Development of a high-throughput microscale cell disruption platform for Pichia pastoris in rapid bioprocess design

The time and cost benefits of miniaturized fermentation platforms can only be gained by employing complementary techniques facilitating high-throughput at small sample volumes. Microbial cell disruption is a major bottleneck in experimental throughput and is often restricted to large processing volumes. Moreover, for rigid yeast species such as Pichia pastoris, no effective high-throughput disruption methods exist. This study describes the development of an automated, miniaturized, high-throughput, non-contact, scalable platform based on Adaptive Focused Acoustics (AFA) to disrupt P. pastoris and recover intracellular heterologous protein. Augmented modes of AFA were established by investigating vessel designs and a novel enzymatic pre-treatment step. Three different modes of AFA were studied and compared to the performance high pressure homogenization. For each of these modes of cell disruption, response models were developed to account for five different performance criteria. Using multiple responses not only demonstrated that different operating parameters are required for different response optima, with highest product purity requiring suboptimal values for other criteria, but also allowed for AFA-based methods to mimic large-scale homogenization processes. These results demonstrate that AFA-mediated cell disruption can be used for a wide range of applications including buffer development, strain selection, fermentation process development and whole bioprocess integration. This article is protected by copyright. All rights reserved

Cell free protein synthesis: a viable option for stratified medicines manufacturing?

Stratified medicines are defined as medicines which target diseases where the patients have been preselected for treatment based on their response to a diagnostic test. The pipeline of these medicines cover a wide range of different treatment types including cell and gene therapies, vaccines based on peptides or proteins; and protein based therapies. These increasingly diverse and by definition smaller market size products require improved agility and productivity in process design if manufacture and supply of affordable medicines is to be achieved. In this paper we review the current state of cell free synthesis (CFS), the new technologies and strategies being developed and its application to the production of stratified medicines; focusing on the production of protein based therapeutic products

Versatile cell-free protein synthesis systems based on chinese hamster ovary cells

We present an alternative production platform for the synthesis of complex proteins. Apart from conventionally applied protein production using engineered mammalian cell lines, this protocol describes the preparation and principle of cell-free protein synthesis systems based on CHO cell lysates. The CHO cell-free system contains endogenous microsomes derived from the endoplasmic reticulum, which enables a direct integration of membrane proteins into a nature like milieu and the introduction of posttranslational modifications. Different steps of system development are described including the cultivation of CHO cells, cell harvesting and cell disruption to prepare translationally active CHO cell lysates. The requirements for DNA templates and the generation of linear DNA templates suitable for the CHO cell-free reaction is further depicted to underline the opportunity to produce different protein variants in a short period. This experimental setup provides a basis for hig h-throughput applications. The productivity of the CHO cell-free systems is further increased by using a non-canonical translation initiation due to the attachment of an internal ribosomal entry site of the Cricket paralysis virus (CRPV IRES) to the 5´ UTR of the desired gene. In this way, a direct interaction of the IRES structure with the ribosome facilitates a translation factor independent initiation of translation. Cell-free reactions were performed in fast and efficient batch reactions leading to protein yields up to 40 μg/mL. The reaction format was further adjusted to a continuous exchange CHO cell-free reaction (CHO CECF) to prolong reaction time and thereby increase the productivity of the cell-free systems. Finally, protein yields up to 1 g/L were obtained. The CHO CECF system represents a sophisticated resource to address structural and functional aspects of difficult-to-express proteins in fundamental and applied research

Analysis of recombinant glycoproteins by mass spectrometry

The advent of new technologies for analysis of biopolymers by mass spectrometry has revolutionised strategies for recombinant protein characterization. The principal recent developments have been matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. Using these tools, accurate molecular mass determinations can now be obtained routinely - often using minute (picomole - femtomole) quantities of protein or protein fragments. These techniques have proved indispensible for detailed characterization of the post-translational modifications of recombinant proteins produced by eukaryotic systems. Glycosylation is arguably the most important and complex of these modifications and has prompted widespread use of these new techniques. In this mini-review article I describe recent advances in the use of mass spectrometry for analysis of recombinant glycoproteins

Getting the glycosylation right: Implications for the biotechnology industry

Glycosylation is the most extensive of all the posttranslational modifications, and has important functions in the secretion, antigenicity and clearance of glycoproteins. In recent years major advances have been made in the cloning of glycosyltransferase enzymes, in understanding the varied biological functions of carbohydrates, and in the accurate analysis of glycoprotein heterogeneity. In this review we discuss the impact of these advances on the choice of a recombinant host cell line, in optimizing cell culture processes, and in choosing the appropriate level of glycosylation analysis for each stage of product development

Synthetic biology for the rapid, precise and compliant detection of microbes

Since the turn of the millennium, an extensive range of applications spanning across science and technology have been turning to synthetic biology for inspiration and innovation in order to enhance, accelerate and permeate their specific fields. The field of microbial detection is no different. Modern synthetic biology offers pioneering approaches to accelerate and fine-tune the detection process, subsequently enabling clinicians to offer fast, targeted treatments. This, essentially, will dramatically reduce medical burden across a wide array of diseases and will provide a crucial step towards limiting the use of antibiotic treatment to completely necessary cases only. In this chapter, some of the key synthetic biology-inspired approaches that are transforming the field of microbial detection are explored, with an emphasis on what the near future holds