Please select what you would like included for printing: Copy the text below and then paste that into your favorite email application This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply Service map data © OpenStreetMap contributors Erin and Erik Luckau like to keep visual reminders of their four sons with them at work the Luckaus have something different — 3D-printed figurines of their boys playing a game of chicken fight The figurines are a product of 3D Aloha Studios the new business that the Luckaus opened in Makawao in April and the first of what they hope will be several 3D-photo studios in Hawaii “Nowadays people take so many photographs on their phone they don’t really look at them,” said Erik Luckau “But with this on your desk or on your kitchen cabinet The Luckaus got a firsthand experience with the technology while on a business trip to Seattle in August 2016 Erin Luckau and the boys were walking around Seattle when they came across a store that did 3D printing She decided to take the boys in for a photo and it’s fun to see them growing,” she said Erin Luckau “couldn’t get it out of my head” and started making some calls The Luckaus took a trip to Berkeley to meet the inventors behind the technology learned more about the 3D printing process and decided to go into business Last year they purchased their first 3D scanner a circular apparatus that consists of several towers equipped with 90 cameras that capture subjects from all angles they had always liked the mission of the Ark Encounter that features a life-sized replica of Noah’s Ark as described in the Bible They proposed setting up the scanner at the park so visitors could purchase and print 3D figurines of themselves They would own the business and handle maintenance of the scanner; the park would operate it the goal had always been to move back home to Maui After getting married in 2004 and having their first child and in April they opened 3D Aloha Studios in Makawao just across the street from Casanova Italian Restaurant While the process of creating the figurines takes time Customers step into the center of the 3D scanner the Luckaus snap some photos and then a computer spends eight hours pulling the 2D images together into a single 3D image It takes three to four hours to then print the figurines out of sandstone polymer but it’s durable,” Erik Luckau said “That’s why we fell in love with it but you don’t want a crumbly product that fades.” A close look at the figurines at 3D Aloha Studios reveals details as intricate as tattoos on a man’s arm the patterns and wrinkles on a pair of dark shirts and even a slight gap in the smile of one of the Luckau boys who had lost a tooth “We like to say that this is the future of photography,” Erik Luckau said explaining that the 3D figurines can capture body language and people’s relationship to each other “at a deeper level than anything’s possible before.” Local orders are printed in Honolulu and shipped to the customer The figurines can also be printed at a facility closest to the customer Depending on how many orders the Luckaus have it can take two to three weeks to arrive at the customer’s doorstep “If you don’t want one of yourself there’s probably someone who wants one of you,” Erin Luckau said The new business is a change of pace for the Luckaus Erik Luckau was an attorney for The AES Corp. while Erin Luckau worked as a curatorial assistant at the National Gallery of Art in Washington they also ran a gourmet cheese-and-wine shop While both said they loved their former jobs the trade-offs of living on Maui and getting more time with their kids — now ages 5 our life just got centered around the idea that you only have your kids for so long,” Erik Luckau said “And if you’re busy during those 10 years is the same reason we do this business — because we want to help other people capture the memories of their kids while they can.” Erin Luckau said they also “want to show our kids what it’s like being an entrepreneur.” The new business also fulfills Erin Luckau’s dreams of working in an art gallery 3D Aloha Studios also features local artists or fish printing pieces by Makawao artist Brian Heustis of Maui Fish Printing; oil paintings by Kihei artist and former Canadian windsurfing champion Bill Sanders; and photography by Lara Fortier The Luckaus hope to eventually expand their business to other locations Up to four people can be in the photo; babies or small pets that can be held in a person’s arms are not counted Large items such as a surfboard or bigger pets do count Prices range from $125 for a 5-inch statue of one person to $995 for a 9-inch statue of four people Erik Luckau explained that they don’t get the discount that comes with ordering in bulk; it’s like printing 100 shirts