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Improving Product Safety Profiles: Host Cell Lines Deficient in CMP-N-Acetylneuraminic Acid Hydroxylase (CMAH) and Alpha-1-3-Galactosyltransferase (GGTA1)

By: Mascarenhas, J., Achtien, K., Richardson, S., Sealover, N., Kaiser, J., Borgschulte, T., George, H., Kayser, K. and Lin, N, Cell Sciences and Development, SAFC Sigma Aldrich2909 Laclede Avenue, Saint Louis, MO 63103, USA

Introduction

Post-translational modifications have been shown to affect the bioactivity, clearance rates, immunogenicity and safety profiles of therapeutic glycoproteins. For example anti- N-glycolylneuraminic acid (Neu5Gc or NGNA) antibodies in humans can interact with Neu5Gc sialylated therapeutic proteins (for example: Cetuximab) produced in non-human mammalian expression systems causing clinical complications. This is because of an inactivating mutation in humans of the gene cytidine monophosphate-N-acetylneuraminic acid hydroxylase (Cmah), an enzyme responsible for Neu5Gc biosynthesis from the N-acetylneuraminic acid (Neu5Ac or NANA) form of sialic acid. Cmah is expressed in CHO cells and the Neu5Gc glycan moiety has been detected on several biologics produced in CHO. Another example of an immunogenic modification on N-glycans is the addition of a terminal galactose-α-1-3 galactose moiety (or α-Gal) mediated by the α-1,3 galactosyltransferase (GGTA1) gene. r-Proteins that carry this epitope can elicit strong immunogenic or anaphylactic reactions due to the presence of circulating anti-α Gal antibodies present in most humans. Contrary to previous assumptions a CHO ortholog of the GGTA1 gene has been reported (Bosques et al), and antigenic α-Gal epitopes detected on the glycosylated proteins.

glycosylation-engineering-targets

Figure 1. Glycosylation Engineering Targets: Elimination of Non-Human Immunogenic Sugar Moieties.

In this work, we describe the targeted deletion of the genes Cmah and GGTA1 in the CHO host cell line (SAFC CHOZN® GS-/-), thus allowing for the production of therapeutic proteins lacking the Neu5Gc and α-Gal immunogenic species respectively. While changes in process and culture conditions can be used to manipulate the levels Neu5Gc and α-Gal immunogenic species, we have taken the approach of the complete elimination of these species by disrupting the relevant genes. These results are the first steps towards host cell engineering to improve product quality, specifically the safety profiles of proteins produced in them.

Materials and Methods

Cell Culture and Fed Batch Assay

CHOZN® GS-/- host cells were transfected and single cell clones generated expressing two different monoclonal antibodies (IgG SO57, and IgG E), an Fc-Fusion protein and FLAG tagged Interferon-gamma (IFNg-FLAG). Fed-batch assays were carried out in duplicate 50-ml TPP™ bioreactor tubes and replenished with glucose and proprietary feeds on days 3 and 6. Cultures were maintained in Ex-CELL® CHO CD Fusion (Sigma-Aldrich, Cat No 14365C).

Glycoform Analysis and Total Sialic Acid Analysis

% Relative of total glycoform distribution was determined either on intact protein using a SEC-MS (Waters Acquity UPLC®/Q-TOF Premier™) method, or on released glycans as described in Figure 3. Total sialic acid analysis was performed on purified glycoproteins. Acid hydrolysis was performed by incubation in 2 M Acetic Acid for 1.5h @ 80°C. This was followed by incubation in 1,2-diamino-4,5-methylenoxybenzene (DMB) for 3h @ 50°C and the derivatized sialic acid was analyzed using HPLC-FLD.

Cell line generation of Cmah (-/-) and GGTA (-/-) Cell Line

Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (Cmah) and α-1,3 galactosyltransferase (GGTA1) biallelic knockouts in a CHOZN® GS-/- host cell lines were produced by zinc finger nuclease (ZFN) mediated targeted gene deletions. Single cell clones were generated using the limiting dilution method and gene modifications confirmed by sequencing.

Results and Discussion

Analytical Detection of α-Gal and Neu5Gc

analytical-detection-gal-and-neu5gc

Figure 2-3: Two different methods were used to detect presence of alpha-Gal. First, was binding of the lectin Bandeiraea simplicifolia Isolectin B4 to total surface glycans followed by FACS analysis. This lectin has a major affinity for terminal α-Dgalactosyl residues. Compared to a CHOK1 cell line, an NS0 cell line had almost 100 % positive staining for lectin binding and no staining at all for a monoclonal antibody IgG SO57 producing cell line in a CHOZN® DHFR-/- background. Cell lines producing two different recombinant proteins, IgG SO57 and an Fc-fusion protein in a CHOZN® GS-/- background were analyzed at Procognia, Israel for N-glycans as described in the Figure 3. No alpha gal was detected in any of the samples.

