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Case Study // OCT 6 2014

A strategy for more uniform glycoproteins: Keep it simple

A cell line engineering strategy for simplifying N-glycosylation of recombinant proteins

"For small-scale transfections, the cells were plated in a 6-well plate 48 h before transfection at ~150,000 cells per well. They were transfected using the TransIT®-293 Transfection Reagent (Mirus Bio LLC) according to the manufacturer's instructions."

-Meuris et al. 1

Background

In this Journal Club case study we review a cell line engineering strategy, referred to as GlycoDelete, that aims to improve biotherapeutic protein manufacturing by decreasing glycan heterogeneity.

GlycoDelete engineering of mammalian cells simplifies N-glycosylation of recombinant proteins
1 Meuris L, Santens F, Elson G, Festjens N, Boone M, Dos Santos A, Devos S, Rousseau F, Plets E, Houthuys E, Malinge P, Magistrelli G, Cons L, Chatel L, Devreese B, Callewaert N. Nat Biotechnol. 2014 May;32(5):485-9. doi: 10.1038/nbt.2885. Epub 2014 Apr 20. PMID: 24752077

Glycosylation has multiple impacts on the biophysical properties of proteins. In particular, for protein-based drugs such as therapeutic antibodies, the diversity of mammalian glycoforms leads to heterogeneity in protein folding, stability, and immunogenicity. Through glycan engineering it may be possible to make the properties of biotherapeutic proteins more uniform and minimize intra-batch and batch-to-batch variability.

The process of N-linked glycosylation (the attachment of carbohydrate chains to asparagine residues of proteins) begins in the lumen of the endoplasmic reticulum and continues in the Golgi apparatus. In the Golgi, some mannose residues are removed, and the glycans are subsequently modified by a series of glycosylation enzymes, beginning with N-acetylglucosaminyltransferase I (GnTI). Following modification by GnTI, glycoproteins are further remodeled to produce a heterogeneous mix of glycans which are categorized as complex or hybrid type glycans. Each stage in the multi-step process of glycosylation occurs with some degree of variability. This leads to many different glycoforms for each protein. The GlycoDelete strategy cuts down on this complexity by first pruning back the long, branched polysaccharides added in the Golgi, and and then re-growing simpler and shorter glycans.

GlycoDelete Strategy

In this study, Meuris et al. generated recombinant glycoproteins with smaller and more homogeneous glycans by re-engineering the N-glycosylation machinery in HEK 293 cells. Once glycoproteins enter the ER decorated with oligomannose, the GlycoDelete approach essentially creates a detour in the normal glycosylation pathway that follows three steps: (1) bypass the enzymes that create complex and hybrid type glycans, (2) cleave off the existing oligomannose to leave only a single sugar, and (3) add back the minimal desirable glycan phenotype. The authors accomplished the first step in this process by selecting a HEK 293 cell line that completely lacked GnTI, such that proteins expressed in this line would sidestep the enzymes that lead to modification with complex or hybrid type N-glycans. Secondly, this parental GnTI deficient cell line was stably transfected with a Golgi-tethered fungal enzyme, endo-β-N-acetylglucosaminidase (endoT). This enzyme targets the base of the glycan trunk to cleave the entire glycan group off of the protein, leaving only an N-acetylglucosamine (GlcNAc) stump. Lastly, this GlcNAc stump can be further modified by endogenous galactosyltransferases and sialyltransferases in the Golgi to add galactose and sialic acid residues, respectively, in order to generate a short, linear trisaccharide. It is important to note that these smaller glycans still exhibited terminal sialic acid residues, which are desireable on biotherapeutic proteins due to the effects of sialic acid on serum half-life and immunogenicity. (Highly sialylated proteins tend to only produce a very weak host-based immune response.)

To select for cells stably transfected with the enzyme endoT and possessing the desired glycan phenotype, the authors treated the cells with the cytotoxic lectin, concanavalin A. The parental GnTI deficient cells still displayed the glycan ligand required for ConA binding, whereas the GlycoDelete cells lacked the oligomannose required for recognition by ConA and were resistant to the cytotoxic levels of ConA.

GlycoDelete cells were transiently transfected with two therapeutic proteins, GM-CSF or anti-CD20 in order to characterize the glycan properties of overexpressed glycoproteins. Mass spectrometry revealed that in both cases, the expressed proteins existed in only three glycoforms that primarily exhibited small, sialylated trisaccharide N-glycans. When compared to normally glycosylated native proteins, both of the overexpressed GlycoDelete proteins showed similar stability, antigenicity and function. One notable exception was that GlycoDelete anti-CD20 exhibited lower binding affinity for Fcɣ receptors—a property that may offer safety benefits for neutralizing antibodies. Anti-CD20 also showed reduced initial serum clearance, which could have implications for dosage of therapeutics produced with the GlycoDelete platform. Surprisingly, the GlycoDelete cells showed similar cell growth and gene expression profiles as the parental cell line indicating that this dramatic change to the proteome had minimal detectable effects on overall cell physiology.

Implications

Mammalian glycoproteins exist as many differently glycosylated variants. GlycoDelete cells, by contrast have only three primary glycoforms. By simplifying the diversity of glycans on overexpressed proteins, this promising approach paves the way for cell lines that generate more homogeneous biotherapeutics.

Future work should address the repeatability of this approach with a variety of different protein targets. Additional questions raised include how the GlycoDelete platform affects process repeatibility, purification, and ultimately the efficacy of various biotherapeutics with simplified glycans.

View more information on large scale protein production.

Mirus Bio reagents useful in cell line engineering and protein manufacturing:

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