Host cell proteins in monoclonal antibody processing: Control, detection, and removal.

Host cell proteins (HCPs) are process-related impurities in a therapeutic protein expressed using cell culture technology. This review presents biopharmaceutical industry trends in terms of both HCPs in the bioprocessing of monoclonal antibodies (mAbs) and the capabilities for HCP clearance by downstream unit operations. A comprehensive assessment of currently implemented and emerging technologies in the manufacturing processes with extensive references was performed. Meta-analyses of published downstream data were conducted to identify trends. Improved analytical methods and understanding of "high-risk" HCPs lead to more robust manufacturing processes and higher-quality therapeutics. The trend of higher cell density cultures leads to both higher mAb expression and higher HCP levels. However, HCP levels can be significantly reduced with improvements in operations, resulting in similar concentrations of approx. 10 ppm HCPs. There are no differences in the performance of HCP clearance between recent enhanced downstream operations and traditional batch processing. This review includes best practices for developing improved processes.

Macrophage-Specific, Mafb-Deficient Mice Showed Delayed Skin Wound Healing.

Macrophages play essential roles throughout the wound repair process. Nevertheless, mechanisms regulating the process are poorly understood. MAFB is specifically expressed in the macrophages in hematopoietic tissue and is vital to homeostatic function. Comparison of the skin wound repair rates in macrophage-specific, MAFB-deficient mice (Mafbf/f::LysM-Cre) and control mice (Mafbf/f) showed that wound healing was significantly delayed in the former. For wounded GFP knock-in mice with GFP inserts in the Mafb locus, flow cytometry revealed that their GFP-positive cells expressed macrophage markers. Thus, macrophages express Mafb at wound sites. Immunohistochemical (IHC) staining, proteome analysis, and RT-qPCR of the wound tissue showed relative downregulation of Arg1, Ccl12, and Ccl2 in Mafbf/f::LysM-Cre mice. The aforementioned genes were also downregulated in the bone marrow-derived, M2-type macrophages of Mafbf/f::LysM-Cre mice. Published single-cell RNA-Seq analyses showed that Arg1, Ccl2, Ccl12, and Il-10 were expressed in distinct populations of MAFB-expressing cells. Hence, the MAFB-expressing macrophage population is heterogeneous. MAFB plays the vital role of regulating multiple genes implicated in wound healing, which suggests that MAFB is a potential therapeutic target in wound healing.

Transcription factor MafB in podocytes protects against the development of focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is a common cause of steroid-resistant nephrotic syndrome. Spontaneous remission of FSGS is rare and steroid-resistant FSGS frequently progresses to renal failure. Many inheritable forms of FSGS have been described, caused by mutations in proteins that are important for podocyte function. Here, we show that a basic leucine zipper transcription factor, MafB, protects against FSGS. MAFB expression was found to be decreased in the podocytes of patients with FSGS. Moreover, conditional podocyte-specific MafB-knockout mice developed FSGS with massive proteinuria accompanied by depletion of the slit diaphragm-related proteins (Nphs1 and Magi2), and the podocyte-specific transcription factor Tcf21. These findings indicate that MafB plays a crucial role in the pathogenesis of FSGS. Consistent with this, adriamycin-induced FSGS and attendant proteinuria were ameliorated by MafB overexpression in the podocytes of MafB podocyte-specific transgenic mice. Thus, MafB could be a new therapeutic target for FSGS.

Role of MafB in macrophages.

The transcription factor MafB regulates macrophage differentiation. However, studies on the phenotype of Mafb-deficient macrophages are still limited. Recently, it was shown that the specific expression of MafB permits macrophages to be distinguished from dendritic cells. In addition, MafB has been reported to be involved in various diseases related to macrophages. Studies using macrophage-specific Mafb-deficient mice show that MafB is linked to atherosclerosis, autoimmunity, obesity, and ischemic stroke, all of which exhibit macrophage abnormality. Therefore, MafB is hypothesized to be indispensable for the regulation of macrophages to maintain systemic homeostasis and may serve as an innovative target for treating macrophage-related diseases.

