Evaluation of single-use tangential flow filtration technology for purification of activated polysaccharides used in conjugate vaccine manufacturing.

Over the past decade, single-use tangential flow filtration (TFF) technologies have emerged to reduce system preparation time, promote fast and flexible product change over, and ultimately shorten process development and manufacturing time/cost. In this study, the performance of a recently developed Pellicon® single-use TFF capsule was compared against traditional Pellicon® cassettes by assessing TFF process performance (such as flux, residuals clearance, and yield) and post-purification product attributes (such as concentration and mass-weighted average molecular weight). Good scaling was shown by comparing process performance and product attributes across different scales and formats. Additionally, similar TFF process performance and post-purification product attributes were observed for the single-use capsule compared to the reusable TFF cassettes. The capsule requires a smaller flush than the cassette, and it is easier to use since it does not require a compression holder or pre-sanitization. The results provide insight into the application of the single-use TFF capsule and scalability of TFF processes for the purification of conjugate vaccines.

Impact of Drug Conjugation and Loading on Target Antigen Binding and Cytotoxicity in Cysteine Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) consist of a target-specific antibody that is covalently conjugated to a drug via a linker. ADCs are designed to deliver cytotoxic drugs (payloads), specifically to cancer cells, while minimizing systemic toxicity. Conventional cysteine conjugation typically results in the formation of ADC molecules containing a heterogeneous mixture of 2, 4, 6, and 8 drug-loaded species. The drug-to-antibody ratio (DAR) of the mixture represents the weighted average of these species. In this report, we have investigated the impact of the hydrophobicity of payloads and the overall drug loading on the in vitro binding and cytotoxicity of ADC species. Several ADCs were prepared by conventional cysteine conjugation using different payloads. ADC species with different DAR values were purified from the ADC mixture and characterized by standard analytical techniques. These ADC species were evaluated for target antigen binding using an immunoassay, enzyme-linked immunosorbent assay (ELISA). The potency was assessed using a cell-based cytotoxicity assay. These structure-function studies lead to a better understanding of factors that impact the in vitro target binding and cytotoxicity of ADC species. ADC species containing hydrophobic payloads with high DAR were found to have lower target binding by ELISA compared to that of the unconjugated antibody or the heterogeneous reference ADC with DAR ∼4. Under similar assay conditions, the ADCs conjugated to hydrophilic payloads did not show a significant impact on the target binding. The cytotoxic potency of ADC species increased with increasing level of drug loading in the cell-based cytotoxicity assay.

Analysis of the DNA joining repertoire of Chlorella virus DNA ligase and a new crystal structure of the ligase-adenylate intermediate.

Chlorella virus DNA ligase is the smallest eukaryotic ATP-dependent DNA ligase known; it suffices for yeast cell growth in lieu of the essential yeast DNA ligase Cdc9. The Chlorella virus ligase-adenylate intermediate has an intrinsic nick sensing function and its DNA footprint extends 8-9 nt on the 3'-hydroxyl (3'-OH) side of the nick and 11-12 nt on the 5'-phosphate (5'-PO4) side. Here we establish the minimal length requirements for ligatable 3'-OH and 5'-PO4 strands at the nick (6 nt) and describe a new crystal structure of the ligase-adenylate in a state construed to reflect the configuration of the active site prior to nick recognition. Comparison with a previous structure of the ligase-adenylate bound to sulfate (a mimetic of the nick 5'-PO4) suggests how the positions and contacts of the active site components and the bound adenylate are remodeled by DNA binding. We find that the minimal Chlorella virus ligase is capable of catalyzing non-homologous end-joining reactions in vivo in yeast, a process normally executed by the structurally more complex cellular Lig4 enzyme. Our results suggest a model of ligase evolution in which: (i) a small 'pluripotent' ligase is the progenitor of the much larger ligases found presently in eukaryotic cells and (ii) gene duplications, variations within the core ligase structure and the fusion of new domains to the core structure (affording new protein-protein interactions) led to the compartmentalization of eukaryotic ligase function, i.e. by enhancing some components of the functional repertoire of the ancestral ligase while disabling others.

Role of nucleotidyltransferase motifs I, III and IV in the catalysis of phosphodiester bond formation by Chlorella virus DNA ligase.

