RESUMO
Charge heterogeneity among therapeutic monoclonal antibodies (mAbs) is considered an important critical quality attribute and requires careful characterization to ensure safe and efficacious drug products. The charge heterogeneity among mAbs is the result of chemical and enzymatic post-translational modifications and leads to the formation of acidic and basic variants that can be characterized using cation exchange chromatography (CEX). Recently, the use of mass spectrometry-compatible salt-mediated pH gradients has gained increased attention to elute the proteins from the charged stationary phase material. However, with the increasing antibody product complexity, more and more selectivity is required. Therefore, in this study, we set out to improve the selectivity by using a solvent-enriched mobile phase composition for the analysis of a variety of mAbs and bispecific antibody products. It was found that the addition of the solvents to the mobile phase appeared to modify the hydrate shell surrounding the protein and alter the retention behavior of the studied proteins. Therefore, this work demonstrates that the use of solvent-enriched mobile phase composition could be an attractive additional method parameter during method development in CEX.
Assuntos
Produtos Biológicos , Concentração de Íons de Hidrogênio , Anticorpos Monoclonais/química , Solventes , Indicadores e Reagentes , Cromatografia por Troca Iônica/métodosRESUMO
Interleukin-2 (IL-2) is a potent molecule in cancer therapy. Clinical application, however, is limited due to its strong side effects during the treatment. We developed an IL-2 variant (IL-2v) immunocytokine to circumvent the drawbacks of the current IL-2 therapy. During the production of the IL-2v immunocytokine in Chinese hamster ovary (CHO) cells, molecules with fragmented IL-2v and therefore reduced cytokine activity can be observed. To control product fragmentation different production process conditions were investigated. By shifting temperature or pH after the cell growth phase to lower values, fragmented species can be reduced from 10% to 12% to about 4%. However, with the adopted process conditions, the effective titer is decreased concomitantly. Moreover, fermentation length and inoculation cell density are parameters to adjust fragmentation and effective titer. A suitable method for efficient process optimization is the design of experiment approach. With this procedure, novel optimal values for temperature, pH value, harvest day, and inoculation cell densities were proposed and tested subsequently. In comparison to the former process, the improved process reduces fragmentation by 66% while keeping the effective titer comparable. In summary, these findings will help to control fragmentation in CHO production processes of different IL-2v or IL-2 containing therapeutic proteins.
Assuntos
Técnicas de Cultura de Células , Interleucina-2/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Animais , Células CHO , Cricetulus , Humanos , Interleucina-2/genética , Estabilidade Proteica , Proteínas Recombinantes de Fusão/genéticaRESUMO
The success of recombinant monoclonal immunoglobulins (IgG) is rooted in their ability to target distinct antigens with high affinity combined with an extraordinarily long serum half-life, typically around 3 weeks. The pharmacokinetics of IgGs is intimately linked to the recycling mechanism of the neonatal Fc receptor (FcRn). For long serum half-life of therapeutic IgGs, the highly pH-dependent interaction with FcRn needs to be balanced to allow efficient FcRn binding and release at slightly acidic pH and physiological pH, respectively. Some IgGs, like the antibody briakinumab has an unusually short half-life of â¼8 days. Here we dissect the molecular origins of excessive FcRn binding in therapeutic IgGs using a combination of hydrogen/deuterium exchange mass spectrometry and FcRn affinity chromatography. We provide experimental evidence for a two-pronged IgG-FcRn binding mechanism involving direct FcRn interactions with both the Fc region and the Fab regions of briakinumab, and correlate the occurrence of excessive FcRn binding to an unusually strong Fab-FcRn interaction.
Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Receptores Fc/metabolismo , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados , Sítios de Ligação , Medição da Troca de Deutério/métodos , Meia-Vida , Humanos , Concentração de Íons de Hidrogênio , Espectrometria de Massas/métodos , Modelos Moleculares , Ligação Proteica , Estabilidade ProteicaRESUMO
Crystallographic evidence suggests that the pH-dependent affinity of IgG molecules for the neonatal Fc receptor (FcRn) receptor primarily arises from salt bridges involving IgG histidine residues, resulting in moderate affinity at mildly acidic conditions. However, this view does not explain the diversity in affinity found in IgG variants, such as the YTE mutant (M252Y,S254T,T256E), which increases affinity to FcRn by up to 10×. Here we compare hydrogen exchange measurements at pH 7.0 and pH 5.5 with and without FcRn bound with surface plasmon resonance estimates of dissociation constants and FcRn affinity chromatography. The combination of experimental results demonstrates that differences between an IgG and its cognate YTE mutant vary with their pH-sensitive dynamics prior to binding FcRn. The conformational dynamics of these two molecules are nearly indistinguishable upon binding FcRn. We present evidence that pH-induced destabilization in the CH2/3 domain interface of IgG increases binding affinity by breaking intramolecular H-bonds and increases side-chain adaptability in sites that form intermolecular contacts with FcRn. Our results provide new insights into the mechanism of pH-dependent affinity in IgG-FcRn interactions and exemplify the important and often ignored role of intrinsic conformational dynamics in a protein ligand, to dictate affinity for biologically important receptors.
Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Receptores Fc/metabolismo , Cromatografia de Afinidade , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Concentração de Íons de Hidrogênio , Imunoglobulina G/genética , Cinética , Mutação de Sentido Incorreto , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Receptores Fc/química , Receptores Fc/genética , Ressonância de Plasmônio de SuperfícieRESUMO
The recycling of immunoglobulins by the neonatal Fc receptor (FcRn) is of crucial importance in the maintenance of antibody levels in plasma and is responsible for the long half-lives of endogenous and recombinant monoclonal antibodies. From a therapeutic point of view there is great interest in understanding and modulating the IgG-FcRn interaction to optimize antibody pharmacokinetics and ultimately improve efficacy and safety. Here we studied the interaction between a full-length human IgG(1) and human FcRn via hydrogen/deuterium exchange mass spectrometry and targeted electron transfer dissociation to map sites perturbed by binding on both partners of the IgG-FcRn complex. Several regions in the antibody Fc region and the FcRn were protected from exchange upon complex formation, in good agreement with previous crystallographic studies of FcRn in complex with the Fc fragment. Interestingly, we found that several regions in the IgG Fab region also showed reduced deuterium uptake. Our findings indicate the presence of hitherto unknown FcRn interaction sites in the Fab region or a possible conformational link between the IgG Fc and Fab regions upon FcRn binding. Further, we investigated the role of IgG glycosylation in the conformational response of the IgG-FcRn interaction. Removal of antibody glycans increased the flexibility of the FcRn binding site in the Fc region. Consequently, FcRn binding did not induce a similar conformational stabilization of deglycosylated IgG as observed for the wild-type glycosylated IgG. Our results provide new molecular insight into the IgG-FcRn interaction and illustrate the capability of hydrogen/deuterium exchange mass spectrometry to advance structural proteomics by providing detailed information on the conformation and dynamics of large protein complexes in solution.
Assuntos
Anticorpos Monoclonais/química , Antígenos de Histocompatibilidade Classe I/química , Imunoglobulina G/química , Receptores Fc/química , Animais , Células CHO , Cricetulus , Deutério , Células HEK293 , Humanos , Hidrogênio , Espectrometria de Massas/métodos , Modelos MolecularesRESUMO
In-depth analytical characterization of biotherapeutics originating from different production batches is mandatory to ensure product safety and consistent molecule efficacy. Previously, we have shown unintended incorporation of tyrosine (Tyr) and leucine/isoleucine (Leu/Ile) at phenylalanine (Phe) positions in a recombinant produced monoclonal antibody (mAb) using an orthogonal MASCOT/SIEVE based approach for mass spectrometry data analysis. The misincorporation could be avoided by sufficient supply of phenylalanine throughout the process. Several non-annotated signals in the primarily chromatographic peptide separation step for apparently single PheâTyr sequence variants (SVs) suggest a role for isobar tyrosine isoforms. Meta- and ortho-Tyr are spontaneously generated during aerobic fed-batch production processes using Chinese hamster ovary (CHO) cell lines. Process induced meta- and ortho-Tyr but not proteinogenic para-Tyr are incorporated at Phe locations in Phe-starved CHO cultures expressing a recombinant mAb. Furthermore, meta- and ortho-Tyr are preferably misincorporated over Leu. Structural modeling of the l-phenylalanyl-tRNA-synthetase (PheRS) substrate activation site indicates a possible fit of non-cognate ortho-Tyr and meta-Tyr substrates. Dose-dependent misincorporations of Tyr isoforms support the hypothesis that meta- and ortho-Tyr are competing, alternative substrates for PheRS in CHO processes. Finally, easily accessible at-line surrogate markers for PheâTyr SV formation in biotherapeutic production were defined by the calculation of critical ratios for meta-Tyr/Phe and ortho-Tyr/Phe to support early prediction of SV probability, and finally, to allow for immediate process controlled PheâTyr SV prevention.