Transcription factor MafB is a marker of tumor-associated macrophages in both mouse and humans.

The transcription factor MafB is specifically expressed in macrophages. We have recently demonstrated that MafB is expressed in anti-inflammatory alternatively activated M2 macrophages in vitro. Tumor-associated macrophages (TAMs) are a subset of M2 type macrophages that can promote immunosuppressive activity, induce angiogenesis, and promote tumor cell proliferation. To examine whether MafB express in TAMs, we analyzed green fluorescent protein (GFP) expression in Lewis lung carcinoma tumors of MafB-GFP knock-in heterozygous mice. FACS analysis demonstrated GFP fluorescence in cells positive for macrophage-markers (F4/80, CD11b, CD68, and CD204). Moreover, quantitative RT-PCR analysis with F4/80+GFP+ and F4/80+GFP- sorted cells showed that the GFP-positive macrophages express IL-10, Arg-1, and TNF-α, which were known to be expressed in TAMs. These results indicate that MafB is expressed in TAMs. Furthermore, immunostaining analysis using an anti-MAFB antibody revealed that MAFB is expressed in CD204-and CD68-positive macrophages in human lung cancer samples. In conclusion, MafB can be a suitable marker of TAMs in both mouse and human tumor tissues.

Mice harboring an MCTO mutation exhibit renal failure resembling nephropathy in human patients.

Multicentric carpotarsal osteolysis (MCTO) is a condition involving progressive osteolysis of the carpal and tarsal bones that is associated with glomerular sclerosis and renal failure (MCTO nephropathy). Previous work identified an autosomal dominant missense mutation in the transactivation domain of the transcription factor MAFB as the cause of MCTO. Several methods are currently used for MCTO nephropathy treatment, but these methods are invasive and lead to severe side effects, limiting their use. Therefore, the development of alternative treatments for MCTO nephropathy is required; however, the pathogenesis of MCTO in vivo is unclear without access to a mouse model. Here, we report the generation of an MCTO mouse model using the CRISPR/Cas9 system. These mice exhibit nephropathy symptoms that are similar to those observed in MCTO patients. MafbMCTO/MCTO mice show developmental defects in body weight from postnatal day 0, which persist as they age. They also exhibit high urine albumin creatinine levels from a young age, mimicking the nephropathic symptoms of MCTO patients. Characteristics of glomerular sclerosis reported in human patients are also observed, such as histological evidence of focal segmental glomerulosclerosis (FSGS), podocyte foot process microvillus transformation and podocyte foot process effacement. Therefore, this study contributes to the development of an alternative treatment for MCTO nephropathy by providing a viable mouse model.

Postnatal lethality and chondrodysplasia in mice lacking both chondroitin sulfate N-acetylgalactosaminyltransferase-1 and -2.

Chondroitin sulfate (CS) is a sulfated glycosaminoglycan (GAG) chain. In cartilage, CS plays important roles as the main component of the extracellular matrix (ECM), existing as side chains of the major cartilage proteoglycan, aggrecan. Six glycosyltransferases are known to coordinately synthesize the backbone structure of CS; however, their in vivo synthetic mechanism remains unknown. Previous studies have suggested that two glycosyltransferases, Csgalnact1 (t1) and Csgalnact2 (t2), are critical for initiation of CS synthesis in vitro. Indeed, t1 single knockout mice (t1 KO) exhibit slight dwarfism and a reduction in CS content in cartilage compared with wild-type (WT) mice. To reveal the synergetic roles of t1 and t2 in CS synthesis in vivo, we generated systemic single and double knockout (DKO) mice and cartilage-specific t1 and t2 double knockout (Col2-DKO) mice. DKO mice exhibited postnatal lethality, whereas t2 KO mice showed normal size and skeletal development. Col2-DKO mice survived to adulthood and showed severe dwarfism compared with t1 KO mice. Histological analysis of epiphyseal cartilage from Col2-DKO mice revealed disrupted endochondral ossification, characterized by drastic GAG reduction in the ECM. Moreover, DKO cartilage had reduced chondrocyte proliferation and an increased number of apoptotic chondrocytes compared with WT cartilage. Conversely, primary chondrocyte cultures from Col2-DKO knee cartilage had the same proliferation rate as WT chondrocytes and low GAG expression levels, indicating that the chondrocytes themselves had an intact proliferative ability. Quantitative RT-PCR analysis of E18.5 cartilage showed that the expression levels of Col2a1 and Ptch1 transcripts tended to decrease in DKO compared with those in WT mice. The CS content in DKO cartilage was decreased compared with that in t1 KO cartilage but was not completely absent. These results suggest that aberrant ECM caused by CS reduction disrupted endochondral ossification. Overall, we propose that both t1 and t2 are necessary for CS synthesis and normal chondrocyte differentiation but are not sufficient for all CS synthesis in cartilage.

