Protein Conjugation Reactions Research Articles

Analyzing protein conjugation reactions for antibody-drug conjugate synthesis using polarized excitation emission matrix spectroscopy.

Antibody-drug conjugates (ADCs) are promising anticancer therapeutics, which offer important advantages compared to more classical therapies. There are a variety of ADC critical quality attributes (CQAs) such as the protein structure, aggregation, and drug-to-antibody ratio (DAR), which all impact on potency, stability, and toxicity. Production processes can destabilize antibodies via a variety of physical and chemical stresses, and or by increased aggregation after conjugation of hydrophobic drugs. Thus, a proper control strategy for handling, production, and storage is necessary to maintain CQA levels, which requires the use of in-process quality measurements to first identify, then understand, and control the variables which adversely affect ADC CQAs during manufacturing.Here, we show how polarized excitation emission matrix (pEEM) spectroscopy, a sensitive, nondestructive, and potentially fast technique, can be used for rapidly assessing aggregation and DAR in a single measurement. pEEM provides several sources of information for protein analysis: Rayleigh scatter for identifying aggregate/particle formation and fluorescence emission to assess chemical and structural changes induced by attachment of a linker and/or a small molecule drug payload. Here, we used a nontoxic ADC mimic (monoclonal antibody with linker molecule) to demonstrate efficacy of the measurement method. Emission changes caused via light absorption by the attached linker, allowed us to predict DAR with good accuracy using fluorescence signal from the final purified products (6% relative error of prediction [REP]) and also from unpurified alkylation intermediates (11% REP). pEEM changes could also be correlated with size (hydrodynamic radius, Rh ) and aggregate content parameters obtained from dynamic light scattering and size exclusion chromatography (SEC). For the starting material and purified product samples, pEEM correlated better with Rh (R2 = 0.99, 6% REP) than SEC determined aggregate content (18% REP). Combining both fluorescence and light scatter signals also enabled in-process size quantification (6% REP). Overall, combining polarized measurements with EEM and Rayleigh scatter provides a single measurement, multi-attribute test method for ADC manufacturing.

Conjugation of Different Aflatoxin Derivatives to Proteins and Their Use as Heterologous Antigens in Immunoassay Development

AbstractAnalysis of small molecule contaminants using biochemical methods like ELISA or biosensors often require their conjugation to a bigger molecule. Protein conjugates of aflatoxins (AFs), potent carcinogenic mycotoxins, are widely used in antibody and analytical assay development. There are more than 20 naturally occurring AF derivatives four of which are regulated by international authorities. AFB1, the most abundant AF derivative, is also the most commonly utilized one in protein conjugation reactions. In this study, we demonstrated that the use of different AF derivatives can is an aid for modifying the conjugation efficacy to obtain optimal hapten densities. With a Mannich‐type reaction scheme, AFG1 and AFG2 conjugates showed more than 3‐fold higher hapten densities compared with AFB1 and AFB2 conjugates. We also showed that the use of heterologous antigens in AF immunoassay development decreases the production costs without affecting assay sensitivity.

'Clickable lectins': bioorthogonal reactive handles facilitate the directed conjugation of lectins in a modular fashion.

Lectins are carbohydrate-binding proteins with specificity for their target ligands. They play diverse roles in cellular recognition and signalling processes, as well as in infections and cancer metastasis. Owing to their specificity, lectins find application in biotechnology and medicine, e.g. for blood group typing, purification of glycoproteins or lipids and as markers that target cancer cells. For some applications, lectins are immobilized on a solid support, or they are conjugated with other molecules. Classical protein conjugation reactions at nucleophilic amino acids such as cysteine or lysine are often non-selective, and the site of conjugation is difficult to pre-define. Random conjugation, however, can interfere with protein function. Therefore, we sought to equip lectins with a unique reactive handle, which can be conjugated with other molecules in a pre-defined manner. We site-specifically introduced non-canonical amino acids carrying bioorthogonal reactive groups into several lectins. As a proof of principle, we conjugated these 'clickable lectins' with small molecules. Furthermore, we conjugated lectins with different ligand specificities with one another to produce superlectins. The 'clickable lectins' might be useful for any process where lectins shall be conjugated with another module in a selective, pre-defined and site-specific manner.

Modeling the Kinetics of Protein Conjugation Reactions

AbstractOver the past decades, the pharmaceutical industry has shown a continuous infatuation for therapeutic proteins. In order to constantly improve their efficacy, chemical reactions involving the conjugation of the therapeutic protein with various chemical reagents have been developed. Little efforts have been put forth to simulate the kinetics of protein conjugation and the literature dealing with kinetic models is rather scarce as compared to the abundant references about conjugation reactions in general. In this work, techniques to measure the kinetics of reaction, model the conjugation between add‐on molecules and proteins, and evaluate the model parameters are discussed.