but each one with a custom design He said they’re working on getting the prices down but pointed out that private photography sessions can also cost hundreds of dollars and I do imagine prices will go down over time,” he said * Colleen Uechi can be reached at cuechi@mauinews.com A grant information session for Maui County’s recycling grants program will be at 2 p.m In light of increased property values driving up tax payments the Maui County Council’s budget committee has .. Copyright © 2025 Maui News Publishing Company LTD | https://www.mauinews.com | 100 Mahalani Street Already seen as market leaders in their respective sectors Lhyfe and ENERPARC share the development and implementation of individual and customised solutions this project is intended to serve industry filling stations and municipalities in the region The successful transformation of industry and the transport sector towards climate neutrality depends to a large extent on renewable hydrogen Establishing decentralised production sites at an early stage and making them available to local customers will play a key role in supporting this transformation the establishment of local production sites should serve to reduce emerging import dependencies of the European Union in the long term will produce up to 1 200 kg of green hydrogen a day Construction of the site will begin in the summer of 2022 with the first deliveries scheduled for the end of 2023 Lhyfe inaugurated the world’s first industrial production site for renewable hydrogen from wind turbines the company announced its first collaboration in Germany with its participation in Deutsche Bahn’s H2goesRail project The company already has around sixty projects in France and abroad and this collaboration with one of the major developers of solar energy in Germany opens up particularly exciting prospects in Germany Connecting decision makers to a dynamic network of information Bloomberg quickly and accurately delivers business and financial information 2025 at 12:22 PM EDTBookmarkSaveTakeaways NEWRenewable energy and climate technology companies that flocked to the US to profit from the subsidies provided by the Biden administration’s Inflation Reduction Act are reassessing whether the US remains the optimal place to run a low-carbon business Volume 9 - 2022 | https://doi.org/10.3389/fmolb.2022.866843 This article is part of the Research TopicStructure-Function Metrology of ProteinsView all 13 articles Monoclonal antibodies (mAbs) are widely used as analytical components in immunoassays to detect target molecules in applications such as clinical diagnostics Functional groups are often conjugated to lysine or cysteine residues to aid immobilization of mAbs or to enable their detection in an antibody antigen complex Good assay performance depends on the affinity and specificity of the mAbs for the antigen The conjugation reaction however can cause higher order structural (HOS) changes and ultimately affect the assay performance four differently conjugated mAbs were selected as model systems and characterized by mass spectrometry intact protein analysis by liquid-chromatography mass-spectrometry (LC-MS) was performed to determine the amount and distribution of conjugation Hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments were carried out for the structural characterization of the conjugated mAbs Immunoassay experiments were performed to monitor the effects of conjugation on the binding properties of the antibodies selected Good agreement between the mass spectrometry and binding experiment results was found it was noted that the overall structural flexibility of the antibodies increases upon cysteine conjugation and decreases for lysine conjugation The conjugation of mAbs with bulky functional groups tends to decrease the deuterium uptake kinetics due to induced steric effects this study shows correlations between conjugation structure and function of immunoassay antibodies and the benefits of mass spectrometry to improve understanding of the conjugation reaction and provide insights that can predict immunoassay performance intact protein analysis by LC-MS and HDX-IM-MS with the UDMSE approach were applied to characterize conjugated antibodies A panel of commercially available mAbs (Humira (Adalimumab) Xolair (Omalizumab) and Herceptin (Trastuzumab)) were selected to undergo conjugation with biotin and fluorescein at lysine (Lys or K) or cysteine (Cys or C) residues using different linker reagents and chemistries The selection of the most appropriate combination “conjugation:antibody:antigen” for MS analysis was performed by immunoassay Four conjugates were selected: fluorescein-isothiocyanate (FITC) conjugated Adalimumab at Lys residues (Adalimumab-Lys) biotinylated Nivolumab via an