MafB is a critical regulator of complement component C1q.

The transcription factor MafB is expressed by monocytes and macrophages. Efferocytosis (apoptotic cell uptake) by macrophages is important for inhibiting the development of autoimmune diseases, and is greatly reduced in Mafb-deficient macrophages. Here, we show the expression of the first protein in the classical complement pathway C1q is important for mediating efferocytosis and is reduced in Mafb-deficient macrophages. The efferocytosis defect in Mafb-deficient macrophages can be rescued by adding serum from wild-type mice, but not by adding serum from C1q-deficient mice. By hemolysis assay we also show that activation of the classical complement pathway is decreased in Mafb-deficient mice. In addition, MafB overexpression induces C1q-dependent gene expression and signals that induce C1q genes are less effective in the absence of MafB. We also show that Mafb-deficiency can increase glomerular autoimmunity, including anti-nuclear antibody deposition. These results show that MafB is an important regulator of C1q.

MafB deficiency accelerates the development of obesity in mice.

MafB, a transcription factor expressed selectively in macrophages, has important roles in some macrophage-related diseases, especially in atherosclerosis. In this study, we investigated the mechanism by which hematopoietic-specific MafB deficiency induces the development of obesity. Wild-type and hematopoietic cell-specific Mafb-deficient mice were fed a high-fat diet for 10 weeks. The Mafb-deficient mice exhibited higher body weights and faster rates of body weight increase than control mice. The Mafb-deficient mice also had a higher percentage of body fat than the wild-type mice, due to increased adipocyte size and serum cholesterol levels. Reverse transcription-PCR analysis showed a reduction in apoptosis inhibitor of macrophage (AIM) in Mafb-deficient adipose tissue. AIM is known as an inhibitor of lipogenesis in adipocytes and is expressed in adipose tissue macrophages. Collectively, our data suggest that Mafb deficiency in hematopoietic cells accelerates the development of obesity.

Ground-based assessment of JAXA mouse habitat cage unit by mouse phenotypic studies.

The Japan Aerospace Exploration Agency developed the mouse Habitat Cage Unit (HCU) for installation in the Cell Biology Experiment Facility (CBEF) onboard the Japanese Experimental Module ("Kibo") on the International Space Station. The CBEF provides "space-based controls" by generating artificial gravity in the HCU through a centrifuge, enabling a comparison of the biological consequences of microgravity and artificial gravity of 1 g on mice housed in space. Therefore, prior to the space experiment, a ground-based study to validate the habitability of the HCU is necessary to conduct space experiments using the HCU in the CBEF. Here, we investigated the ground-based effect of a 32-day housing period in the HCU breadboard model on male mice in comparison with the control cage mice. Morphology of skeletal muscle, the thymus, heart, and kidney, and the sperm function showed no critical abnormalities between the control mice and HCU mice. Slight but significant changes caused by the HCU itself were observed, including decreased body weight, increased weights of the thymus and gastrocnemius, reduced thickness of cortical bone of the femur, and several gene expressions from 11 tissues. Results suggest that the HCU provides acceptable conditions for mouse phenotypic analysis using CBEF in space, as long as its characteristic features are considered. Thus, the HCU is a feasible device for future space experiments.