Characterization of Reactions between Water-Soluble Trialkylphosphines and Thiol Alkylating Reagents: Implications for Protein-Conjugation Reactions.

Water-soluble trialkylphosphines such as tris(carboxyethyl)phosphine (TCEP) and trishydroxypropyl phosphine (THPP) are effective agents for reducing disulfide bonds in proteins and are increasingly becoming the reagents of choice for bioconjugation strategies that modify cysteine (thiol containing) amino acids. These reducing agents are often considered as being chemically compatible with Michael acceptors such as maleimides and, as such, are often not removed prior to performing protein conjugation reactions. Here, we demonstrate the rapid and irreversible reaction of both TCEP and THPP with derivatives of the commonly employed thiol alkylating groups, maleimide and vinyl sulfone. Mechanistic investigations revealed distinct differences between the reactions of TCEP and THPP with maleimide, leading to the production of either nonproductive ylenes or succidimidyl derivatives, respectively. Importantly, we also demonstrate the incorporation of nonproductive ylenes formed between maleimide and TCEP into the Pneumococcal capsular polysaccharide Pn6b following strategies employed toward the production of conjugate vaccines.

Abacavir forms novel cross‐linking abacavir protein adducts in patients

Abacavir (ABC), a nucleoside-analogue reverse transcriptase inhibitor, is associated with severe hypersensitivity reactions that involve the activation of CD8+ T cells in a HLA-B*57:01-restricted manner. Recent studies have claimed that non-covalent interactions of ABC with HLA-B*57:01 are responsible for the immunological reactions associated with ABC. However, the formation of haemoglobin-ABC aldehyde (ABCA) adducts in patients exposed to ABC suggests that protein conjugation might represent a pathway for antigen formation. To further characterize protein conjugation reactions, we used mass spectrometric methods to define ABCA modifications of human serum albumin and glutathione S-transferase Pi in vitro and in patients commencing ABC therapy. ABCA formed a novel intramolecular cross-linking adduct on human serum albumin in patients and in vitro via Michael addition, followed by nucleophilic adduction of the aldehyde with a neighbouring protein nucleophile. Adducts were detected on lysine, histidine, and cysteine residues in subdomain IB of human serum albumin. Only a cysteine adduct and a putative cross-linking adduct were detected on glutathione S-transferase Pi. Modelling the docking of ABCA with HLA B*57:01 confirmed that ABCA has a strong binding affinity when bound covalently to Ser116, a key residue with regards to recognition by ABC-specific CD8+ T cells. These findings reveal that ABC forms novel types of haptens in all patients taking the drug. It is therefore vital that the immunological consequences of such haptenation pathways are explored in the in vitro models that have been used by various groups to define a new mechanism of drug hypersensitivity for ABC.

Abacavir Forms Novel Cross-Linking Abacavir Protein Adducts in Patients

Abacavir (ABC), a nucleoside-analogue reverse transcriptase inhibitor, is associated with severe hypersensitivity reactions that are thought to involve the activation of CD8+ T cells in a HLA-B*57:01-restricted manner. Recent studies have claimed that noncovalent interactions of ABC with HLA-B*57:01 are responsible for the immunological reactions associated with ABC. However, the formation of hemoglobin-ABC aldehyde (ABCA) adducts in patients exposed to ABC suggests that protein conjugation might represent a pathway for antigen formation. To further characterize protein conjugation reactions, we used mass spectrometric methods to define ABCA modifications in patients receiving ABC therapy. ABCA formed a novel intramolecular cross-linking adduct on human serum albumin (HSA) in patients and in vitro via Michael addition, followed by nucleophilic adduction of the aldehyde with a neighboring protein nucleophile. Adducts were detected on Lys159, Lys190, His146, and Cys34 residues in the subdomain IB of HSA. Only a cysteine adduct and a putative cross-linking adduct were detected on glutathione S-transferase Pi (GSTP). These findings reveal that ABC forms novel types of antigens in all patients taking the drug. It is therefore vital that the immunological consequences of such pathways of haptenation are explored in the in vitro models that have been used by various groups to define new mechanisms of drug hypersensitivity exemplified by ABC.

One-pot squaric acid diester mediated aqueous protein conjugation

A water-soluble squaric acid dialkyl diester derivative is presented, which enables one-pot, two-step amine-selective protein conjugation reactions with (functional) amines in water. This reagent not only allows all-aqueous protein modifications, but also tolerates e.g. hydroxyl groups and can also be used for the modification of proteins with water-insoluble amines.

Analysis of the dispersity in carbohydrate loading of synthetic glycoproteins using MALDI-TOF mass spectrometry

Statistical correlation of mass spectrum peak broadening with product dispersity in protein conjugation reactions allows more detailed characterization of putative therapeutic conjugates.

