Degradation of recombinant proteins by Chinese hamster ovary host cell proteases is prevented by matriptase-1 knockout.

An increasing number of nonantibody format proteins are entering clinical development. However, one of the major hurdles for the production of nonantibody glycoproteins is host cell-related proteolytic degradation, which can drastically impact developability and timelines of pipeline projects. Chinese hamster ovary (CHO) cells are the preferred production host for recombinant therapeutic proteins. Using protease inhibitors, transcriptomics, and genetic knockdowns, we have identified, out of the >700 known proteases in rodents, matriptase-1 as the major protease involved in the degradation of recombinant proteins expressed in CHO-K1 cells. Subsequently, matriptase-1 was deleted in CHO-K1 cells using "transcription activator-like effector nucleases" (TALENs) as well as zinc-finger nucleases (ZFNs). This resulted in a superior CHO-K1 matriptase (KO) cell line with strongly reduced or no proteolytic degradation activity toward a panel of recombinantly expressed proteins. The matriptase KO cell line was evaluated in spike-in experiments and showed little or no degradation of proteins incubated in culture supernatant derived from the KO cells. This effect was confirmed when the same proteins were recombinantly expressed in the KO cell line. In summary, the combination of novel cell line engineering tools, next-generation sequencing screening methods, and the recently published Chinese hamster genome has enabled the development of this novel matriptase KO CHO cell line capable of improving expression yields of intact therapeutic proteins.

Improving expression of recombinant human IGF-1 using IGF-1R knockout CHO cell lines.

Chinese Hamster Ovary (CHO) cells are widely used for the large-scale production of recombinant biopharmaceuticals. However, attempts to express IGF-1 (a mutated human Insulin-like growth factor 1 Ea peptide (hIGF-1Ea mut)) in CHO cells resulted in poor cell growth and low productivity (0.1-0.2 g/L). Human IGF-1 variants negatively impacted CHO cell growth via the IGF-1 receptor (IGF-1R). Therefore knockout (KO) of the IGF-1R gene in two different CHO cell lines as well as knockdown (KD) of IGF-1R in one CHO cell line were performed. These cell line engineering approaches decreased significantly the hIGF-1 mediated cell growth inhibition and increased productivity of both KO CHO cell lines as well as of the KD CHO cell line. A productivity increase of 10-fold at pool level and sevenfold at clone level was achieved, resulting in a titer of 1.3 g/L. This data illustrate that cell line engineering approaches are powerful tools to improve the yields of recombinant proteins which are difficult to produce in CHO cells.

Morphological development of thick-tufted layer v pyramidal cells in the rat somatosensory cortex.

The thick-tufted layer V pyramidal (TTL5) neuron is a key neuron providing output from the neocortex. Although it has been extensively studied, principles governing its dendritic and axonal arborization during development are still not fully quantified. Using 3-D model neurons reconstructed from biocytin-labeled cells in the rat somatosensory cortex, this study provides a detailed morphological analysis of TTL5 cells at postnatal day (P) 7, 14, 21, 36, and 60. Three developmental periods were revealed, which were characterized by distinct growing rates and properties of alterations in different compartments. From P7 to P14, almost all compartments grew fast, and filopodia-like segments along apical dendrite disappeared; From P14 to P21, the growth was localized on specified segments of each compartment, and the densities of spines and boutons were significantly increased; From P21 to P60, the number of basal dendritic segments was significantly increased at specified branch orders, and some basal and oblique dendritic segments were lengthened or thickened. Development changes were therefore seen in two modes: the fast overall growth during the first period and the slow localized growth (thickening mainly on intermediates or lengthening mainly on terminals) at the subsequent stages. The lengthening may be accompanied by the retraction on different segments. These results reveal a differential regulation in the arborization of neuronal compartments during development, supporting the notion of functional compartmental development. This quantification provides new insight into the potential value of the TTL5 morphology for information processing, and for other purposes as well.