The therapeutic efficacy and pharmacokinetics of antibody−drug conjugates (ADCs) in general, and antibody−oligonucleotide conjugates (AOCs) in particular, depend on the drug-to-antibody ratio (DAR) distribution and average value. The DAR is considered a critical quality attribute, and information pertaining to it needs to be gathered during ADC/AOC development, production, and storage. However, because of the high structural complexity of ADC/AOC samples, particularly in the initial drug-development stages, the application of the current state-of-the-art mass spectrometric approaches can be limited for DAR analysis. Here, we demonstrate a novel approach for the analysis of complex ADC/AOC samples, following native sizeexclusion chromatography Orbitrap Fourier transform mass spectrometry (FTMS). The approach is based on the integration of the proteoform-level mass spectral peaks in order to provide an estimate of the DAR distribution and its average value with less than 10% error. The peak integration is performed via a truncation of the Orbitrap's unreduced time-domain ion signals (transients) before mass spectra generation via FT processing. Transient recording and processing are undertaken using an external data acquisition system, FTMS Booster X2, coupled to a Q Exactive HF Orbitrap FTMS instrument. This approach has been applied to the analysis of whole and subunit-level trastuzumab conjugates with oligonucleotides. The obtained results indicate that ADC/AOC sample purification or simplification procedures, for example, deglycosylation, could be omitted or minimized prior to the DAR analysis, streamlining the drug-development process.
BACKGROUND Different strategies can be used to improve the tumor:non‐tumor ratio of radiolabeled antibodies in immunotargeting. One approach is to use secondary antibodies to clear out redundant, circulating primary antibodies. In the current study, the in vitro complex formation and in vivo clearing capabilities and metabolism of the monoclonal antibody TS1 and its monoclonal anti‐idiotype, αTS1, were studied. METHODS Complex formation studies were performed using polyacrylamide gel electrophoresis (PAGE), gel permeation chromatography, and electron microscopy. The clearance and metabolism of the complexes were studied in nude mice. RESULTS PAGE and gel permeation chromatography showed that more than 70% of the antibodies formed complexes. The electron microscopy studies revealed that the complexes formed between TS1 and αTS1 are mainly ring‐shaped (66.6–73.4%), comprising 4 to > 8 antibodies. These rings consist of equal numbers of idiotype and anti‐idiotype. The most commonly observed complexes were tetrameric rings (26.8–40.5%), hexameric rings (10.7–11.9%), and rings containing more than eight monoclonal antibodies (6.6–14‐4%). The in vivo study illustrated that within 24 hours 80% of the total nuclide content had been degraded and excreted via the urine, compared with 25% for similarly treated mice that did not receive any anti‐idiotype. CONCLUSIONS Interestingly, the electron microscopy study demonstrated that dimers were rare (0.4–1.2%), probably reflecting a location of epitopes incompatible with tight, sterically constrained dimeric interactions; insufficient flexibility of the immunoglobulin G1 subtype hinge regions; or both. The anti‐idiotypic clearing mechanisms proved efficient in nude mice. In vivo metabolic studies indicate that the accumulation and degradation of TS1/αTS1 immune complexes, to a large extent, take place in the liver, where a substantial amount was detected as soon as 1 hour after anti‐idiotype injection. Cancer 2002;94:1306–13. © 2002 American Cancer Society. DOI 10.1002/cncr.10301
BackgroundOptimization of conditions during recombinant protein production for improved yield is a major goal for protein scientists. Typically this is achieved by changing single crucial factor settings one at a time while other factors are kept fixed through trial-and-error experimentation. This approach may introduce larger bias and fail to identify interactions between the factors resulting in failure of finding the true optimal conditions.ResultsIn this study we have utilized design of experiments in order to identify optimal culture conditions with the aim to improve the final yield of the anti-keratin 8 scFv TS1-218, during expression in P. pastoris in shake flasks. The effect of: pH, temperature and methanol concentration on the yield of TS1-218 using buffered minimal medium was investigated and a predictive model established. The results demonstrated that higher starting pH and lower temperatures during induction significantly increased the yield of TS1-218. Furthermore, the result demonstrated increased biomass accumulation and cell viability at lower temperatures which suggested that the higher yield of TS1-218 could be attributed to lower protease activity in the culture medium. The optimal conditions (pH 7.1, temperature 11°C and methanol concentration 1.2%) suggested by the predictive model yielded 21.4 mg TS1-218 which is a 21-fold improvement compared to the yield prior to optimization.ConclusionThe results demonstrated that design of experiments can be utilized for a rapid optimization of initial culture conditions and that P. pastoris is highly capable of producing and secreting functional single-chain antibody fragments at temperatures as low as 11°C.
The monoclonal antibody TS1 against cytokeratin 8 and its antiidiotype alphaTS1 have been used for immunotargeting and therapy of carcinomas in experimental tumor model systems. The interaction surfaces between mab TS1, the cytokeratin 8 epitope, and its anti-idiotypic antibody, alphaTS1, were studied in detail in order to make future veneering of the interactions possible. The V-genes of TS1 and alphaTS1 were cloned and sequenced and the CDRs and the framework residues were identified. Amino acids participating in the interactions were identified following chemical modifications of residues in non-protected and protected molecules of cytokeratin 8, alphaTS1 and TS1. From the sequences, the three-dimensional structures were generated using computer modelling of the antibody variable regions. Several charged amino acid, histidine and tyrosine residues were displayed in the antibody surfaces implicated in the interactions and chemical modification confirmed the importance of these amino acids. The cytokeratin 8 epitope has previously been identified by Johansson et al. and it displays negatively charged amino acid residues which could be identified in the chemical modification. It was also revealed that the TS1 binding to cytokeratin 8 and alphaTS1 respectively are partly overlapping; a histidine identified in TS1 is probably involved only in the interaction with alphaTS1. Furthermore, the chemical modification demonstrated that exchanging aspartic-glutamic acids to asparagine-glutamine residues in TS1 increased the binding of TS1 to cytokeratin 8, indicating that there is at least one acidic amino acid that is an obstacle in the TS1-CK8 binding. The detailed assembly of the interaction surfaces will facilitate the future use of site directed mutagenesis to improve the TS1-CK8 association rate and the clearing of TS1 with alphaTS1 in vivo.
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