N-hydroxy succinimide (NHS) activated ester reacting at Lys residues (Nivolumab-Lys) fluorescein labelled Omalizumab using fluorescein-5-malimide at Cys residues (Omalizumab-Cys) and biotinylated Trastuzumab at Cys residues using a dibromomaleimide (DBM) linker (Trastuzumab-Cys) These conjugates will be referred in this paper as Adalimumab-Lys Omalizumab-Cys and Trastuzumab-Cys respectively indicating the type of mAb and the conjugation at Cys or Lys residues For the structural characterization of the changes induced upon conjugation of the Omalizumab antibody and Trastuzumab antibody native and conjugated forms were directly compared by HDX-MS a more complex experimental design was applied to simultaneously define structural differences between native and conjugated mAbs at the antigen binding site (paratope) which consists of a set of three complementarity-determining regions (CDR) in both the heavy (HC) and light chain (LC) the native mAbs were analyzed and compared to the native and conjugated mAb in complex with their respective antigens: tumor-necrosis factor α (TNFα) for Adalimumab and the programmed cell death protein 1 (PD-1) for Nivolumab HABA/Avidin reagent and deuterium oxide were purchased from Sigma-Aldrich (Gillingham biotin-PEG2-malimide and FITC were purchased from Fisher Scientific (Loughborough Fluorescein-5-maleimide was purchased from TCI (Tokyo Nivolumab and Omalizumab were purchased from Medizone (Oberhaching All native and conjugated mAbs were provided as solutions in 100 mM potassium phosphate buffer pH 7.5 by Fleet Bioprocessing: 34 µM of Omalizumab 20 µM Omalizumab-Cys and 15 µM Trastuzumab-Cys The recombinant human proteins TNFα (Fisher Scientific Germany) were solubilized at 1 g/L (57.5 µM) in water A tetrapeptide (PPPI) was synthesized and purchased from Thermo Fisher Scientific (Loughborough The peptide standard glu-1-fibrinopeptide B was purchased from Sigma-Aldrich (Gillingham Optigrade HPLC Special Grade acetonitrile and ultra-pure water (18 MΩ cm−1) were used 96 well polystyrene microtitre plates were purchased from Greiner Bio-One (Stonehouse Sure Blue Reserve TMB substrate solution was purchased from Insight Biotechnology (Wembley Bioaffy CD1000 and Rexxip buffers were purchased from Gyros Protein Technologies AB (Uppsala anti-omalizumab and anti-adalimumab were purchased from Bio Rad (Watford The preparation of mAb conjugates was carried out using well-established literature methods and is described in supporting information S1-1 Trastuzumab-Cys was conjugated by mild TCEP reduction and reaction with dibromomaleimide-biotin; the aim was to conjugate a single biotin across one reduced inter-chain disulfide residue Omalizumab-Cys was conjugated by forcing conditions using TCEP reduction and reaction with fluorescein-maleimide aiming for conjugation to all reduced inter-chain disulfide residues Adalimumab-Lys and Nivolumab-Lys were conjugated with FITC and biotin-XX-NHS respectively both aiming for high incorporations of label For the intact protein analysis of native and conjugated mAbs the mAb stock solutions were diluted to a 6 µM mAb concentration in 100 mM Tris (pH 7.4) Deglycosylation of the samples was performed by adding 2 µL peptide:N-glycosidase (PNGase F United Kingdom) to 18 µL of 6 μM mAb followed by incubation at 37°C for 24 h The completion of the deglycosylation of native mAbs was confirmed by LC-MS and deconvoluted masses of glycosylated and deglycosylated mAbs are provided in supporting information S1-2 For the middle-down LC-MS analysis of native and conjugated mAbs 10 µL DTT (50 mM) was added to 10 µL of the deglycosylated mAb sample and incubated at room temperature for 30 min to separate the HC and LC the deglycosylated and deglycosylated plus reduced mAb samples were diluted to a mAb concentration of 3 μM and 5% acetonitrile 10.5 pmol of sample was injected onto a MAbPac Reversed Phase HPLC Column (2.1 × 100 mm Thermo Fisher Scientific) in a Vanquish UHPLC system (Thermo Fischer Scientific Chromatographic separation was achieved using a linear gradient starting from 85% A/15% B to 70% A/30% B over 1 min to 60% A/40% B over 13 min at a flow rate of 250 μL/min and 65°C column temperature MS experiments were performed using a Q-Exactive Plus Orbitrap instrument with a HESI-II probe source (Thermo Fisher Scientific Germany) in positive nanoelectrospray ionization (nESI) mode using a 320°C source temperature Data were acquired in 35 K resolution mode over a range of 1,000–5,000 m/z (high mass range (HMR) mode) Protein deconvolution was performed using BioPharma finder v 2.0 software (Thermo Fisher Scientific Deconvolution was performed in the 10–180 kDa range a range of 4–10 minimum charge states and a target mass of 147 kDa Antibodies and proteins