With the strong growth in biologics, large molecules, and biopharmaceutical therapeutics in recent years, the pharmaceutical and biotech industries are increasingly turning toward peptides and proteins in the search for drug discovery targets. While both possess numerous properties that offer significant therapeutic potential, there are fundamental differences between the two compounds. This article examines some similarities and differences between proteins and peptides in light of potential market applications, manufacturing techniques, and the regulatory environment…
Tag: <span>therapeutic proteins</span>
Therapeutic proteins manufactured in cellular systems contain residual DNA derived from host cell substrates used in production. Risk assessment of the residual host cell DNA is necessary, as some of these DNA sequences may be potentially infectious or oncogenic. Oncogenic potential lies in transmission of the activated oncogenes to subjects receiving the product, thereby inducing oncogenic events. Therefore, it becomes essential for drug manufacturers to show clearance of genomic DNA (oncogenic sequences as well) throughout production processes and to confirm low levels of residual DNA in the final drug substance. This study attempted to estimate the oncogenes in the total residual DNA using a highly sensitive, specific, and robust method—quantitative polymerase chain reaction (qPCR). Routinely, total residual DNA is estimated using either the 18S ribosomal (r)DNA gene or Alu equivalent multicopy gene sequence as qPCR targets. We have determined the copy numbers of these qPCR targets along with the oncogene (Ras gene) and housekeeping genes (ACTB and GAPDH) and established a ratio of their presence in protein samples. Another objective of the study was to estimate the level of oncogenes from several in-process step samples in the manufacturing and purification process and check the clearance of total residual DNA including oncogenes. Upon quantification, the proportions of oncogenes present were one tenth of the quantified residual DNA levels (Ras gene:18S RNA) in the purification stage samples, providing information that the therapeutic protein product was safe from the presence of oncogenes in residual DNA by a factor of ten…
Agarose-based chromatography beads were first introduced by Stellan Hjertén in 1962. Fifty years later, beaded agarose has become the dominant resin for protein purification and is extensively used, ranging from research-scale in sub mL volumes to full-scale manufacturing in > 500 L chromatography columns. Recent resin development work has focused on increasing capacity and selectivity through different grafting technologies and ligand developments.
Stirred tank single-use bioreactors (SUBs) have been widely adopted for production of biopharmaceuticals such as monoclonal antibodies in mammalian cell culture. However, they are seldom used for commercial production of biologics with microbial fermentation. SUBs offer time-saving advantages because they do not require significant downtime for cleaning and sterilization, so finding a SUB that can perform well with high cell density microbial fermentation processes has the potential to increase the number of production runs. Therefore, for this study, a His-tagged protease inhibitor was chosen as a model protein to demonstrate that the Sartorius Biostat STR® MO, a SUB recently developed for microbial fermentation, is suited for recombinant protein production by high cell density Escherichia coli fermentation processes.
At 50 L scale, the SUB achieved good process control and allowed an oxygen uptake rate (OUR) of up to 240 mmoles/L/h. The fermentation runs produced up to 5.8 g/L of the soluble recombinant protein and a dry cell weight of >60 g/L at the end of fermentation. Additionally, the SUB showed a similar fermentation profile when compared with data from parallel runs in 15 L sterilise-in-place (SIP) vessels using identical media and process parameters. This study indicates that with a minimum investment of capital resources, stirred tank SUBs could be used in pilot-scale manufacturing with high cell density microbial fermentations to potentially shorten the timelines and costs of advancing therapeutic proteins to clinic.
Since the first approval for human use of a recombinant protein therapeutic, this sector of the pharmaceutical market has grown rapidly. The first approved protein therapeutics were small, non-glycosylated proteins such as insulin and human growth hormone; they were produced in bacterial systems. With the advent of mammalian cell-based production systems, it became possible to produce more complex, glycosylated proteins for use as recombinant therapeutics…
The analytical characterization of recombinant protein therapeutic drug products has broadened to include the use of more sophisticated technologies. The expansion of technical abilities has translated into increasing the depth and breadth of our knowledge and understanding of the drug product intended for commercialization. With the availability of more precise methods, the regulatory expectations for understanding the characteristics of a protein therapeutic drug product are increasing. A thorough understanding of a therapeutic protein’s biochemical and biophysical characteristics is necessary to support investigational new drug (IND) applications and other drug regulatory filings…
Current expression technologies have enabled the production of thousands of recombinant proteins in diverse production hosts. Therapeutic recombinant proteins have been engineered for a variety of purposes including reduced antigenicity, longer half-life, simplified process development, and increased affinity. Protein engineering has relied on various high throughput methods (e.g., directed evolution, phage display) to identify candidate proteins with the desired therapeutic properties. The physiological and biochemical diversity of native and engineered proteins reflects on the abundance of production hosts, expression tools, and different approaches for protein purification. Notably, a key step in high-throughput protein production is purification, which is a bottleneck where large numbers of samples are involved. Universal purification methods that can be applied to virtually any protein, and that are amenable to automation, can be used to address this problem…
Today concentrated efforts are underway to improve the bioactivity of therapeutic proteins with the aim of reducing: (i) the number and concentration of the applied doses of the therapeutic protein, (ii) undesired side effects, and (iii) the cost of a therapy. A very promising strategy is to optimise the glycosylation of these biotherapeutics. A novel expression platform, GlycoExpressâ„¢, has been developed to produce proteins with fully human glycosylation, optimised sialylation, and improved bioactivity…