A new outlook towards kidney injuries

Acute and chronic progression of injury to the kidney leads to the failure of the renal system and has become an increasingly important cause of morbidity and mortality. Present diagnosis detects the condition only after irreversible loss of 70 percent of kidney function. Current research is focused only on the clinical manifestations after the kidney injuries and not towards the exact cause of the condition. Here we propose a new outlook- that there is an involvement of a pathogen in the pathogenesis of kidney injuries. Basis for our proposal is given by the similarity of the pathogenesis events occurring between a classical example of hepatitis and kidney injuries. Furthermore, literature regarding the role of early kidney injury biomarkers in innate immunity indicates the involvement of the pathogen. Research in this theme possesses a strong possibility in the development of therapeutic, preventive and management strategies for the acute and chronic kidney injuries

Site-Specific Modification Of Proteins Mediated By Transglutaminase

Transglutaminase (TGase) catalyzes an acyl transfer reaction between the [gamma]-carboxamide group of glutamine (Gln) and the [epsilon]-amino group of lysine (Lys) residues to form a stable amide bond. The TGase reaction can be used for bioconjugation of an amino-derivative of poly-ethylene glycol (PEG) to protein drugs, leading to PEGylated proteins that display increased bioactivity and stability. The procedure was shown to lead to site-specific bioconjugation of few proteins, thus offering a valid alternative to the chemical methods of PEGylation in current use (1). Moreover, TGase can be used for site-specifically labeling proteins with fluorescent groups at the level of Gln or Lys for diagnostic applications. The TGase attack is not entirely dependent on a specific consensus sequence around the Gln or Lys residues. However, a correlation between the TGase-mediated sites of PEGylation and the chain flexibility has been observed (2). Our study was focused to elucidate the molecular features favoring the TGase mediated site-specific reactions on two extensively
studied model proteins, i.e. apomyoglobin (apoMb) and lysozyme. Besides amino-PEG, we used dansyl-cadaverine as an acyl acceptor
and N-carbobenzoxy-Gln-Gly-OH as acyl donor for the TGase reactions. The sites of protein modification were determined by fingerprinting and ESI mass spectrometry. Myoglobin in its holo form is not susceptible to TGase reactions due to its rigid conformation,but the apo form was conjugated with PEG and fluorescent labels at the level of helix F (chain segment 82–99). The NMR study on apoMb had earlier demonstrated increased flexibility of the helix F (3) and, moreover, several proteases cleave the 153-residue chain of apoMb at the level of helix F only (4). Therefore, the chain region attacked by both TGase and proteases is a flexible or unfolded site(s). Lysozyme in its disulfide crosslinked native state is highly resistant to proteases and TGase attack. However, the more dynamic three-disulphide derivative of lysozyme, lacking the Cys6–Cys127 disulfide bridge, is susceptible to TGase-mediated reactions, as well as limited proteolysis. These results indicate that the sites of TGase reactions and the sites of limited proteolysis have a clear analogy for their presence in flexible/disordered regions of protein substrates. Overall, our studies clearly demonstrate that TGase-mediated reactions occur only at disordered chain regions, as evidenced by the correlation between sites of the TGase reaction and sites of enhanced chain flexibility, the latter deduced from the crystallographic Bfactor

sniper shot pegylation tgase mediated site specific conjugation of peg to proteins