were stored separately at 4°C until analysis All native and conjugated antibodies were diluted to a final protein concentration of 10 µM with 50 mM potassium phosphate buffer For the direct comparison between native and conjugated mAbs two sets of samples were prepared: Omalizumab vs The more complex HDX-MS experiment for the structural characterization of Adalimumab-Lys and Nivolumab-Lys included four samples the native mAb in complex with antigen (Adalimumab:TNFα ratio 1:2) and the conjugated mAb in complex with antigen (Adalimumab-Lys:TNFα The peptide PPPI as internal standard was added to each sample at 10 µM Sample handling and mixing steps were performed using a first-generation LEAP PAL system set (LEAP Technologies 15 µL sample was diluted 10-fold either in 50 mM potassium phosphate buffer pH 7.4 for the generation of peptide maps or in 50 mM potassium phosphate buffer (pH 7.0) prepared in D2O for exchange experiments The HDX time course for each HDX-MS data set is summarized in supporting information S2 Each exchange time point was run in triplicate and quenched by a 2-fold dilution with 50 µL of 1 M TCEP 8 M Urea in 100 mM potassium phosphate buffer (pH 2.5) at 4°C for 10 min 95 µL of quenched sample was injected onto a refrigerated nano-ACQUITY UPLC system with HDX technology (Waters MA) for on-line pepsin digestion and chromatographic separation Protein digestion was performed on an Enzymate BEH pepsin column (300 Å MA) at a flow rate of 70 μL/min (0.5% v/v formic acid) and a pressure of 4,800 psi The digestion pressure was held by using a PEEK restrictor placed after the trap column Digestion temperature was maintained at 15°C for Adalimumab Nivolumab and at 20°C for Omalizumab the proteolytic peptides were trapped and desalted for 3.8 min using an ACE C18 guard cartridge (5 μm 2.1 mm) and chromatographically separated on an ACE Excel 2 Super C18 column (2 μm United Kingdom) at a flow rate of 100 μL/min using a 7 min linear gradient from 8% B to 35% B ESI-IM-MS experiments were performed on a Synapt G2Si Q-TOF-MS instrument (Waters MA) in positive nanoelectrospray ionization mode 80 V source offset and 250°C desolvation temperature For the peptide map generation, the instrument was operated in the UDMSE mode, which is MSE with applied ion mobility and optimized collision energies based on measured ion arrival times. Optimized collision energies were from Distler and co-workers (Distler et al., 2014) Deuterium exchange experiments were acquired in IM-MS mode Data acquisition in both modes UDMSE and IM-MS was carried out over a range of 50–2000 m/z a scan time of 0.2 s using the same ion mobility settings as follows: 650 m/s wave velocity 450 µsec mobility trapping release time and a wave velocity ramp of 800–500 m/sec Data was lock mass corrected against m/z 785.8426 of glu-1-fibrinopeptide B (100 fmol solution in 50% methanol Fluorescein incorporations were determined by UV-vis spectrophotometry using an 495 nm extinction coefficient of 150.5 (mg/ml)−1 cm−1 and 280:495 Rz of 0.34 Biotin incorporations were determined by HABA/Avidin colorimetric assay following the instructions provided by the reagent supplier using a 500 nm extinction coefficient of 34,000 M−1 cm−1 A description of the different immunoassay formats is provided in the supporting information S1-5 A total of seven different assay formats (Fc binding Fc capture and streptavidin capture) were used to characterize the conjugate performance relative to each other and the unconjugated mAbs An advantage of conjugating at the inter-chain disulfide bridges is that it expected to have lower impact on antigen binding due to its distance from the mAb binding site Amount of mAb conjugation by intact LC-MS protein analysis and colorimetry For the Omalizumab antibody, conjugation was achieved by using a commercially available linker fluorescein-5-maleimide. By LC-MS intact protein analysis, no residual intact antibody was detected, and the free HC and LC were observed to contain three and one conjugate molecules respectively (Table 1, supporting information S1-6: Figure 1) This indicated that all eight sulfhydryl groups of the four inter-chain disulfide bonds were conjugated as anticipated and that the structure of Omalizumab-Cys was mainly stabilized through non-covalent interactions between the mAb chains Relative differences in HDX-MS between native (reference state) and cysteine conjugated mAb samples Trastuzumab with DBM-biotin (A) and Omalizumab with fluorecein-5-maleimide (B) are illustrated on the crystal structure of the generic IgG1 Fc domain (PDB: 5vgp) and the specific Fab domains PDB: 6 mh2 (A) and PDB: 2xa8 (B) Increased (blue) and decreased (red) HDX-MS rates as relative changes from the native mAbs are indicated by the color