Assuntos
Anticorpos Monoclonais/biossíntese , Células CHO/enzimologia , Células CHO/metabolismo , Fenilalanina-tRNA Ligase/metabolismo , Proteínas Recombinantes/biossíntese , Tirosina/metabolismo , Animais , Anticorpos Monoclonais/genética , Domínio Catalítico , Cricetulus , Feminino , Leucina/metabolismo , Modelos Moleculares , Fenilalanina-tRNA Ligase/química , Conformação Proteica , Proteínas Recombinantes/genéticaRESUMO
Host cell protein (HCP) characterization is a crucial quality parameter for biotherapeutic drug safety and stability. With a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach, we identified ubiquitin in ultrafiltration/diafiltration (UF/DF) pools of one of our monoclonal antibody (mAb) products. Since ubiquitin occurs physiologically as a post-translational modification (PTM) involved in many cellular functions, we suspected the possibility that if identified as an HCP, it may occur as a covalent modification on the mAb. In fact, in this study we characterized and quantified the ubiquitin modification on the Fc domain of mAbX by data dependent acquisition (DDA) and data independent acquisition (DIA) - MS workflows. Covalent binding and site localization were confirmed by identifying a characteristic diglycine motif on the modified peptide. Initially observed reduced detectability of ubiquitin in samples prepared with native digestion was attributed to impaired digestion and subsequent removal along with the mAb in the precipitation step. Our work has contributed to a better understanding of ubiquitin as an HCP considering its specific features such as occurrence in different topologies and provided insight into how covalent binding to a drug product can affect its identification by MS when native digestion conditions are used.
Assuntos
Anticorpos Monoclonais , Espectrometria de Massas em Tandem , Ubiquitina , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Células CHO , Cricetulus , Espectrometria de Massa com Cromatografia Líquida , Processamento de Proteína Pós-Traducional , Espectrometria de Massas em Tandem/métodos , Ubiquitina/química , Ubiquitina/metabolismoRESUMO
This work illustrates the benefits and limitations of using ultra-short reversed phase liquid chromatography (RPLC) columns for the characterization of various complex bispecific antibodies after prolonged thermal stress at the middle-up level of analysis. First, we have demonstrated that alternative organic modifiers, such as isopropanol, can be used in RPLC mode without generating excessive pressure, thanks to the prototype 10 × 2.1 mm, 2.7 µm particle column. However, compared to acetonitrile, the selectivity was not improved, at least for the selected biopharmaceutical products. Importantly, very fast separations (sub-1 min) of high quality were systematically obtained for the different samples when using a spectroscopic detector, but a severe loss of performance was observed with mass spectrometry (MS) detection due to dispersion effects. Based on these results, there is a clear need to improve the interfacing between LC and MS (shorter/thinner tubing) to mitigate band broadening.
RESUMO
Multiply deprotonated anions [M - nH](n-) of large peptide mellitin, ubiquitin, and beta-casein proteins were subjected to laser irradiation at 260 nm in a quadrupole ion trap. For all compounds, the predominant event consecutive to laser irradiation was the detachment of an electron. The subsequent isolation and collisional activation of the oxidized [M - nH]((n-1)-*) resulted in extensive fragmentation of the peptide backbone. For mellitin peptide, nearly a complete series of c(*), z, and a(*), x product ions were observed. Applied to proteins, this technique, coined as activated-electron photodetachment dissociation (activated-EPD), achieved much more extensive sequence coverage than regular collision activated dissociation (CAD) on the even-electron components. Furthermore, the activated-EPD spectrum of beta-casein displayed phosphorylated fragment ions which suggest that the method is able to preserve part of the labile bonds of post-translational modifications. Activated-EPD is, therefore, a promising complementary technique to other dissociation techniques governed by radicals, i.e., electron capture dissociation (ECD), electron transfer dissociation (ETD), and electron detachment dissociation (EDD), for the structural characterization of large peptides and small proteins.