Commercially available recombinant protein drugs often cause immune reactions in the body which reduces their efficiency. Protein drugs can be PEGylated, (attachment of polyethylene glycol) to overcome this problem. PEGylation increases bioavailability by reduced immune reactions and decreased renal clearance [1]. So far the traditional approaches for PEGylation involve harsh reaction conditions which provide a heterogeneous product (PEG attached randomly to different sites) along with the formation of several byproducts. Due to heterogeneity, the PEGylated protein drug faces challenge for FDA approval. Therefore, there is an immense need to develop an approach which could generate a homogeneous PEGylated protein drug.The transglutaminase (TGase) is an enzyme which catalyzes specifically the formation of a covalent bond (-CONH-) between the glutamine residue and the amine group of the lysine. This TGase reaction can be engineered by substituting the lysine with primary amines, which results in the formation of a similar covalent bond between the glutamine and the primary amine [2]. We utilized the specificity of TGase by using primary PEG-amines for conjugation with the glutamine present in the model protein (apomyoglobin). The reaction conditions were optimized in order to get a mono-conjugated PEGylated derivative. The site of conjugation was determined by affinity purification of the modified peptides and characterized by the ESI Q-TOF mass spectrometer. Therefore, we were able to develop a site-specific PEGylated apomyoglobin without byproducts, which eventually reduced the derivative purification steps as compared with the traditional PEGylation approaches. This strategy was further implemented on commercial pharmaceutical proteins

Generating glycan variants for biological activity testing by means of parallel experimental design and multivariate analysis

For more than 20 years, the industry has mainly invested in productivity enhancements. Recently, the focus of cell-culture process development began to shift. The modulation of quality attributes of recombinant therapeutic protein has gained substantial interest as demonstrated by the plethora of recent publications describing the effect of cell culture media on post-translational modifications of recombinant proteins1. Focusing on glycosylation, our team has developed a toolbox of media design beyond the commonly known media components and a rational high-throughput experimental design method. We identified and tested a large variety of novel cell culture compatible chemical components in industrial relevant Chinese hamster ovary cell lines (CHO) expressing recombinant antibodies and antibody fusion molecules. The compounds were evaluated in five different parallel 96-DWP fed-batch experiments, considering their mode of biological action. Viable cell density, viability and product titer were monitored and purified supernatants underwent N-glycan analysis by 2AB-UPLC and site-specific glycan-peptide analysis. Multivariate analysis identified the best performing glycosylation modulators, which were confirmed in spin tubes. Intracellular nucleotide and nucleotide sugar levels were analyzed by capillary electrophoresis, the gene expression by next-generation sequencing technologies, and the impact of the generated glycan variants on the biological activity was assessed. Non-targeted metabolite profiling was carried out to build a multivariate model linking metabolites with the glycan fingerprint. The screening experiments in 96-DWP produced a large glycosylation distribution diversity2,3. Subsequent D-optimal quadratic design in shake tubes confirmed the outcome of the selection process and provided a solid basis for sequential process development at a larger scale. The glycosylation profile with respect to the glycosylation specifications was greatly improved in shake tube experiments: 75% of the conditions were equally close or closer to the specifications than the best 25% in 96-deepwell plates. Further enhancement enabled us to generate extreme glycosylation variants, including high mannose, afucosylated, galactosylated as well as sialic acid species of both a mAb and an antibody fusion molecule with three N-glycosylation sites. The glycan variants induced significant responses in the respective in vitro biological activity assays. Moreover, metabolites correlating with time-dependent glycan profiling data were pinpointed and the glycan distribution of an external data set predicted. Our data highlight the great potential of cell culture medium optimization to modulate product quality and show the feasibility of the generation of a wide range of glycan variants suitable for biological activity testing. [1] Brühlmann D, Jordan M, Hemberger J, Sauer M, Stettler M and Broly H, Tailoring recombinant protein quality by rational media design, Biotechnology Progress 2015, 31:615–629. [2] Brühlmann D, Muhr A, Parker R, Vuillemin T, Bucsella B, Torre S, La Neve F, Lembo A, Haas T, Sauer M, Souquet J, Broly H, Hemberger J, Jordan M, Cell culture media supplemented with raffinose reproducibly enhances high mannose glycan formation, Journal of Biotechnology 2017, 252:32-42. [3] Brühlmann D, Sokolov M, Butté A, Sauer M, Hemberger J, Souquet J, Broly H, Jordan M, Parallel experimental design and multivariate analysis provides efficient screening of cell culture media supplements to improve Biosimilar product quality, Biotechnology and Bioengineering 2017, 114(7):1363-1631

