Antibodies are widely used affinity molecules in many fields of biological science and their use in therapy is constantly growing. The most common tool used for purification of antibodies is Protein A affinity chromatography, a method that offers high productivity and pure protein product. However, when eluting the captured antibodies from the column, low pH is needed which can be deleterious for certain antibodies and Fc-fusion proteins. We have addressed this issue by protein engineering. Through a combination of selection and directed mutagenesis the behavior of an IgG-binding domain from Protein A was improved resulting in ZCa, that allows for considerably milder elution of the captured antibody, since efficient release can be achieved by merely adding sodium chloride. This results in the release of IgG1 at pH 6 and antibodies of subclasses IgG2 and IgG4 at neutral pH. In contrast to elution at low pH using a commercial Protein A resin, this mild elution has been shown to eliminate the formation of antibody aggregates in the capture step. A tetrameric version of ZCa has demonstrated a binding capacity comparable to commonly used Protein A resins, including exceptional selectivity and recovery. Further, the resin was proved to be highly robust by processing high antibody titers for a great number of cycles in a continuous antibody-production and purification process at pilot scale, rendering high yields. The mild and efficient purification strategy based on ZCa has the potential to enable the development of a broader range of antibodies, which cannot tolerate the current acidic conditions used in antibody capture.

Biopharmaceutical manufacturing from a microbial host has many advantages over traditional CHO systems and processing differences in primary recovery provide opportunities to explore alternate techniques that leverage the physical properties of the organism. High titers generated in the Pelican Expression Technology platform are retained within the cell post-fermentation and primary recovery methods can vary depending on the characteristics of the strain and protein stability. We demonstrated that a unique approach using osmotic shock is not only viable for manufacturing but may be a preferential method of protein release.

There is a constant need in the pharmaceutical industry to accelerate the production of antibodies and other large molecules in the early stages of R&D. Here we describe a high-throughput cell line generation process that negates the need to perform multiple transfections per expressed protein. Our results show that this process can be applied to a large number of samples with high protein yield and protein quality.

Genome-integrated as well as growth-decoupled E. coli expression systems enable continuous protein production. Efficient implementation requires suitable process strategies for cultivation and product recovery and purification. The presentation will show two case studies inclusive an economic evaluation with standard fed batch as benchmark.

The conventional His tag often leads to reduced expression level and cleavage is expensive, slow and often the required N-terminus is not achieved. An expression and purification system based on a circular permutated Caspase which generates an authentic N-terminus irrespective of the amino acid is presented.

This presentation will highlight some challenges and effective solutions from early to late stage of mAb development. Some major challenges  related to cell line development, upstream development, downstream development, analytical methods, DS/DP stability are discussed. Effective solutions are assessed and applied.

The cost and required time to conduct reliable process understanding and characterization limits the bioprocess understanding. The talk highlights several upstream showcases in which the combination of hybrid modeling and advanced design space screening methods increased process understanding while simultaneously significantly reduced the required experiments by up to 70%. The chosen model structure also enabled the usage of those models during up-scaling and easy implementation for process monitoring and control.

Single Pass Tangential Flow Filtration (SPTFF) systems can provide inline concentration of monoclonal antibodies and other biotherapeutics, both to resolve bottlenecks in existing processes and as part of the development of integrated continuous downstream processes. We have developed a model that can be used to optimize the design of SPTFF modules, specifically accounting for the concentration dependence of the antibody viscosity as well as the variation in flow rate and antibody concentration with position. Model simulations and experiments were used to examine the effects of channel width, length, and cassette staging (number of parallel cassettes in each stage) on SPTFF performance for specific process objectives.

Chromatography has been established as a central step in the purification of proteins, although this is often cost-determining and also has some disadvantages in terms of process engineering. As an alternative, magnetic separation is presented here, in which magnetic nanoparticles serve as a separation phase that can bind the target molecule directly in unclarified cell culture supernatants or cell lysates. In our case, depending on the system, cost-intensive functionalization of the particles can be omitted and a pilot-scale technical implementation is presented.

HIC is typically used as a polishing step to remove impurities in a non-platform protein purification process. In select instances, it has been employed as a capture step for novel biotherapeutics. In this work, we demonstrated that combining two kosmotropic salts can increase the DBC on HIC resin significantly compared to single salt systems. We demonstrated this improvement with three different dual salt systems using two model proteins.

In this case study, host cell protein reduction challenges were encountered with a recombinant protein conjugate expressed in CHO cells. A baseline process using conventional chromatography and filtration techniques was rapidly developed to support toxicology and Phase 1 production. However, HCP levels of 10,000-20,000 ng/mg were measured at the intermediate stage (pre-conjugation) of the process. Here we discuss the systematic approach to reduce HCP levels to within specification using strategies amenable to manufacturing.

The transformation from batch to integrated continuous bioprocessing only “shrinks” the unit-operations while necessary auxiliaries are drastically increased. This leads either to necessarily large hold tanks or high personal costs. To solve this bottleneck, we developed a device for a continuous on-demand reconstitution of media and buffers directly from solids.