The Atg12-Atg5 Conjugate Has a Novel E3-like Activity for Protein Lipidation in Autophagy

Autophagy is a bulk degradation process in eukaryotic cells; autophagosomes enclose cytoplasmic components for degradation in the lysosome/vacuole. Autophagosome formation requires two ubiquitin-like conjugation systems, the Atg12 and Atg8 systems, which are tightly associated with expansion of autophagosomal membrane. Previous studies have suggested that there is a hierarchy between these systems; the Atg12 system is located upstream of the Atg8 system in the context of Atg protein organization. However, the concrete molecular relationship is unclear. Here, we show using an in vitro Atg8 conjugation system that the Atg12-Atg5 conjugate, but not unconjugated Atg12 or Atg5, strongly enhances the formation of the other conjugate, Atg8-PE. The Atg12-Atg5 conjugate promotes the transfer of Atg8 from Atg3 to the substrate, phosphatidylethanolamine (PE), by stimulating the activity of Atg3. We also show that the Atg12-Atg5 conjugate interacts with both Atg3 and PE-containing liposomes. These results indicate that the Atg12-Atg5 conjugate is a ubiquitin-protein ligase (E3)-like enzyme for Atg8-PE conjugation reaction, distinctively promoting protein-lipid conjugation.

Novel preparation and characterization of a trastuzumab???streptavidin conjugate for pre-targeted radionuclide therapy

This study describes a novel and convenient route for the preparation of a trastuzumab-streptavidin conjugate such as might be used in a pre-targeting system and its in-vitro and in-vivo evaluation. Trastuzumab was irradiated with UV light in the presence of stannous ions to reduce a number of the disulfide bridges to free thiol groups. A range of irradiation times were studied in order to quantify the number of thiols produced and to optimize the reduction process. The conjugate was then prepared by reaction with succinimidyl 4-(N-maleimidomethy cyclohexane)-1-carboxylate (SMCC)-linked streptavidin. Initial conjugation reactions in phosphate buffer were inefficient, producing low conjugate yields, but conjugation reactions in triethanolamine-based buffer showed greatly increased conjugation yields. A high purity product (approximately 100%) was obtained following purification by gel-filtration HPLC as determined by subsequent size exclusion HPLC analysis. The conjugate was shown to possess an essentially identical immunoreactivity to that of the native, unconjugated antibody and an unaltered biotin binding stoichiometry. Shedding and internalization by Her-2-expressing cells were low and the uptake in vivo by Her-2-expressing xenografts in nude mice was similar to that of labelled antibody. Our results demonstrate a new, simple and effective method for the successful synthesis of antibody-streptavidin conjugates which could also be applied to many other heterodimeric protein conjugation reactions.

A comparison between SDS-PAGE and size exclusion chromatography as analytical methods for determining product composition in protein conjugation reactions

Horseradish peroxidase (HRP) was conjugated with bovine serum albumin (BSA) or human α 1-proteinase inhibitor (α 1-PI). The enzyme was maleimidylated using N-succinimidyl 4-( N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and then allowed to react with thiolated BSA or reduced α 1-PI. The conjugation products were analysed both by SDS-PAGE and size exclusion chromatography (SEC) on Sephadex G200. The two methods of evaluating conjugative processes were compared with respect to information provided in relation to the behaviour of the products in solution. The results showed that neither SDS-PAGE nor SEC alone provides sufficient information about conjugate structure. The basic conjugate units observed in electrophoresis tend to form dimeric or higher-order aggregates under gel chromatographic conditions.

The use of MALDI mass spectrometry to characterize synthetic protein conjugates

MALDI mass spectrometry has been used to study the conjugation of two small molecules to hen egg white lysozyme and human serum albumin. Hapten densities were determined with both proteins. Synthetic peptide mixtures, designed to eliminate the need for subsequent sequencing experiments, were used to assess the potential reactivities of various amino acid side chains in proteins. Selective hydrolysis reactions were also used to differentiate ester and amide conjugates, which are expected to have quite different in vivo stabilities. Finally, MALDI was used to follow the kinetics of protein conjugation reactions, even for reactions having initial rates differing by as much as three orders of magnitude.

Use of the Npys thiol protection in solid phase peptide synthesis. Application to direct peptide-protein conjugation through cysteine residues.

The protection of the thiol function of cysteine with the 3-nitro-2-pyridylsulfenyl (Npys) group has been successfully applied in the solid phase synthesis of nine peptides. A reexamination of the chemical stability of the protecting group has shown that, while Npys is essentially suitable for standard Boc/benzyl synthesis conditions, it is inadequate for the Fmoc strategy. Its proven stability to "high" HF acidolysis can not be extended to "low-high" conditions without significant thiol deprotection. On the other hand, the Npys group is quite compatible with standard photolytical cleavage conditions. The stability of Npys to HF and its thiol-activating character allow its application in peptide-carrier protein conjugation reactions by specific coupling through cysteine residues in the peptide.