Levels of Neu5Gc are rProtein and Clone Dependent

levels-of-neu5Gc-rprotein-clone-dependent

Figure 4-5: Total sialic acid analysis was performed on lead clones producing different recombinant proteins in a CHOZN® GS -/- host cell line. While an Fc-fusion protein had the highest reported NANA levels, NGNA was only detected in an IFNg-FLAG producing cell line and in a IgG E (known to have glycosylation in the Fab region as well as the Fc region) producing cell line. Three different clones producing IgG E were analyzed and NGNA detected in the clone with the highest NANA levels.

Increasing Higher Order Glycosylation Using Glyco Protein Quality Supplement

higher-order-glycosylation

Figure 6-7. Increased substrate for sialic acid/alpha-gal by increasing galactosylation (more G1/G2), by adding Protein Quality Supplement (PQS) during the fed-batch process on day 2. No Neu5Gc or alpha-gal was detected even with the increased G2F in either the IgG SO57 or the fusion protein expressing clones.

Figure 8-9. Increased substrate for sialic acid by increasing galactosylation (more G1/G2) in IgG E expressing Clone 70G9, by adding Protein Quality Supplement (PQS) during the fed-batch process on day 2 using two different process conditions. Values indicate glycan modifications at two sites, one on the Fc region and one on the Fab region. An overall increase in higher order glycosylation was seen under Process B and with addition of PQS. NGNA was detected under these conditions, levels however were below the limit of quantitation (BQL).  

Genetic Disruption of Cmah and GGTA1 Using ZFNs

Workflow of Cmah GGTA1 Double KO

workflow-of-cmah

Figure 10.

Genotypic characterization of the Cmah (-/-) and GGTA (-/-) Cell Line

genotypic-characterization

Figure 10-11: The Cmah (-/-) and GGTA (-/-) Cell Line was created by sequential knock out of the Cmah and GGTA1 gene in the SAFC CHOZN® GS-/- host cell line. RNA of ZFN pair designed against Exon 5 of the CHO Cmah gene was transfected and single cell cloning followed by genetic characterization led to the isolation of 5 different biallelic Cmah (-/-) clones. One of the clones was subsequently transfected with ZFN pairs designed against Exon 9 of the CHO GGTA1 gene and GGTA1 (-/-) clones were identified. Figure 11, shows the modified genotype of the Cmah (-/-) and GGTA (-/-) Cell Line compared to the wild-type sequence.  

Conclusions

  • Immunogenicity issues may arise with the presence of non-human glycan epitopes on glycoprotein therapeutics such as Galactose alpha-1,3-galactose (α-Gal), or the sialic acid derivative N-Glycolylneuraminic acid (Neu5Gc).
  • While the genes responsible for the generation of these two epitopes were found to be present in SAFC CHOZN® GS-/- host cell line, α-Gal was non-detectable in the four different recombinant proteins produced in the CHOZN® GS-/- host cell line background by the current analytical characterization methods.
  • NGNA was only detected in an SAFC CHOZN® GS-/- host cell line producing IFNg-FLAG and in an IgG E (known to have glycosylation in the Fab region as well as the Fc region) producing cell line.
  • ZFN mediated targeted deletion of the genes Cmah and GGTA1 in the CHO host cell line (SAFC CHOZN® GS-/-), was performed allowing for the production of therapeutic proteins lacking the Neu5Gc and α-Gal immunogenic species respectively in this cell line.
  • Further characterization of the Cmah (-/-) and GGTA (-/-) cell line as well as different analytical methods to probe for the presence or absence of the α-Gal moiety are ongoing.
  • These results are the first steps towards host cell engineering to improve product quality specifically the safety profiles of proteins produced in them.

Acknowledgements

Isil Yasa and Kevin Ray, Analytical R&D Sigma Aldrich
Yehudit Amor, Ph.D, Procognia Israel Ltd.

Materials

     

References

  1. Michael C. Borys, et al. Biotechnology and Bioengineering, Vol. 105, No. 6, April 15, 2010
  2. Carlos J Bosques, et al. Nature Biotechnology, Vol. 28, No. 11, November, 2010

 

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