Site-specific modification of proteins by transglutaminase

Site-specific modification of proteins by transglutaminase Transglutaminase (TGase; EC 2.3.2.13) catalyzes the reaction between the γ-amido group of a protein-bound Gln residue (–CONH2, the acceptor) and an amino group (–NH2, the donor) of an alkyl-amine (protein-CONH2 + H2N-ligand → protein-CONH-ligand + NH3). In the case of protein substrates, TGase causes an intra- and inter-molecular crosslinking of proteins by formation of an isopeptide bond involving the side chains of Gln and Lys residues. The acyl donor can be also a small amido-ligand mimicking the Gln residue, so that TGase allows a useful and interesting variability of substrates, thus leading to the modification of proteins at the level of Gln or Lys residues using appropriate substrate reagents. Recently, the microbial TGase from S. mobaraensis has attracted a strong interest for protein modification, considering its stability, high reactivity and small size. The X-ray structure of this TGase has been solved and shown to contain an active site given by a triad Cys-His-Asp in analogy to a protease. A striking result of recent studies is that reactions mediated by TGase can be site-specific with some proteins, sometimes leading to the modification of only one Gln residue among the many Gln residues of a protein substrate. On the other hand, there is only a moderate specificity for Lys residues. With the view to shed light into the molecular features dictating the site-specific reaction(s) of TGase, in this Thesis a number of TGase-mediated reactions have been studied using proteins of known structure and dynamics, as apomyoglobin (apoMb), egg-white lysozyme (LYS) and bovine pancreatic ribonuclease A (RNase). Amino- as well amido-ligands have been used in the TGase-mediated reactions, so that it was possible to analyse the specificity of modification of both Gln and Lys in the examined proteins. We have found an almost strict specificity of TGase-mediated reactions at the level Gln91 of apoMb, a residue embedded in the highly flexible or unfolded helix-F of the holo protein, as given by previous NMR measurements and limited proteolysis data. Also a Gln-mimicking ligand can be covalently linket by TGase at a Lys residue of the same chain region. Thus, we concluded that local enhanced flexibility or even fully local unfolding dictates the site-specific reaction with TGase. While RNase can be selectively modified by using an amido-ligand at the level of the ɛ-amino group of Lys1, a similar Lys1 of LYS was instead fully unreactive. It was possible to relate this finding to the flexibility and rigidity of the N-terminal region of RNase and LYS, respectively, on the basis of the crystallographically determined B-factor values (a measure of chain flexibility) of these two proteins. A nicked species of RNase with the single peptide bond Asn34-Leu35 cleaved (RNase Th1) and a LYS derivative with a single disulfide bridge reduced among the four of native LYS (LYSCM6, 127) were shown to be much more reactive in the TGase-mediated reactions than the parent intact proteins, in agreement with their enhanced flexibility or partial unfolding. Moreover, we could demonstrate that the sites or regions susceptible to TGase reactions are also prone to limited proteolysis phenomena, implying that both TGase and a protease require some local unfolding for a site-specific enzymatic reaction. Indeed, this in keeping with view that the biorecognition phenomenon is similar for both enzymes, considering also the fact that TGase acts as a reverse protease (amide synthesis instead of hydrolysis). An interesting outcome of these studies resides in the fact that we can envisage a novel enzymatic method of covalent coupling of an amino-polymer as poly(ethylene)glycol (PEG) to specific Gln residue(s) of proteins of pharmaceutical interest. Indeed, using TGase and an amino derivative of PEG (PEG-NH2), it was possible to prepare homogeneous PEGylated derivatives of apoMb, human growth hormone (hGH) and granulocyte colony-stimulating factor (G-CSF). Overall, we have interpreted our findings as indicating that the selective TGase-mediated reactions require a flexible or unfolded polypeptide substrate. Therefore, it is possible to predict the sites of TGase attack on a protein substrate, provided that its structure and dynamics are known. Considering the increasing relevance of PEGylated protein drugs and the high regulatory demands for their approval, it can be anticipated that the innovative methods for the site-specific PEGylation of proteins using TGase will be considered a useful advance in the methodologies used for protein modification.Modifica sito-specifica di proteine