ATP-dependent DNA ligases catalyze the sealing of 5'-phosphate and 3'-hydroxyl termini at DNA nicks by means of a series of three nucleotidyl transfer steps. Here we have analyzed by site-directed mutagenesis the roles of conserved amino acids of Chlorella virus DNA ligase during the third step of the ligation pathway, which entails reaction of the 3'-OH of the nick with the DNA-adenylate intermediate to form a phosphodiester and release AMP. We found that Asp65 and Glu67 in nucleotidyltransferase motif III and Glu161 in motif IV enhance the rate of step 3 phosphodiester formation by factors of 20, 1000 and 60, respectively. Asp29 and Arg32 in nucleotidyltransferase motif I enhance the rate of step 3 by 60-fold. Gel shift analysis showed that mutations of Arg32 and Asp65 suppressed ligase binding to a pre-adenylated nick, whereas Asp29, Glu67 and Glu161 mutants bound stably to DNA-adenylate. We infer that Asp29, Glu67 and Glu161 are involved directly in the step 3 reaction. In several cases, the effects of alanine or conservative mutations on step 3 were modest compared to their effects on the composite ligation reaction and individual upstream steps. These results, in concert with available crystallographic data, suggest that the active site of DNA ligase is remodeled during the three steps of the pathway and that some of the catalytic side chains play distinct roles at different stages.

Conserved residues in domain Ia are required for the reaction of Escherichia coli DNA ligase with NAD+.

NAD(+)-dependent DNA ligases are present in all bacteria and are essential for growth. Their unique substrate specificity compared with ATP-dependent human DNA ligases recommends the NAD(+) ligases as targets for the development of new broad-spectrum antibiotics. A plausible strategy for drug discovery is to identify the structural components of bacterial DNA ligase that interact with NAD(+) and then to isolate small molecules that recognize these components and thereby block the binding of NAD(+) to the ligase. The limitation to this strategy is that the structural determinants of NAD(+) specificity are not known. Here we show that reactivity of Escherichia coli DNA ligase (LigA) with NAD(+) requires N-terminal domain Ia, which is unique to, and conserved among, NAD(+) ligases but absent from ATP-dependent ligases. Deletion of domain Ia abolished the sealing of 3'-OH/5'-PO(4) nicks and the reaction with NAD(+) to form ligase-adenylate but had no effect on phosphodiester formation at a preadenylated nick. Alanine substitutions at conserved residues within domain Ia either reduced (His-23, Tyr-35) or abolished (Tyr-22, Asp-32, Asp-36) sealing of a 5'-PO(4) nick and adenylyl transfer from NAD(+) without affecting ligation of pre-formed DNA-adenylate. We suggest that these five side chains comprise a binding site for the nicotinamide mononucleotide moiety of NAD(+). Structure-activity relationships were clarified by conservative substitutions.

Role of nucleotidyl transferase motif V in strand joining by chlorella virus DNA ligase.

ATP-dependent DNA ligases, NAD(+)-dependent DNA ligases, and GTP-dependent RNA capping enzymes are members of a covalent nucleotidyl transferase superfamily defined by a common fold and a set of conserved peptide motifs. Here we examined the role of nucleotidyl transferase motif V ((184)LLKMKQFKDAEAT(196)) in the nick joining reaction of Chlorella virus DNA ligase, an exemplary ATP-dependent enzyme. We found that alanine substitutions at Lys(186), Lys(188), Asp(192), and Glu(194) reduced ligase specific activity by at least an order of magnitude, whereas substitutions at Lys(191) and Thr(196) were benign. The K186A, D192A, and E194A changes had no effect on the rate of single-turnover nick joining by preformed ligase-adenylate but affected subsequent rounds of nick joining at the ligase adenylation step. Conservative substitutions K186R, D192E, and E194D partially restored activity, whereas K186Q, D192N, and E194Q substitutions did not. Alanine mutation of Lys(188) elicited distinctive catalytic defects, whereby single-turnover nick joining by K188A-adenylate was slowed by an order of magnitude, and high levels of the DNA-adenylate intermediate accumulated. The rate of phosphodiester bond formation at a pre-adenylated nick (step 3 of the ligation pathway) was slowed by the K188A change. Replacement of Lys(188) by arginine reversed the step 3 arrest, whereas glutamine substitution was ineffective. Gel-shift analysis showed that the Lys(188) mutants bound stably to DNA-adenylate. We infer that Lys(188) is involved in the chemical step of phosphodiester bond formation.