bar and are shown between the lowest and highest time points from 5 min to 4 h (A) and 15 min to 8 h (B) Light chain (LC) domains located in Fab are VL and CL Heavy chain (HC) domains are VH and CH1 of Fab and CH2/CH3 of Fc The hinge region with inter-chain disulfide bonds is unresolved in crystal 3D structures and is sketched by the dashed line connecting the CH1 and CH2 of HC Missing cysteine residues of the HC in the hinge region are indicated as asterisks Missing sequence information for deuterium uptake kinetics are highlighted in green Glycans are indicated as yellow sticks and cysteine residues forming disulfide bonds as spheres Because mAb chains as LC and HC are mainly cross-linked using the DBM linker the conjugation distribution between HC and LC is not clearly determinable the main presence of conjugated intact and HC-HC-LC populations proves that inter-chain disulfide bonds are the target of the conjugation approach Relative differences in HDX-MS between native (reference state) and lysine conjugated mAb samples are illustrated on the crystal structure for Nivolumab of the generic IgG4 Fc domain (PDB: 4c54) and the Nivolumab specific Fab domain PDB: 5ggq (A) and for Adalimumab on the IgG1 Fc domain (PDB: 5vgp) and Adalimumab specific Fab PDB: 3wd5 (B) Increased (blue) and decreased (red) HDX-MS rates as relative changes from the native mAbs are indicated by the color bar and are shown for the highest incubation time 8 h (A) and 4 h (B) Glycans are indicated as yellow sticks and lysine residues as spheres Relative performance of conjugated mAbs against the native mAbs as measured by different immunoassay formats a mixture of species with 28–42 conjugated residues was observed which implies that potentially all Lys residues might be conjugated to different degrees The most abundant conjugated forms of Nivolumab-Lys showed 35–36 biotin molecules Intact LC-MS analysis of the Adalimumab-Lys conjugate showed the presence of a mixture of conjugates containing 2-6 FITC molecules per antibody with the most abundant form containing 2-4 incorporated FITC molecules This result was in good agreement with the fluorescein incorporation as determined by UV-vis colorimetry the biotin incorporation for Nivolumab determined by HABA/Avidin colorimetric assay was grossly under-estimated possibly due to a limitation of the assay due to steric effects which is likely to be a universal limitation of this colorimetric assay it was shown after reduction and LC-MS analysis of Adalimumab-Lys the only indication of fluorescein conjugation was present on the LC which interacts with Cys223 of the CH1 domain showing decreased deuterium uptake kinetics for adjacent residues 218–220 Increased deuterium uptake kinetics were observed in the regions CL (121–125) CH1 (171–177) and CH2 (239–255) located distant from the hinge region consistent with the extension of the typical disulfide bond distance via the incorporated succinimide ring during conjugation which results in opening up the surrounding protein structure the conjugation of Omalizumab via the classical maleimide linker fluorescein-5-maleimide caused reduction of the disulfide bonds and an increased molecular distance between cysteine residues with a consequent loss of structure and increased flexibility The higher order structure of Adalimumab-Lys conjugate with only two FITC molecules on the LC was much less impacted by the conjugation, most likely due to the lower level of conjugation as was observed from the intact mass spectrometry analysis (Figure 2B) CL) shows slightly decreased deuterium uptake kinetics in peptides which contain Lys residues as K42/45 in the N-terminus and K183/188/190 in the C-terminus Because of the proximity of these Lys residues to each other it was difficult to determine the site of conjugation without any further experimentation No significant structural differences were observed for the HC of the Fab region (VH CH1) when comparing the native and conjugated antibody for CDR-H1 and -H3 increased deuterium uptake kinetics were observed suggesting a loss of structure that may also have an impact on the binding properties of the antibody The structural differences in the CH2 of the Fc domain are similar to the Nivolumab-Lys conjugate indicating that the conjugation has an indirect impact on the mAb structure According to intact protein MS experiments For the Lys conjugates a correlation between the amount of conjugation and the higher order structural differences observed was clearly demonstrated by HDX-MS experiments (Figure 2) an increase in conjugation at Lys residues resulted in a decreased deuterium uptake kinetics across the whole protein structure The range of recognition values is based on a typical immunoassay CV of ±10% As recognition