con transglutaminasi La transglutaminasi (TGasi; EC 2.3.2.13) catalizza la reazione tra il gruppo γ-ammidico di un residuo di Gln (-CONH2, accettore) ed un gruppo amminico (-NH2, donatore) di una alchil-ammina (proteina-CONH2 + H2N-ligando → proteina-CONH-ligando + NH3). Con substrati proteici la TGasi determina una reticolazione intra- e inter-molecolare di proteine mediante la formazione di un legame isopeptidico tra le catene laterali dei residui di Gln e Lys. Il donatore acilico può essere anche un ammido-ligando in grado di mimare la catena laterale di Gln e, pertanto, la TGase consente una modifica di proteine a livello dei residui di Gln o Lys utilizzando opportuni reagenti. Recentemente, la TGase microbica da S. mobaraensis ha suscitato un notevole interesse per la modifica enzimatica di proteine, considerando la sua stabilità, elevata reattività e piccole dimensioni. La struttura ai raggi-X di questa TGase ha rivelato la presenza di un sito attivo costituito da una triade Cys-His-Asp in analogia al sito attivo di una proteasi. Recenti studi hanno dimostrato che le reazioni mediate da TGase possono essere sito-specifiche, portando talvolta alla modifica di un solo residuo Gln tra i molti residui Gln di un substrato proteico. D'altra parte, è stata accertata solo una moderata specificità per i residui di Lys. Con lo scopo di chiarire i motivi strutturali che determinano la specificità di azione della TGase, in questa Tesi sono state studiate una serie di reazioni TGase-mediate utilizzando proteine di struttura e dinamica note, come apomioglobina (apoMb), lisozima da bianco d’uovo (LYS) e ribonucleasi pancreatica bovina (RNase). Sono stati utilizzati sia ammino- che ammido-ligandi nelle reazioni con TGase ed in tal modo è stato possibile analizzare la specificità di modifica sia a livello di Gln che di Lys con le proteine esaminate. E’ stata accertata una specificità rigorosa delle reazioni TGase-mediate a livello di Gln91 di apoMb, un residuo localizzato nel segmento corrispondente all’elica F della proteina nativa e risultato molto flessibile o unfolded in apoMb da precedenti misure NMR e da dati di proteolisi limitata. Anche un ligando in grado di mimare il residuo di Gln può essere legato da TGase a livello di una Lys incorporata nello stesso segmento flessibile. Pertanto, è stato concluso che una elevata flessibilità locale o unfolding determina la reazione sito-specifica di TGase. Mentre con la RNase si può ottenere con TGase ed un ammido-ligando la specifica modifica a livello del gruppo ɛ-aminico di Lys1, la simile Lys1 del LYS non reagisce affatto in simili condizioni. E 'stato possibile mettere in relazione questo fatto con la flessibilità e la rigidità della regione N-terminale di RNase e LYS, rispettivamente, sulla base dei valori del fattore-B (correlato alla flessibilità della catena polipeptidica) di queste due proteine ottenuti da dati cristallografici. Un derivato di RNase con il singolo legame peptidico Asn34-Leu35 idrolizzato (RNase Th1) ed un derivato di LYS con un ponte disolfuro ridotto (LYSCM6, 127) sono risultati molto più reattivi nelle reazioni con TGase rispetto alle corrispondenti proteine native, in accordo con la loro maggiore flessibilità o parziale denaturazione. Inoltre, abbiamo potuto dimostrare che i siti o regioni suscettibili di reazioni con TGase subiscono anche fenomeni di proteolisi limitata, significando che sia la TGase che una proteasi necessitano di un local unfolding per determinare una reazione enzimatica sito-specifica. In effetti, questo è linea con il fatto che il bioriconoscimento del substrato è simile per entrambi gli enzimi, considerando il fatto che la TGase è una proteasi inversa (sintesi invece che idrolisi di una ammide). Un risultato interessante di questi studi risiede nel fatto che si apre la strada ad un nuovo metodo enzimatico di binding covalente di poli(etilene)glicole (PEG) a livello di specifici residui di Gln in proteine di interesse farmaceutico. Infatti, utilizzando TGase ed un ammino-derivato di PEG (PEG-NH2), è stato possibile preparare derivati peghilati omogenei di apoMb, ormone della crescita umano (hGH) e granulocyte colony-stimulating factor (G-CSF). In generale, i risultati ottenuti hanno indicato chiaramente che le reazioni selettive di TGase richiedono un substrato flessibile o unfolded. Pertanto, è possibile prevedere i siti di attacco di TGase su un substrato proteico, se sono note la sua struttura e dinamica. Considerando la crescente importanza dei farmaci proteici peghilati e le speciali richieste di omogeneità dettate dalle autorità regolatorie, si può prevedere che la peghilazione sito-specifica con TGase di proteine di interesse farmaceutico troverà utili applicazioni