Assuntos
Elétrons , Processos Fotoquímicos , Proteínas/química , Sequência de Aminoácidos , Ânions/química , Espectrometria de Massas , Dados de Sequência Molecular , Fragmentos de Peptídeos/químicaRESUMO
Monoclonal antibodies (mAbs) and proteins containing antibody domains are the most prevalent class of biotherapeutics in diverse indication areas. Today, established techniques such as immunization or phage display allow for an efficient generation of new mAbs. Besides functional properties, the stability of future therapeutic mAbs is a key selection criterion which is essential for the development of a drug candidate into a marketed product. Therapeutic proteins may degrade via asparagine (Asn) deamidation and aspartate (Asp) isomerization, but the factors responsible for such degradation remain poorly understood. We studied the structural properties of a large, uniform dataset of Asn and Asp residues in the variable domains of antibodies. Their structural parameters were correlated with the degradation propensities measured by mass spectrometry. We show that degradation hotspots can be characterized by their conformational flexibility, the size of the C-terminally flanking amino acid residue, and secondary structural parameters. From these results we derive an accurate in silico prediction method for the degradation propensity of both Asn and Asp residues in the complementarity-determining regions (CDRs) of mAbs.
Assuntos
Asparagina/química , Ácido Aspártico/química , Região Variável de Imunoglobulina/química , Relação Estrutura-Atividade , Inteligência Artificial , Asparagina/metabolismo , Ácido Aspártico/metabolismo , Região Variável de Imunoglobulina/metabolismo , Redes e Vias Metabólicas , Modelos Moleculares , Conformação Molecular , Proteólise , Curva ROCRESUMO
Sequence variants in recombinant biopharmaceuticals may have a relevant and unpredictable impact on clinical safety and efficacy. Hence, their sensitive analysis is important throughout bioprocess development. The two stage analytical approach presented here provides a quick multi clone comparison of candidate production cell lines as a first stage, followed by an in-depth analysis including identification and quantitation of aberrant sequence variants of selected clones as a second stage. We show that the differential analysis is a suitable tool for sensitive and fast batch to batch comparison of recombinant proteins. The optimized approach allows for detection of not only single amino acid substitutions in unmodified peptides, but also substitutions in posttranslational modified peptides such as glycopeptides, for detection of truncated or elongated sequence variants as well as double amino acid substitutions or substitution with amino acid structural isomers within one peptide. In two case studies we were able to detect sequence variants of different origin down to a sub percentage level. One of the sequence variants (Thr â Asn) could be correlated to a cytosine to adenine substitution at DNA (desoxyribonucleic acid) level. In the second case we were able to correlate the sub percentage substitution (Phe â Tyr) to amino acid limitation in the chemically defined fermentation medium.
Assuntos
Anticorpos Monoclonais/química , Imunoglobulina G/química , Software , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/isolamento & purificação , Sequência de Bases , Células CHO , Cromatografia em Gel , Cricetinae , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoglobulina G/genética , Imunoglobulina G/isolamento & purificação , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fenilalanina/genética , Mutação Puntual , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Padrões de Referência , Análise de Sequência de DNA , Análise de Sequência de Proteína/métodos , Espectrometria de Massas em Tandem/normas , Tripsina/químicaRESUMO
We compare product-ion mass spectra produced by electron detachment dissociation (EDD) and electron photodetachment dissociation (EPD) of multi-deprotonated peptides on a Fourier transform and a linear ion trap mass spectrometer, respectively. Both methods, EDD and EPD, involve the electron emission-induced formation of a radical oxidized species from a multi-deprotonated precursor peptide. Product-ion mass spectra display mainly fragment ions resulting from backbone cleavages of C(alpha)-C bond ruptures yielding a and x ions. Fragment ions originating from N-C(alpha) backbone bond cleavages are also observed, in particular by EPD. Although EDD and EPD methods involve the generation of a charge-reduced radical anion intermediate by electron emission, the product ion abundance distributions are drastically different. Both processes seem to be triggered by the location and the recombination of radicals (both neutral and cation radicals). Therefore, EPD product ions are predominantly formed near tryptophan and histidine residues, whereas in EDD the negative charge solvation sites on the backbone seem to be the most favorable for the nearby bond dissociation.