Review

Glycosylation of recombinant proteins: Problems and prospects

L-asparaginase (ASNase) has played a key role in the management of acute lymphoblastic leukaemia (ALL). As an amidohydrolase, it catalyzes the hydrolysis of L-asparagine, a crucial step in the treatment of ALL. Various ASNase variants have evolved from diverse sources since it was first used in paediatric patients in the 1960s. This review describes the available ASNase and approaches being used to develop ASNase as a biobetter candidate.

The review discusses the Glycosylation and PEGylation techniques, which are frequently used to develop biobetter versions of the majority of the therapeutic proteins. Further, it explores current ASNase biobetters in therapeutic use and discusses the protein engineering and chemical modification approaches that were employed to reduce immunogenicity, extend protein half-life, and enhance protease stability of ASNase. Emerging strategies like immobilization and encapsulation are also highlighted as potential pathways for improving ASNase properties.

The purpose of the development of ASNase biobetter is to achieve a novel therapeutic candidate that could improve catalytic efficiency, in vivo stability with minimum glutaminase (GLNase) activity and toxicity. Modification of ASNase by immobilization and encapsulation or by fusion technologies like Albumin fusion, Fc fusion, ELP fusion, XTEN fusion, etc. can be exploited to develop a novel biobetter candidate suitable for therapeutic approaches.

This review emphasizes the importance of biobetter development for therapeutic proteins like ASNase. Improved ASNase molecules have the potential to significantly advance the treatment of ALL and have broader implications in the pharmaceutical industry.

The production of recombinant proteins is a rapidly expanding industrial field. Several proteins that previously were extracted from their natural source by expensive and time-consuming processes can now be produced in various organisms, including bacteria, yeast, insect cells, and mammalian cells. These traditional systems have their advantages and disadvantages, but one of the major problems is related to post-translational modifications that mostly differ from those in mammals. The LEXSY system was developed for the expression of recombinant proteins in Leishmania tarentolae a non-pathogenic protozoan species isolated from the lizards Tarentolae annularis and Tarentolae mauritanica it is a biosafety level I protist with high biomass production that is well-suited for cultivation on an industrial scale. The system has advantages for the expression of several proteins, including glycosylated proteins, and for the homogenous glycosylation of mammalian proteins. In this review, we have compared the classical systems and LEXSY, and presents a historical review of heterologous expression in Leishmania. We also list the specific characteristics, advantages, and problems with LEXSY in a work-oriented perspective to facilitate the use of this eukaryotic expression platform.

In recent times, plant molecular farming (PMF) in plant expression hosts has attracted a lot of interest for production of modern human therapeutics. Its advantages include presence of inherent posttranslational modifications, lower cost, safety, ecofriendly, easy scale-up, and fewer regulatory issues. Interestingly, plant hosts impart “natural protection” to human therapeutic recombinant products by default, since they are inherently incapable of harboring/propagating human pathogens. Plants being eukaryotes, it was expected that active versions of human proteins produced within them would resemble natural human proteins. However, it was not found to necessarily be so, owing to inherent differences in the glycosylation patterns of the two hosts. A prerequisite for successful therapeutic applications of PMF-based biopharmaceuticals would be to have them glycosylated in human compatible precise patterns. Glycoengineering technology, wherein the genes of plant specific glycosyltransferases are replaced with their human counterparts, is proving to be a practical approach in modifying the PMF-based products in order to tailor them for human utility. A few such “humanized” products, termed “Biosimilars” and “Biobetters,” actually display the desired effectiveness and functional value. Human Growth Hormone (hGH), surface antigen of Hepatitis B (HBsAg), Hepatitis vaccine ZMapp, and FDA approved enzyme ELELYSO have already been harvested from plant expression systems. This chapter mainly discusses the glycosylation motifs of human biopharmaceutics expressed in plant host systems, the pros combined with cons of using such hosts, the possibility of utilizing plant cell culture, and finally the scope of developing better, cheaper and “humanized” products via glycoengineering methods.

In recent times, plant molecular farming (PMF) in plant expression hosts has attracted a lot of interest for production of modern human therapeutics. Its advantages include presence of inherent posttranslational modifications, lower cost, safety, ecofriendly, easy scale-up, and fewer regulatory issues. Interestingly, plant hosts impart “natural protection” to human therapeutic recombinant products by default, since they are inherently incapable of harboring/propagating human pathogens. Plants being eukaryotes, it was expected that active versions of human proteins produced within them would resemble natural human proteins. However, it was not found to necessarily be so, owing to inherent differences in the glycosylation patterns of the two hosts. A prerequisite for successful therapeutic applications of PMF-based biopharmaceuticals would be to have them glycosylated in human compatible precise patterns. Glycoengineering technology, wherein the genes of plant specific glycosyltransferases are replaced with their human counterparts, is proving to be a practical approach in modifying the PMF-based products in order to tailor them for human utility. A few such “humanized” products, termed “Biosimilars” and “Biobetters,” actually display the desired effectiveness and functional value. Human Growth Hormone (hGH), surface antigen of Hepatitis B (HBsAg), Hepatitis vaccine ZMapp, and FDA approved enzyme ELELYSO have already been harvested from plant expression systems. This chapter mainly discusses the glycosylation motifs of human biopharmaceutics expressed in plant host systems, the pros combined with cons of using such hosts, the possibility of utilizing plant cell culture, and finally the scope of developing better, cheaper and “humanized” products via glycoengineering methods.

The growing adoption of single-use technologies in biopharmaceutical manufacturing has primarily been driven by the well-recognized benefits of single-use solutions against their traditional stainless steel counterparts, as described in this article. The current expansion of single-use technologies in most of the process steps of biomanufacturing operations has been accelerated by the reduction of cell culture and downstream purification volumes under 2000 L scale, and by the recent technology developments in terms of bags, tubing, filters, sterile connectors, fluid transfer systems, and sensors, both of which make possible the concept of a complete single-use biomanufacturing suite. Major suppliers of single-use components are transitioning from supplying products to designing application-based integrated solutions tailored at the various unit operations that constitute traditional biotech upstream processing, cell culture, downstream purification, and final fill and finish. Also, single-use technology qualification and further process validation become an integrated approach where validation studies commonly used for already existing plastic components such as filters can be extended to the newest single-use process components, such as bags, thermoplastic tubing, sterile connectors, and sensors. The article concludes with a discussion of future outlooks on next-generation single-use product designs and their influence on the complete layout of modern bioproduction plants.

Large proteins are usually expressed in eukaryotic systems, while smaller ones are expressed in prokaryotic systems. For proteins that require glycosylation, mammalian cells, fungi, or the baculovirus system is chosen. The least expensive, easiest and quickest expression of proteins can be carried out in Escherichia coli. However, this bacterium cannot express very large proteins. Also, for S–S-rich proteins and proteins that require posttranslational modifications, E. coli is not the system of choice. The two most utilized yeasts are Saccharomyces cerevisiae and Pichia pastoris. Yeasts can produce high yields of proteins at low cost, proteins larger than 50 kDa can be produced, signal sequences can be removed, and glycosylation can be carried out. The baculoviral system can carry out more complex posttranslational modifications of proteins. The most popular system for producing recombinant mammalian glycosylated proteins is that of mammalian cells. Genetically modified animals secrete recombinant proteins in their milk, blood, or urine. Similarly, transgenic plants such as Arabidopsis thaliana and others can generate many recombinant proteins.