotransferase exactly paralleled the accumulation of reactive glycans during growth and development. The expressions of N-glycan core Fucalpha1,6GlcNAc and Fucalpha1,3GlcNAc and their respective fucosyl transferase activities were also synchronous, but their developmental regulation differed from one another. Fucalpha1, 6GlcNAc was expressed maximally during growth but declined during development. In contrast core Fucalpha1,3GlcNAc epitopes were expressed almost exclusively during development. These findings provide direct evidence for a novel type of O-phosphofucosylation, demonstrate the existence of an O- fucosyl transferase, and identify two different types of core fucosylation in the N-glycans of Dictyostelium.      

Solid-state 13C NMR spectroscopy of a 13C carbonyl-labeled polypeptide

High resolution structural elucidation of macromolecular structure by solid-state nuclear magnetic resonance requires the preparation of uniformly aligned samples that are isotopically labeled. In addition, to use the chemical shift interaction as a high resolution constraint requires an in situ tensor characterization for each site of interest. For ¹³C in the peptide backbone, this characterization is complicated by the presence of dipolar coupled ¹⁴N from the peptide bond. Here the ¹³C₁-Gly₂ site in gramicidin A is studied both as a dry powder and in a fully hydrated lipid bilayer environment. Linewidths reported for the oriented samples are a factor of five narrower than those reported elsewhere, and previous misinterpretations of the linewidths are corrected. The observed frequency from oriented samples is shown to be consistent with the recently determined structure for this site in the gramicidin backbone. It is also shown that, whereas a dipolar coupling between ¹³C and ¹⁴N is apparent in dry preparations of the polypeptide, in a hydrated bilayer the dipolar coupling is absent, presumably due to a `self-decoupling' mechanism.     

Cholera-toxin-dependent ADP-ribosylation of the adenylate cyclase regulatory protein in turkey erythrocyte membranes

Incubation of turkey erythrocyte membranes with cholera toxin and [³²P]NAD caused toxin-dependent incorporation of ³²P into a 42,000 M_r peptide which could be distinguished from toxin-independent ³²P incorporation into other membrane proteins. The radiolabeled 42,000 M_r peptide could be extracted from the membranes using Lubrol PX. When toxin-treated membranes were incubated with isoproterenol and GMP before detergent solubilization, the 42,000 M_r labeled peptide was adsorbed by GTP-γ-agarose which, with the same conditions, adsorbed the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide and guanine nucleotide regulatory protein activity were coeluted from the affinity matrix by guanylyl-β,γ-imidodiphosphate, GDP, and GMP. Guanosine 5′-O-(2-thiodiphosphate), an analog of GDP which blocks guanine nucleotide- and fluoride-stimulated adenylate cyclase activity, caused elution of labeled peptide which exhibited no regulatory protein activity. Our data support the view that the 42,000 M_r peptide is part of the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide allows identification of both active and inactive regulatory protein and should be useful in monitoring the purification of the regulatory protein from turkey erythrocytes.     

Thy-1-Mediated phosphatidylinositol turnover in cultured rat glomerular mesangial cell

Thy-1 glycoprotein is expressed in rat glomerular mesangial cells, and anti-Thy-1 nephritis induced by anti-Thy-1 antibodies is a model of human renal diseases. In this study, we examined Thy-1-mediated biological reactions in cultured rat glomerular mesangial cells utilizing two anti-Thy-1 monoclonal antibodies (mAbs), 1-22-3 and OX-7. Incubation of the cells with these mAbs resulted in increased inositol trisphosphate (IP₃) levels. The rise in IP₃ produced by mAb 1-22-3 was greater than that produced by mAb OX-7 at the same dose. Incubation of mesangial cells with these mAbs resulted in an increase in the intracellular free calcium concentration ([Ca²⁺]i). mAb 1-22-3 induced a sustained increase in [Ca²⁺]i, while that induced by mAb OX-7 lasted 1-2 min, then decreased to the basal level. An transient increase in [Ca²⁺]i was also observed in Ca²⁺-free medium, indicating that these [Ca²⁺]i increases are due to release of Ca²⁺ from internal stores by IP₃ without calcium flux across cell membrane. When cells were pretreated with protein tyrosine kinase (PTK) inhibitors (herbimycin A or genistein), Thy-1-mediated increases in [Ca²⁺]i were inhibited. These data suggest that Thy-1 induces the production of IP₃ (including inositol 1,4,5-triphosphate, an intracellular Ca²⁺-releasing factor) and that PTKs may contribute to the Thy-1-mediated elevation of [Ca²⁺]i which presumably results from phospholipase C activation following Thy-1-mediated signaling in rat mesangial cells. © 1996 Wiley-Liss, Inc.     