values are calculated from two assay results (conjugated the potential error around the recognition value was calculated as follows: recognition value x 0.818 (0.9/1.1) to recognition value x 1.222 (1.1/0.9) For the determination of relative Fc recognition of Adalimumab-Lys an anti-Fc antibody was used to estimate the differences in the Fc binding between native and conjugated mAb a specific anti-complex mAb for Adalimumab-Lys and Omalizumab-Cys which detects only the Ab-Ag complex and not Ab or Ag alone was applied to analyze the relative antigen recognition between native and conjugated mAb the antigen recognition of Nivolumab-Lys was determined by using a competitive assay where the conjugate binds the immobilized antigen and displacement occurs by varying free antigen concentrations With increasing concentrations of free antigen the conjugate is displaced from the immobilized antigen resulting in a signal decrease Nivolumab conjugates were then ranked by calculating the free antigen concentration required to produce 50% reduction in the signal In the case of the Trastuzumab-Cys conjugate the biotin label is used in the streptavidin capture assay to validate the Fc binding and antigen affinity and thus the signals relate to the Trastuzumab conjugate with the least modification of the variable region (100%) out of a mAb conjugate panel The relative Fc binding for Adalimumab-Lys is largely unchanged compared to the native mAb showing that the conjugation does not have a significant impact on the Fc domain whereas the relative antigen recognition is slightly reduced to 91% indicating conjugation might be more present in the Fab region of the mAb The results for Nivolumab-Lys show a great contrast in the sense that the Fc binding is reduced to 61.1% whereas the sensitivity in a competitive assay format is actually increased to 132.5% compared to the native mAb As the Fc binding is significantly reduced due to the conjugation it is assumed that the conjugation has predominantly modified the Fc region results from the competitive assay indicate some modification of the binding site Whilst this would typically be expected to reduce the utility of the conjugate slight changes to antigen recognition appear to have increased the ease with which displacement by free antigen occurs increasing the sensitivity of the end assay and indicating a change in the Ab-Ag interaction Trastuzumab-Cys shows the best antigen affinity within a conjugated mAb panel whereas the Fc binding assay result is significantly reduced to 67% implying that the conjugation has modified mainly the Fc region Omalizumab-Cys shows a significant reduction in antigen recognition Whilst there is a detectable reduction in Fc recognition this is relatively small compared to the change in antigen recognition these results suggest that both Fab and Fc regions of Omalizumab-Cys have been modified with the Fab modification being most significant in potential assay performance terms Monoclonal antibodies are used as analytical tools in immunoassays to immobilize or detect antigens or molecules of interest conjugation is carried out through a chemical reaction the effects of conjugation regarding amount and location on the structure and functionality of the mAb which ultimately may affect immunoassay performance the conjugation reaction efficiency was studied by intact MS analysis and the impact of conjugation at either Lys or Cys residues on the mAb higher order structure and its binding properties was assessed by HDX-MS and immunoassay respectively The results of the mass spectrometric characterization of different mAb conjugates correlate very well with the mAb function and binding affinity The cysteine conjugation occurs in a specific manner as the inter-chain disulfide bonds are typically targeted the Cys residues of a disulfide bond are dithiosuccinimide-bridged maintaining the disulfide bonding but with an extension of the typical distance When using a classical maleimide linker such as fluorescein-5-maleimide for the cysteine conjugation of Omalizumab the rebuilding of disulfide bridges is not possible the overall mAb structure for Trastuzumab and Omalizumab conjugates show predominantly increased deuterium uptake kinetics due to extended bond distances (Trastuzumab) or reduced disulfide bonds (Omalizumab) resulting in more flexibility in structure protein regions in close proximity to the hinge region and inter-chain disulfide bonds show decreased deuterium uptake kinetics due to the conjugation with bulky groups The intact protein analysis results correlate well with the HDX-MS results where an increased amount of conjugation per mAb was also associated with a relative decrease in deuterium uptake kinetics the most accessible inter-chain