In-vivo biological activity and glycosylation analysis of a biosimilar recombinant human follicle-stimulating hormone product (Bemfola) compared with its reference medicinal product (GONAL-f).

Recombinant human follicle-stimulating hormone (r-hFSH) is widely used in fertility treatment. Although biosimilar versions of r-hFSH (follitropin alfa) are currently on the market, given their structural complexity and manufacturing process, it is important to thoroughly evaluate them in comparison with the reference product. This evaluation should focus on how they differ (e.g., active component molecular characteristics, impurities and potency), as this could be associated with clinical outcome. This study compared the site-specific glycosylation profile and batch-to-batch variability of the in-vivo bioactivity of Bemfola, a biosimilar follitropin alfa, with its reference medicinal product GONAL-f. The focus of this analysis was the site-specific glycosylation at asparagine (Asn) 52 of the α-subunit of FSH, owing to the pivotal role of Asn52 glycosylation in FSH receptor (FSHR) activation/signalling. Overall, Bemfola had bulkier glycan structures and greater sialylation than GONAL-f. The nominal specific activity for both Bemfola and GONAL-f is 13,636 IU/mg. Taking into account both the determined potency and the nominal amount the average specific activity of Bemfola was 14,522 IU/mg (105.6% of the nominal value), which was greater than the average specific activity observed for GONAL-f (13,159 IU/mg; 97.3% of the nominal value; p = 0.0048), although this was within the range stated in the product label. A higher batch-to-batch variability was also observed for Bemfola versus GONAL-f (coefficient of variation: 8.3% vs 5.8%). A different glycan profile was observed at Asn52 in Bemfola compared with GONAL-f (a lower proportion of bi-antennary structures [~53% vs ~77%], and a higher proportion of tri-antennary [~41% vs ~23%] and tetra-antennary structures [~5% vs <1%]). These differences in the Asn52 glycan profile might potentially lead to differences in FSHR activation. This, together with the greater bioactivity and higher batch-to-batch variability of Bemfola, could partly explain the reported differences in clinical outcomes. The clinical relevance of the differences observed between GONAL-f and Bemfola should be further investigated

Glycan and antennarity and sialylation distribution at Asn52.

<p><b>A)</b> Comparison of glycan distribution at Asn52 between Bemfola and GONAL-f (individual species) <b>B)</b> Comparison of antennarity and sialylation at Asn52 between Bemfola and GONAL-f. Asn, asparagine.</p