Manufacturing of recombinant proteins in microbial systems, in particular in E. coli, generally requires thorough process development due to the absence of a platform process. The caspase-based fusion process (CASPON (R)) offers a platform manufacturing process using special protease cleavable fusion-tags. These tags allow the implementation of His-tag based affinity chromatography for facile target protein capture and offer expression and solubility enhancing capabilities. The tags are intended as N-terminal fusion motifs that can be fully cleaved using a modified caspase-2 protease, the CASPON (R) enzyme. Here, we systematically explore the influence of various physicochemical parameters on its enzymatic activity. This characterization was performed in parallel in two independent research laboratories using different assays, i.e. a Forster resonance energy transfer-based assay using small peptide substrates and a reversed phase high performance liquid chromatography method using model proteins. Both assays demonstrated great agreement and reveal that CASPON (R) enzyme is highly active at a wide range of temperatures, pH and is resistant to a variety of chemical substances that are commonly employed in bioprocessing, e.g. NaCl, kosmotropes, chaotropes and others. The presence of imidazole at concentrations commonly found in the elution fraction of immobilized metal affinity chromatography does not seem to affect the activity of CASPON (R) enzyme, enabling its use directly after the capture step in downstream processing (DSP). A case study of five biopharmaceuticals is presented. The platform process exhibits consistently high performance in up- and downstream processing, achieving high soluble titers, high yield and purity. The presence of host cell proteins during the DSP in particular was investigated in-depth using process proteomics, revealing that a core of host cell proteins is process dependent and can commonly be expected to be found in the platform DSP. The information presented here can serve as a guide on how to implement the CASPON (R) platform process for the production of various recombinant proteins.

Continuous precipitation coupled with continuous tangential flow filtration is a cost-effective alternative for the capture of recombinant antibodies from crude cell culture supernatant. The removal of surge tanks between unit operations, by the adoption of tubular reactors, maintains a continuous harvest and mass flow of product with the advantage of a narrow residence time distribution (RTD). We developed a continuous process implementing two orthogonal precipitation methods, CaCl2 precipitation for removal of host-cell DNA and polyethylene glycol (PEG) for capturing the recombinant antibody, with no influence on the glycosylation profile. Our lab-scale prototype consisting of two tubular reactors and two stages of tangential flow microfiltration was continuously operated for up to 8 days in a truly continuous fashion and without any product flow interruption, both as a stand-alone capture and as an integrated perfusion-capture. Furthermore, we explored the use of a negatively charged membrane adsorber for flow-through anion exchange as first polishing step. We obtained a product recovery of approximately 80% and constant product quality, with more than two logarithmic reduction values (LRVs) for both host-cell proteins and host-cell DNA by the combination of the precipitation-based capture and the first polishing step.

Precipitation of antibodies by means of suitable precipitation agents is considered as an alternative to state-of-the-art affinity chromatography, although redissolution of the precipitated product with minimal formation of aggregates still poses challenges. One of these is to find an appropriate buffer composition, promoting the rapid redissolution of antibodies at high yields, with minimal impact on product quality. Here we report the development of a robust method for efficient redissolution of two industrially relevant antibodies, namely adalimumab and trastuzumab, which were precipitated by ZnCl2. Using this method, yields over 90% were achieved. Furthermore, the effect of various parameters such as pH and ionic strength on the redissolution were investigated. A mechanism regarding zinc-precipitated proteins, in which the deprotonated buffer species are the main actors responsible for redissolution due to their high affinity for zinc is hypothesized. We further evaluated the impact of ZnCl2 precipitation and redissolution at low pH on high molecular weight impurities and additionally assessed protein stability in these conditions. Glycoanalysis and nano-differential scanning calorimetry confirmed that glycostructure and tertiary and quaternary structure are not affected by the redissolution method.

Fusion protein technologies improve the expression and purification of recombinant proteins, but the removal of the tags involved requires specific proteases. The circularly permuted caspase-2 (cpCasp2) with its specific cleavage site, efficiently generates the untagged protein. While cleavage with cpCasp2 is possible before all 20 proteinogenic amino acids, cleavage before valine, leucine, isoleucine, aspartate and glutamate suffers from slow, and before proline extremely slow, turnover. To make the platform fusion protein process even more general such that any protein with an authentic N-terminus can be produced with high efficiency, the bacterial selection system PROFICS (PRotease Optimization via Fusion-Inhibited Carbamoyltransferase-based Selection) was used to evolve cpCasp2 into a variant with a catalytic turnover two orders of magnitude higher and the ability to cleave before any amino acid. The high specificity and the stability of the original circularly permuted protease was fully retained in this mutant, while the high manufacturability was mostly retained, albeit with decreased soluble titer. Four point-mutations are responsible for this change in activity, two of which are located in or near the binding pocket of the active site. This variant was named CASPON enzyme and is a major component of the CASPase-based fusiON (CASPON) platform technology. Applicability for the production of recombinant proteins was demonstrated by enzymatic removal of the CASPON tag from five model proteins. The CASPON tag enables high soluble expressions, affinity purification and good accessibility for cleavage. The five industry-relevant proteins of interest were FGF2, TNF, GH, GCSF and PTH.

Royal jelly has received attention because of its necessity for the development of queen honeybees as well as claims of benefits on human health; this product of the hypopharyngeal glands of worker bees contains a large number of proteins, some of which have been claimed to have various biological effects only in their glycosylated state. However, although there have been glycomic and glycoproteomic analyses in the past, none of the glycan structures previously defined would appear to have potential to trigger specific biological functions. In the current study, whole royal jelly as well as single protein bands were subject to off-line LC-MALDI-TOF MS glycomic analyses, complemented by permethylation, Western blotting and arraying data. Similarly to recent in-depth studies on other insect species, previously overlooked glucuronic acid termini, sulfation of mannose residues and core β-mannosylation of the N-glycans were found; additionally, a relatively rare zwitterionic modification with phosphoethanolamine is present, in contrast to the phosphorylcholine occurring in lepidopteran species. Indicative of tissue-specific remodelling of glycans in the Golgi apparatus of hypopharyngeal gland cells, only a low amount of fucosylated or paucimannosidic glycans were detected as compared with other insect samples or even bee venom. The unusual modifications of hybrid and multiantennary structures defined here may not only have a physiological role in honeybee development, but represent epitopes recognized by pentraxins with roles in animal innate immunity.