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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)

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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) Improving Product Safety Profiles: Host Cell Lines Deficient in CMP-N-Acetylneuraminic Acid Hydroxylase (CMAH) and Alpha-1-3-Galactosyltransferase (GGTA1) 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 Results and Discussion Genetic Disruption of Cmah and GGTA1 Using ZFNs Post-translational modifications have been shown to affect the bioactivity, clearance rates, immunogenicity and safety Analytical Detection of α-Gal and Neu5Gc profiles of therapeutic glycoproteins. For example anti- N-glycolylneuraminic acid (Neu5Gc or NGNA) antibodies in Workflow of Cmah GGTA1 Double KO Figure 2 Figure 3 humans can interact with Neu5Gc sialylated therapeutic proteins (for example: Cetuximab) produced in non-human Figure 10 CHO K1 NS0 DN20 GS -/- parental cell line 1 Biallelic Cmah KO clone mammalian expression systems causing clinical complications. This is because of an inactivating mutation in humans mAb1 and Fc fusion samples of the gene cytidine monophosphate-N-acetylneuraminic acid hydroxylase (Cmah), an enzyme responsible for Neu5Gc Cmah ZFN RNA transfection GGTA1 ZFN RNA transfection biosynthesis from the N-acetylneuraminic acid (Neu5Ac or NANA) form of sialic acid. Cmah is expressed in CHO cells N-linked glycans cleaved using and the Neu5Gc glycan moiety has been detected on several biologics produced in CHO. Another example of an SSC PNGase F after Trypsin cleavage immunogenic modification on N-glycans is the addition of a terminal galactose-α-1-3 galactose moiety (or α-Gal) mediated Single Cell Cloned and screened by the α-1,3 galactosyltransferase (GGTA1) gene. r-Proteins that carry this epitope can elicit strong immunogenic or Labelling with 2AB Single Cell Cloned and screened for GGTA1 gene KO anaphylactic reactions due to the presence of circulating anti-α Gal antibodies present in most humans. Contrary to FSC previous assumptions a CHO ortholog of the GGTA1 gene has been reported (Bosques et al), and antigenic α-Gal Separation on normal phase (NP) 5 Cmah -/- clones identified column using HPLC and characterized epitopes detected on the glycosylated proteins. Counts 1 GGTA -/- and Cmah -/- Enzyme degradation followed by re- Clone separation on NP column using HPLC. Glycans identified according to standard retention time database Genotypic characterization of the Cmah (-/-) and GGTA (-/-) Cell Line Eliminate -1,3 gal Bandeiraea simplicifolia Isolectin B4 (BSI-B4) (GGTA1) No alpha-Gal detected Figure 11 Levels of Neu5Gc are rProtein and Clone Dependent Figure 4 Figure 5 7 1.8 mol NANA/mol protein mol NGNA/mol protein mol NANA/mol protein mol NGNA/mol protein Cmah: 17 bp deletion, 31 bp deletion 6 1.6 1.4 5 1.2 4 1 3 0.8 Eliminate Neu5Gc 0.6 2 (Cmah) 0.4 1 0.2 Sialic Acid Content on puried rProtein Content Sialic Acid Figure 1. Glycosylation Engineering Targets: Elimination of Non-Human Immunogenic Sugar Sialic Acid Content on purified rProtein n.d n.d 0.01 0.04 0 0 Ggta1: 4 bp deletion, 4bp deletion Moieties. IgG SO57 Fc-Fusion protein E IFNgFlag IgG E Clone 147A2 IgG E Clone 70G9 IgG E Clone 142C9 IgG E Clone 147A2 Figure 10-11: The Cmah (-/-) and GGTA (-/-) Cell Line was created by sequential knock out of the Cmah and GGTA1 In this work, we describe the targeted deletion of the genes Cmah and GGTA1 in the CHO host cell line (SAFC CHOZN® Figure 2-3: Two different methods were used to detect presence of alpha-Gal. First, was binding of the lectin Bandeiraea gene in the SAFC CHOZN® GS-/- host cell line. RNA of ZFN pair designed against Exon 5 of the CHO Cmah gene was GS-/-), thus allowing for the production of therapeutic proteins lacking the Neu5Gc and α-Gal immunogenic species simplicifolia Isolectin B4 to total surface glycans followed by FACS analysis. This lectin has a major affinity for terminal α-D- transfected and single cell cloning followed by genetic characterization led to the isolation of 5 different biallelic Cmah respectively. While changes in process and culture conditions can be used to manipulate the levels Neu5Gc and α-Gal galactosyl residues. Compared to a CHOK1 cell line, an NS0 cell line had almost 100 % positive staining for lectin binding ® (-/-) clones. One of the clones was subsequently transfected with ZFN pairs designed against Exon 9 of the CHO GGTA1 immunogenic species, we have taken the approach of the complete elimination of these species by disrupting the and no staining at all for a monoclonal antibody IgG SO57 producing cell line in a CHOZN DHFR-/- background. Cell lines ® gene and GGTA1 (-/-) clones were identified. Figure 11, shows the modified genotype of the Cmah (-/-) and GGTA (-/-) relevant genes. These results are the first steps towards host cell engineering to improve product quality, specifically producing two different recombinant proteins, IgG SO57 and an Fc-fusion protein in a CHOZN GS-/- background were Cell Line compared to the wild-type sequence. the safety profiles of proteins produced in them. analyzed at Procognia, Israel for N-glycans as described in the Figure 3. No alpha gal was detected in any of the samples. 