In vitro and in vivo efficacy of anti‐chikungunya virus monoclonal antibodies produced in wild‐type and glycoengineered Nicotiana benthamiana plants

Chikungunya virus (CHIKV) is a mosquito‐transmitted alphavirus, and its infection can cause long‐term debilitating arthritis in humans. Currently, there are no licensed vaccines or therapeutics for human use to combat CHIKV infections. In this study, we explored the feasibility of using an anti‐CHIKV monoclonal antibody (mAb) produced in wild‐type (WT) and glycoengineered (?XFT) Nicotiana benthamiana plants in treating CHIKV infection in a mouse model. CHIKV mAb was efficiently expressed and assembled in plant leaves and enriched to homogeneity by a simple purification scheme. While mAb produced in ?XFT carried a single N‐glycan species at the Fc domain, namely GnGn structures, WT produced mAb exhibited a mixture of N‐glycans including the typical plant GnGnXF₃ glycans, accompanied by incompletely processed and oligomannosidic structures. Both WT and ?XFT plant‐produced mAbs demonstrated potent in vitro neutralization activity against CHIKV. Notably, both mAb glycoforms showed in vivo efficacy in a mouse model, with a slight increased efficacy by the ?XFT‐produced mAbs. This is the first report of the efficacy of plant‐produced mAbs against CHIKV, which demonstrates the ability of using plants as an effective platform for production of functionally active CHIKV mAbs and implies optimization of in vivo activity by controlling Fc glycosylation.     

Possible identity of a membrane-bound with a soluble cyclic AMP-independent erythorocyte protein kinase that phosphorylates spectrin

A soluble casein kinase isolated and purified to homogeneity from the human erythrocyte cytosol by phosphocellulose and Sephadex G-200 chromatographies is indistinguishable from the membrane-bound casein (spectrin_kinase according and site-specificity criteria. The soluble enzyme shows an M_r of about 30 000 by gel filtration and comigrates with the purified membrane spectrin kinase as a single polypeptide of 32 000 Da on sodium dodecyl sulfate polyacrylamide gels. The soluble kinase phosphorylates spectrin in situ in spectrin kinase-depleted ghosts and catalyzes the in vitro phosphorylation of partially dephosphorylated spectrin with saturation kinetics identical to those displayed by the membrane spectrin kinase. When component 2 of spectrin that has been phosphorylated with [γ-³²P]ATP by either the soluble or the membrane kinases was subjected to limited proteolysis, the same 21500 Da papain-generated phosphopeptide was found to have been produced by the two enzymes. The same 21 500 Da phosphopeptide was identified after papain digestion of spectrin isolated from intact cells that had been incubated with ³²P_i. However, this particular peptide was not labeled in spectrin that had been phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase. Identical phosphopeptide patterns were obtained by gel filtration and two-dimensional peptide maps of trypsin-cleaved component 2 of spectrin that had been labeled in situ, in intact ghosts or in spectrin kinase-depleted ghosts supplemented with the soluble kinase. These findings indicate a possible identity of the soluble with the membrane-bound casein (spectrin) kinase.     

The adenine pocket of a single catalytic site is derivatized when the bovine heart mitochondrial F1-ATPase is photoinactivated with 4-amino-1-octylquinaldinium

The bovine heart mitochondrial F₁-ATPase (MF₁) is reversibly inhibited in the dark by 4-amino-1-octylquinaldinium (AOQ) with an I_0.5 value of 48 μM. When irradiated in the presence of AOQ, MF₁ is photoinactivated with an apparent K_d of 12 μM. About 1.1 mol of [³H]AOQ were incorporated per mol of MF₁ on complete photoinactivation. Fractionation of a cyanogen bromide digest of MF₁ photolabeled with [³H]AOQ followed by fractionation of peptic digests of partially purified cyanogen bromide fragments led to isolation of two CNBr/peptic fragments labeled with³H. Sequence analysis of the labeled peptides revealed that one contained residues 423–441 of the β subunit. A gap in position 2 of the sequence indicates that βPhe424 is derivatized. The phenyl side-chain of this residue is part of a pocket that binds the adenine moiety of ATP or ADP at catalytic sites. The other peptide, which was labeled to a greater extent, contained residues 342–358 of the β subunit, but in this case, no gap was found in the sequence indicating that the derivatized amino-acid side-chain might not have survived the conditions of automatic Edman degradation. This peptide contains βTyr345, the side-chain of which is also a component of the pocket that binds the adenine moiety of ATP or ADP to catalytic sites. However, for the reason stated, there is no direct evidence that βTyr345 is labeled in this peptide.     

