Practical applicationThis review summarizes recent progress and current status of bioprocess monitoring. There has been an increasing emphasis on the applications of process analytical tools in bioprocessing for biologics manufacturing. The integration of in-line, on-line and at-line sensors and the real-time characterization of the physiological state of cells will lead to robust processes and enhanced product quality. AbstractThe productivity of cell culture manufacturing for biologics has increased momentously in the past decades. Increasingly, the process research efforts are devoted into improving product quality and consistency. Consistent process performance and successful implementation of quality by design (QbD) practice requires well-utilized process analytical technology (PAT). This review summarizes recent progress and current status of bioprocess monitoring. Many sensors for bioprocess monitoring have been available for decades while new ones, especially spectrometric sensors, are making their way into cell culture bioprocesses. On-line sampling devices have grown mature in the past decade thus making many instruments traditionally used for off-line analysis available for at-line use. With a general trend of using better defined medium for cell cultivation and increasing emphasis of process analytical tools, the spectrometric methods are also making headway in cell culture process monitoring. The integration of those sensing technologies will be important to advance the real-time monitoring of the state of cellular physiology for the control for process consistency and product quality.

Nosiheptide-resistance methyltransferase (NHR) of Streptomyces actuosus is a class IV methyltransferase of the SpoU family and methylates 23S rRNA at nucleotide adenosine corresponding to A1067 in Escherichia coli. Such methylation is essential for resistance against nosiheptide, a sulfur peptide antibiotic, which is produced by the nosiheptide-producing strain, S. actuosus. Here, we report the crystal structures of NHR and NHR in complex with SAM (S-adenosyl-l-methionine) at 2.0 and 2.1 A resolution, respectively. NHR forms a functional homodimer, and dimerization is required for methyltransferase activity. The monomeric NHR is comprised of the N-terminal RNA binding domain (NTD) and the C-terminal catalytic domain (CTD). Overall, the structure of NHR suggests that the methyltransferase activity is achieved by "reading" the RNA substrate with NTD and "adding" methyl group using CTD. Comprehensive mutagenesis and methyltransferase activity assays reveal critical regions for SAM binding in CTD and loops (L1 and L3) essential for RNA recognition in NTD. Finally, the catalytic mechanism and structural model that NHR recognizes 23S rRNA is proposed based on the structural and biochemical analyses. Thus, our systematic structural studies reveal the substrate recognition and modification by the nosiheptide-resistance methyltransferase.

Development of serum-free suspension cell culture processes is very important for influenza vaccine production. Previously, we developed a MDCK suspension cell line in a serum-free medium. In the present study, the growth kinetics of suspension MDCK cells and influenza virus production in the serum-free medium were investigated, in comparison with those of adherent MDCK cells in both serum-containing and serum-free medium. It was found that the serum-free medium supported the stable subculture and growth of both adherent and suspension cells. In batch culture, for both cell lines, the growth kinetics in the serum-free medium was comparable with those in the serum-containing medium and a commercialized serum-free medium. In the serum-free medium, peak viable cell density (VCD), haemagglutinin (HA) and median tissue culture infective dose (TCID50) titers of the two cell lines reached 4.51×106 cells/mL, 2.94Log10(HAU/50 μL) and 8.49Log10(virions/mL), and 5.97×106 cells/mL, 3.88Log10(HAU/50 μL), and 10.34Log10(virions/mL), respectively. While virus yield of adherent cells in the serum-free medium was similar to that in the serum-containing medium, suspension culture in the serum-free medium showed a higher virus yield than adherent cells in the serum-containing medium and suspension cells in the commercialized serum-free medium. However, the percentage of infectious viruses was lower for suspension culture in the serum-free medium. These results demonstrate the great potential of this suspension MDCK cell line in serum-free medium for influenza vaccine production and further improvements are warranted.

Charge variants, especially acidic charge variants, of recombinant monoclonal antibodies are the major critical quality attributes in the biotechnology industry due to their potential influence on stability and biological activity. The chemical properties of the acidic charge variants have been challenging to fully characterize, and it is critical for process development and optimization. To completely understand the multiple sources of acidic charge variants, the major charge forms of an IgG1 monoclonal antibody were firstly isolated and then analyzed by a battery of characterization tools. It was found that various degrees of disulfide bond reduction, the deamination of HC-T8 Asn84 and HC-T35 Asn388 and aggregation account for the majority of acidic charge heterogeneity and the terminal galactosylation content was in relation to the acidic charge heterogeneity. The correlation between acidic charge heterogeneity and galactosylation content was further explored by weak cation exchange chromatography with the use of β1-4 galactosidase digestion. The results showed that galactosylation was not the source of the acidic charge variants per se. Meanwhile, to gain insights into the impact on binding affinity of monoclonal antibody to IgE and FcRn, charge variants were also analyzed by competitive ELISA and surface plasmon resonance, respectively. All isolated charge variants had similar affinity binding to IgE and FcRn binding relative to the starting material.

HS628 has been developed as a proposed biosimilar product of originator tocilizumab (Actemra®). An extensive physicochemical and biological characterization was conducted to assess similarity between HS628 and originator tocilizumab. The amino acid sequence was shown to be identical between HS628 and originator tocilizumab. The higher order structure was found to be indistinguishable from originator tocilizumab. Concerning purity and heterogeneity, HS628 was demonstrated to have similar posttranslational modifications, charge heterogeneity, size heterogeneity, and glycosylation to originator tocilizumab. Moreover, HS628 exhibited highly similar binding affinity and antiproliferative activity as well as capability of inhibiting STAT3 phosphorylation compared to originator tocilizumab. Taken together, HS628 can be considered as a highly similar molecule to originator tocilizumab in terms of physicochemical and biological properties.