CMC Regulatory Considerations for Antibody-Drug Conjugates.

Antibody-drug conjugates unite the specificity and long circulation time of an antibody with the toxicity of a chemical cytostatic or otherwise active drug using appropriate chemical linkers to reduce systemic toxicity and increase therapeutic index. This combination of a large biological molecule and a small molecule creates an increase in complexity. Multiple production processes are required to produce the native antibody, the drug and the linker, followed by conjugation of afore mentioned entities to form the final antibody-drug conjugate. The connected processes further increase the number of points of control, resulting in necessity of additional specifications and intensified analytical characterization. By combining scientific understanding of the production processes with risk-based approaches, quality can be demonstrated at those points where control is required and redundant comparability studies, specifications or product characterization are avoided. Over the product development lifecycle, this will allow process qualification to focus on those areas critical to quality and prevent redundant studies. The structure of the module 3 common technical document for an ADC needs to reflect each of the production processes and the combined overall approach to quality. Historically, regulatory authorities have provided varied expectations on its structure. This paper provides an overview of essential information to be included and shows that multiple approaches work as long as adequate cross-referencing is included.

Controlled release of therapeutic antibody formats.

The local administration of antibodies can represent in many cases a significant improvement for antibody-based therapies. The benefits of local delivery include high drug concentrations at the target site, the possibility of lower drug dosing and less systemic drug exposure. Currently, the most relevant delivery sites for therapeutic antibodies are the posterior segments of the eye, mucosal surfaces, the articular joints and the central nervous system (CNS). In addition, the oral and pulmonary route may enable non-invasive systemic antibody delivery. However, local antibody delivery to these sites is characterized by short drug residence times and a low compliance of administration. Controlled release (CR) systems can address these limitations and, thereby, enable and improve local delivery applications by achieving long lasting local drug concentrations, improved efficacy-dosing ratios and reduced treatment-associated side effects. The requirements for CR antibody formulations are more complex compared to conventional CR systems for small molecules, and their development poses an enormous technical challenge. Therefore, the review highlights experiences and challenges gathered in the development of the different CR systems for antibodies to date. Additionally, the unmet technological needs encountered in the field are described. This includes a critical evaluation of the limited capability of various CR systems to preserve antibody stability, delivery site specific considerations, as well as the processability of a CR system with a particular focus on drug loading and injectability. We believe that the success of CR and local delivery approaches could create an enormous added value for patients in the future.

Pharmacokinetics, biocompatibility and bioavailability of a controlled release monoclonal antibody formulation.

The sustained and localized delivery of monoclonal antibodies has become highly relevant, because of the increasing number of investigated local delivery applications in recent years. As the local delivery of antibodies is associated with high technological hurdles, very few successful approaches have been reported in the literature so far. Alginate-based delivery systems were previously described as promising sustained release formulations for monoclonal antibodies (mAbs). In order to further investigate their applicability, a single-dose animal study was conducted to compare the biocompatibility, the pharmacokinetics and the bioavailability of a human monoclonal antibody liquid formulation with two alginate-based sustained delivery systems after subcutaneous administration in rats. 28 days after injection, the depot systems were still found in the subcutis of the animals. A calcium cross-linked alginate formulation, which was injected as a hydrogel, was present as multiple compartments separated by subcutaneous tissue. An in situ forming alginate formulation was recovered as a single compact and cohesive structure. It can be assumed that the multiple compartments of the hydrogel formulation led to almost identical pharmacokinetic profiles for all tested animals, whereas the compact nature of the in situ forming system resulted in large interindividual variations in pharmacokinetics. As compared to the liquid formulation the hydrogel formulations led to lower mAb serum levels, and the in situ forming system to a shift in the time to reach the maximum mAb serum concentration (Tmax) from 2 to 4 days. Importantly, it was shown that after 28 days only marginal amounts of residual mAb were present in the alginate matrix and in the tissue at the injection site indicating nearly complete release. In line with this finding, systemic drug bioavailability was not affected by using the controlled release systems. This study successfully demonstrates the suitability and underlines the potential of polyanionic systems for local and controlled mAb delivery.

Protein-polyanion interactions for the controlled release of monoclonal antibodies.

The objective of the present study was to investigate ionic interactions between alginate and a monoclonal antibody (mAb1) and to utilize those interactions for the sustained release of mAb1. The existence of ionic interactions between alginate and mAb1 was strongly reflected by their rheological behavior. A 3-4 times increase in storage modulus (G') was observed by addition of 30 mg/mL mAb1 to a 20 mg/mL alginate solution. This increase was strongly dependent on pH and ionic strength. In vitro release studies revealed a marked pH-dependence of release rates and the reversibility of alginate-mAb1 complexation under physiological conditions. Two alginate-mAb1 sustained release formulations were developed by an internal gelation technique using CaCO(3) and CaHPO(4) as calcium sources for physical cross-linking. The CaCO(3) formulation provided a stable pH-environment, optimally suited for pH-sensitive proteins. CaHPO(4) led to a lower pH and stronger alginate-mAb1 interactions. The CaHPO(4) cross-linked alginate released mAb1 over a period of 10-15 days. The long release period and changes in viscoelastic properties of alginate, when being mixed with mAb1, indicate the incorporation of mAb1 molecules into a mixed network with alginate. The results of this study demonstrate that ionic interactions between polyanions and mAb1 are present and that they can be exploited for sustained release delivery of mAb1.

P-body formation is a consequence, not the cause, of RNA-mediated gene silencing.

P bodies are cytoplasmic domains that contain proteins involved in diverse posttranscriptional processes, such as mRNA degradation, nonsense-mediated mRNA decay (NMD), translational repression, and RNA-mediated gene silencing. The localization of these proteins and their targets in P bodies raises the question of whether their spatial concentration in discrete cytoplasmic domains is required for posttranscriptional gene regulation. We show that processes such as mRNA decay, NMD, and RNA-mediated gene silencing are functional in cells lacking detectable microscopic P bodies. Although P bodies are not required for silencing, blocking small interfering RNA or microRNA silencing pathways at any step prevents P-body formation, indicating that P bodies arise as a consequence of silencing. Consistently, we show that releasing mRNAs from polysomes is insufficient to trigger P-body assembly: polysome-free mRNAs must enter silencing and/or decapping pathways to nucleate P bodies. Thus, even though P-body components play crucial roles in mRNA silencing and decay, aggregation into P bodies is not required for function but is instead a consequence of their activity.

Heating as a rapid purification method for recovering correctly-folded thermotolerant VH and VHH domains.

BACKGROUND: Recombinant antibodies from Camelidae (VHHs) are potentially useful tools for both basic research and biotechnological applications because of their small size, robustness, easy handling and possibility to refold after chemio-physical denaturation. Their heat tolerance is a particularly interesting feature because it has been recently related to both high yields during recombinant expression and selective purification of folded protein. RESULTS: Purification of recombinant RE3 VHH by heat treatment yielded the same amount of antibody as purification by affinity chromatography and negligible differences were found in stability, secondary structure and functionality. Similar results were obtained using another class of thermotolerant proteins, the single domain VH scaffold, described by Jespers et al. However, thermosensitive VHs could not withstand the heat treatment and co-precipitated with the bacterial proteins. In both cases, the thermotolerant proteins unfolded during the treatment but promptly refolded when moved back to a compatible temperature. CONCLUSION: Heat treatment can simplify the purification protocol of thermotolerant proteins as well as remove any soluble aggregate. Since the re-folding capability after heat-induced denaturation was previously correlated to higher performance during recombinant expression, a unique heating step can be envisaged to screen constructs that can provide high yields of correctly-folded proteins.