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 Conclusions Materials and Methods region) producing cell line. Three different clones producing IgG E were analyzed and NGNA detected in the clone with • Immunogenicity issues may arise with the presence of non-human glycan epitopes on glycoprotein therapeutics such the highest NANA levels. Cell Culture and Fed Batch Assay as Galactose alpha-1,3-galactose (α-Gal), or the sialic acid derivative N-Glycolylneuraminic acid (Neu5Gc). CHOZN® GS-/- host cells were transfected and single cell clones generated expressing two different monoclonal Increasing Higher Order Glycosylation Using Glyco Protein Quality Supplement • While the genes responsible for the generation of these two epitopes were found to be present in SAFC CHOZN® GS- antibodies (IgG SO57, and IgG E), an Fc-Fusion protein and FLAG tagged Interferon-gamma (IFNg-FLAG). Fed-batch /- host cell line, α-Gal was non-detectable in the four different recombinant proteins produced in the CHOZN® GS-/- Figure 7 assays were carried out in duplicate 50-ml TPPTM bioreactor tubes and replenished with glucose and proprietary feeds Figure 6 host cell line background by the current analytical characterization methods. ® 100 80 on days 3 and 6. Cultures were maintained in Ex-CELL CHO CD Fusion (Sigma-Aldrich, Cat No 14365C). mAB1 IgG mAB1 IgG + Glyco PQS Fc Fusion-1 Fc Fusion-1 + Glyco PQS ® 90 70 • NGNA was only detected in an SAFC CHOZN GS-/- host cell line producing IFNg-FLAG and in an IgG E (known to 80 60 70 have glycosylation in the Fab region as well as the Fc region) producing cell line. 50 Glycoform Analysis and Total Sialic Acid Analysis 60 Glycoforms 40 ® Glycoforms 50 • ZFN mediated targeted deletion of the genes Cmah and GGTA1 in the CHO host cell line (SAFC CHOZN GS-/-), was % Relative of total glycoform distribution was determined either on intact protein using a SEC-MS (Waters Acquity 40 30 30 performed allowing for the production of therapeutic proteins lacking the Neu5Gc and α-Gal immunogenic species ® 20 % of Total UPLC /Q-TOF Premier™) method, or on released glycans as described in Figure 3. Total sialic acid analysis was % of Total 20 10 respectively in this cell line. 10 performed on purified glycoproteins. Acid hydrolysis was performed by incubation in 2 M Acetic Acid for 1.5h @ 80°C. n.d 0 0 This was followed by incubation in 1,2-diamino-4,5-methylenoxybenzene (DMB) for 3h @ 50°C and the derivatized High Mannose G0 G1 G2 Core Fucose Sialylated Gal a 1-3, Gal High Mannose G0 G1 G2 Core Fucose Sialylated Gal a 1-3, Gal • Further characterization of the Cmah (-/-) and GGTA (-/-) cell line as well as different analytical methods to probe for sialic acid was analyzed using HPLC-FLD. the presence or absence of the α-Gal moiety are ongoing. Figure 8 Figure 9 • These results are the first steps towards host cell engineering to improve product quality specifically the safety profiles 35 35 Clone 70G9 + Process A Clone 70G9 + Process B Cell line generation of Cmah (-/-) and GGTA (-/-) Cell Line 30 30 of proteins produced in them. Clone 70G9 + Process A + PQS Clone 70G9 + Process B + PQS 25 Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (Cmah) and α-1,3 galactosyltransferase (GGTA1) 25 20 20 ® biallelic knockouts in a CHOZN GS-/- host cell lines were produced by zinc finger nuclease (ZFN) mediated targeted 15 15 gene deletions. Single cell clones were generated using the limiting dilution method and gene modifications confirmed 10 10 % of Total Glycoforms Acknowledgements 5 % of Total Glycoforms 5 by sequencing. N.D N.D BQL BQL 0 0 Isil Yasa and Kevin Ray, Analytical R&D Sigma Aldrich G0F, G0F G1F, G0F G2F,G0F G2FS,G0F G2FS,G1F G2FS2,G0F G2FS2,G1F G2FS2,G1F NGNA G0F, G0F G1F, G0F G2F,G0F G2FS,G0F G2FS,G1F G2FS2,G0F G2FS2,G1F G2FS2,G1F NGNA Yehudit Amor, Ph.D, Procognia Israel Ltd. 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.
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