disulfide of Trastuzumab-Cys modified using the chemistry employed is Cys214 of the CL domain and Cys223 of the CH1 domain implying no significant structural changes at the antigen binding site This also correlates well with the immunoassay results that the selected Trastuzumab-Cys mAb shows the best antigen affinity within a conjugated mAb panel results of intact protein analysis and HDX-MS reveal that all eight sulfhydryl groups of inter-chain disulfide bonds are conjugated and thus the mAb structure relies only on non-covalent interactions between heavy and light chain causing major structural changes with loss of recognition properties shown by immunoassay experiment results for both the Fab and Fc regions the conjugation at lysine residues generated more heterogenous mixtures of different conjugated mAb variants (Nivolumab and Adalimumab) Conjugation with biotin and FITC was chemically performed without reduction and therefore the disulfide bridges were maintained The HDX-MS data showed decreased deuterium uptake kinetics of the conjugated mAbs vs the native form with the relative level of conjugation correlating well with the relative decrease of deuterium uptake kinetics the Nivolumab-Lys is highly conjugated (28–42 biotin molecules per mAb) and the significantly decreased deuterium uptake indicates a potential change of higher order structure that could have a negatively impact on the antigen recognition This was also confirmed by the immunoassay experiment results conjugation of the Adalimumab mAb had less effects on the deuterium uptake kinetics and binding properties of the antibody as was shown by both HDX-MS and immunoassay experiments This possibly due to the 2-6 FITC conjugation mostly occurring on the LC as was shown by intact protein MS analysis Consistent with the immunoassay results which indicate that the conjugation is more present in the Fab region and the Fc part is not significantly impacted this study shows that conjugation can have an impact on the performance of the antibodies used for immunoassay engineering and that mass spectrometry experiments can aid an improved understanding and optimization of the conjugation reaction This will ultimately aid to better performance a thorough characterization of the conjugation reaction will ensure continuity in performance over time and across different batches The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material LL and KG performed antibody characterization by LC-MS CB prepared antibody conjugates and performed colorimetry measurements MQ and AD contributed to conception and design of the study All authors contributed to manuscript revision The work described in this paper was funded (in part) by the United Kingdom government Department for Business Energy and Industrial Strategy (BEIS); and the Innovate United Kingdom Strategic Priorities Fund (Grant number 105569) and AD were employed by the Company Fleet Bioprocessing Ltd The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmolb.2022.866843/full#supplementary-material Site-Specifically Labeled Immunoconjugates for Molecular Imaging-Part 1: Cysteine Residues and Glycans PubMed Abstract | CrossRef Full Text | Google Scholar and Our Laboratory Experience with Peptide/protein Analyses Using ELISA PubMed Abstract | CrossRef Full Text | Google Scholar Bridging Disulfides for Stable and Defined Antibody Drug Conjugates PubMed Abstract | CrossRef Full Text | Google Scholar “Immunoassay Methods,” in Assay Guidance Manual [Internet] MD: Eli Lilly & Company and the National Center for Advancing Translational Sciences) Google Scholar Online Hydrogen-Deuterium Exchange Traveling Wave Ion Mobility Mass Spectrometry (HDX-IM-MS): a Systematic Evaluation PubMed Abstract | CrossRef Full Text | Google 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Copyright © 2022 Luckau, Groves, Blencowe, Scrimshaw, Dent and Quaglia. 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The use distribution or reproduction in other forums is permitted provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited in accordance with accepted academic practice distribution or reproduction is permitted which does not comply with these terms *Correspondence: Luise Luckau, bHVpc2UubHVja2F1QGxnY2dyb3VwLmNvbQ== Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish 2023 at 8:00 AM EDTBookmarkSaveLock This article is for subscribers only.Wind power industry growth dropped to the lowest in three years in 2022 hit by rising costs and shifting government policies That’s according to the latest analysis from clean energy researchers at BloombergNEF While global governments are relying on a rapid scale-up of the wind industry to hit climate goals shrinking profit margins and soaring costs have cooled development