The incorporation of Na2 75SeO3 into sheep erythrocytes

Injection of Na₂ ⁷⁵SeO₃ into untreated, phlebotomized, and phenylhydrazine treated sheet has shown that the rate of⁷⁵SeO₃-incorporation into erythrocytes is dependent on the degree of stimulation. Analysis of labeled erythrocytes by gel filtration and twodimensional electrophoresis has indicated that the transient labeling of a hemoglobin-like peptide is the only protein labeled in addition to glutathione peroxidase.     

Characterization of recombinant scFv antibody reactive with an apical antigen of Eimeria acervulina

The chicken monoclonal antibody (mAb), 6D-12-G10, reacts with an apical complex protein at the anterior tip of E. acervulina sporozoites that inhibits parasite invasion in vitro. Because this mAb was produced at low amount from the original hybridoma cells, an scFv antibody was constructed by amplification of the corresponding V _H and V _L genes and expressed in E. coli. The scFv antibody was produced at a minimum of 7 mg l^–1 and exhibited virtually identical antigen reactivity as the original mAb.     

Phosphohistidine as a stoichiometric intermediate in reactions catalyzed by isoenzymes of wheat germ acid phosphatase.

Burst titration experiments conducted on a highly purified isoenzyme of wheat germ acid phosphatase under conditions where [S]_o > K_m indicate that there is one titratable active site per molecule of enzyme of molecular weight 59,000. The enzyme is labeled to only a small extent with inorganic [³²P]phosphate ion. Incubation of wheat germ acid phosphatase with ³²P-labeled substrates such as p-nitrophenyl phosphate or inorganic pyrophosphate followed by quenching in alkali results in the stoichiometric trapping of a base-stable, acid-labile phosphorylated protein. The extent of ³²P incorporation parallels the degree of purity of the enzyme and corresponds to the incorporation of 1 mol of phosphate per mole of enzyme. The incorporation is eliminated by the simultaneous presence of excess unlabeled phosphate ion (a competitive inhibitor) and is not observed when a noncatalytic protein (such as bovine serum albumin) is substituted for the enzyme. Complete alkaline hydrolysis of the labeled protein results in the recovery of an 85% yield of τ-phosphohistidine, identified by ion-exchange chromatography, high-voltage paper electrophoresis, and comparison with a synthetic sample. A ³²P-labeled tryptic tetradecapeptide was isolated following hydrolysis of the labeled, reduced, and carboxymethylated protein with trypsin at pH 8.3, separation of the labeled peptide, and purification by two methods including a novel variant of a diagonal electrophoresis technique. The end groups and composition of the peptide are reported. The data are consistent with the interpretation that a phosphohistidine-enzyme intermediate is formed as an obligatory intermediate in the catalytic reaction involving this enzyme.     

Site-specific labeling of ‘second generation’ annexin V with 99mTc(CO)3 for improved imaging of apoptosis in vivo

In this study ‘second generation’ AnxV was specifically labeled with ^99mTc in three different ways outside the binding region of the protein to obtain an improved target-to-background activity ratio. The compounds were tested in vitro and in vivo in normal mice and in a model of hepatic apoptosis (anti-Fas mAb). The apoptosis binding was most prominent for the HIS-tagged ‘second generation’ AnxV labeled with ^99mTc(CO)₃ in comparison to ^99mTc-HYNIC-cys-AnxV and ^99mTc(CO)₃-DTPA-cys-AnxV.     

Localized conformational interrogation of antibody and antibody-drug conjugates by site-specific carboxyl group footprinting

Establishing and maintaining conformational integrity of monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) during development and manufacturing is critical for ensuring their clinical efficacy. As presented here, we applied site-specific carboxyl group footprinting (CGF) for localized conformational interrogation of mAbs. The approach relies on covalent labeling that introduces glycine ethyl ester tags onto solvent-accessible side chains of protein carboxylates. Peptide mapping is used to monitor the labeling kinetics of carboxyl residues and the labeling kinetics reflects the conformation or solvent-accessibility of side chains. Our results for two case studies are shown here. The first study was aimed at defining the conformational changes of mAbs induced by deglycosylation. We found that two residues in C_H2 domain (D268 and E297) show significantly enhanced side chain accessibility upon deglycosylation. This site-specific result highlighted the advantage of monitoring the labeling kinetics at the amino acid level as opposed to the peptide level, which would result in averaging out of highly localized conformational differences. The second study was designed to assess conformational effects brought on by conjugation of mAbs with drug-linkers. All 59 monitored carboxyl residues displayed similar solvent-accessibility between the ADC and mAb under native conditions, which suggests the ADC and mAb share similar side chain conformation. The findings are well correlated and complementary with results from other assays. This work illustrated that site-specific CGF is capable of pinpointing local conformational changes in mAbs or ADCs that might arise during development and manufacturing. The methodology can be readily implemented within the industry to provide comprehensive conformational assessment of these molecules.