Nature Biotechnology

Published by Springer Nature

Online ISSN: 1546-1696


Print ISSN: 1087-0156


  • Article

June 1998


32 Reads

James C. Blair
Maintaining the balance of commitment, communication, criticism, conjecture, and competition is the basis for bioentrepreneurial success.

The power of many
  • Article
  • Full-text available

March 2011


235 Reads

Applications of crowdsourcing in commercial biotech remain few and far between, but the approach is proving increasingly popular for solving challenges in basic research. Clare Sansom reports.

An individual approach

November 2005


26 Reads

Reduced side effects and more effective therapies are some of the benefits promised by pharmacogenomics. But to reach these goals, industry will have to marshal a broad range of skills.

When less is more

September 2007


36 Reads

An analysis of recent returns from venture-backed biotech firms reveals that companies receiving the most financing do not necessarily deliver the best returns.

Patient power

September 2012


21 Reads

Patient foundations are not only exploring new funding models but also catalyzing translational research, with notable successes. Jim Kling reports.

Anti-galactose-Î ±-1,3-galactose IgE from allergic patients does not bind α-galactosylated glycans on intact therapeutic antibody Fc domains

July 2011


117 Reads

Jeroen J. Lammerts van Bueren


Theo Rispens





Manufacture of monoclonal antibodies (mAb) and related biologics is performed in eukaryotic cell lines to allow proper glycosylation, which is often critical for function. The first mAbs were produced in the mouse-derived cell lines SP2/0 and NS0, which have the genetic makeup to produce proteins containing nonhuman glycan structures, such as galactose-α-1,3- galactose (α-Gal) and N-glycolylneuramic acid1, that can adversely affect safety and half-life. The best example is Erbitux (cetuximab), produced in SP2/0 cells, clinical use of which has been associated with α-Gal–specific, IgE-mediated anaphylactic responses2. In addition, patients with IgE antibodies to α-Gal may experience allergic reactions to red meat3,4. The quantity of α-Gal required to induce hypersensitivity reactions is not known; however, it is clear that binding of IgE antibodies against α-Gal is an important trigger in this pathology.

Figure 1: Structural complex of CHO Ggta1 exons 8–9 gene product with UDP-2F-Gal donor and LacNAc acceptor substrate. (a) Shown in stereo view is the cartoon representation of the enzyme active site with the side chains of the key residues labeled using the single amino acid code and the numbering based on the bovine α3GalT crystal structure (PDB: 1G93). The UDP-2F-Gal is shown in stick representation in green and the type II LacNAc acceptor substrate is shown in stick representation in yellow. The location of the Mn2+ cation (in purple) is obtained from the bovine α3GalT crystal structure template used to construct the homology model. (b) Sequence alignment of the various α3GalT with the highly conserved critical active site residues highlighted in gray.
Figure 2: MS/MS fragmentation analysis of N-glycan species observed in abatacept. (a–c) Comparison of the MS/MS fragmentation profile between an N-glycan species with a neutral mass of 2,070 Da observed in Orencia (a) and the fragmentation profile of an isobaric hybrid species (b) and an isobaric α-Gal–containing species (c) from our N-glycan fragmentation database. Nomenclature for glycan structures: blue squares, GlcNAc; red triangles, fucose; green circles, mannose; yellow circles, galactose. The additional galactose is depicted in the lower branch to simplify the fragmentation nomenclature. Fragmentation nomenclature is based on ref. 10.
Chinese hamster ovary cells can produce galactose-??-1,3-galactose antigens on proteins

November 2010


525 Reads

Chinese hamster ovary (CHO) cells are widely used for the manufacture of biotherapeutics, in part because of their ability to produce proteins with desirable properties, including 'human-like' glycosylation profiles. For biotherapeutics production, control of glycosylation is critical because it has a profound effect on protein function, including half-life and efficacy. Additionally, specific glycan structures may adversely affect their safety profile. For example, the terminal galactose-α-1,3-galactose (α-Gal) antigen can react with circulating anti α-Gal antibodies present in most individuals. It is now understood that murine cell lines, such as SP2 or NSO, typical manufacturing cell lines for biotherapeutics, contain the necessary biosynthetic machinery to produce proteins containing α-Gal epitopes. Furthermore, the majority of adverse clinical events associated with an induced IgE-mediated anaphylaxis response in patients treated with the commercial antibody Erbitux (cetuximab) manufactured in a murine myeloma cell line have been attributed to the presence of the α-Gal moiety. Even so, it is generally accepted that CHO cells lack the biosynthetic machinery to synthesize glycoproteins with α-Gal antigens. Contrary to this assumption, we report here the identification of the CHO ortholog of N-acetyllactosaminide 3-α-galactosyltransferase-1, which is responsible for the synthesis of the α-Gal epitope. We find that the enzyme product of this CHO gene is active and that glycosylated protein products produced in CHO contain the signature α-Gal antigen because of the action of this enzyme. Furthermore, characterizing the commercial therapeutic protein abatacept (Orencia) manufactured in CHO cell lines, we also identified the presence of α-Gal. Finally, we find that the presence of the α-Gal epitope likely arises during clonal selection because different subclonal populations from the same parental cell line differ in their expression of this gene. Although the specific levels of α-Gal required to trigger anaphylaxis reactions are not known and are likely product specific, the fact that humans contain high levels of circulating anti-α-Gal antibodies suggests that minimizing (or at least controlling) the levels of these epitopes during biotherapeutics development may be beneficial to patients. Furthermore, the approaches described here to monitor α-Gal levels may prove useful in industry for the surveillance and control of α-Gal levels during protein manufacture.

Table 2 . Summary of nuclear transfer results from α1,3GT knockout primary fibroblast cells
Figure 3: Five 1,3GT gene knockout piglets at 2 weeks of age.
Targeted disruption of the 1,3-galactosyltransferase gene in cloned pigs

April 2002


773 Reads

Galactose-alpha1,3-galactose (alpha1,3Gal) is the major xenoantigen causing hyperacute rejection in pig-to-human xenotransplantation. Disruption of the gene encoding pig alpha1,3-galactosyltransferase (alpha1,3GT) by homologous recombination is a means to completely remove the alpha1,3Gal epitopes from xenografts. Here we report the disruption of one allele of the pig alpha1,3GT gene in both male and female porcine primary fetal fibroblasts. Targeting was confirmed in 17 colonies by Southern blot analysis, and 7 of them were used for nuclear transfer. Using cells from one colony, we produced six cloned female piglets, of which five were of normal weight and apparently healthy. Southern blot analysis confirmed that these five piglets contain one disrupted pig alpha1,3GT allele.

Figure 1. Organization of the genomic loci of ovine (A) GGTA1 or (B) PrP genes and the promoterless targeting vectors used for disruption. Numbering of the exons in GGTA1 is based on the mouse; translation initiates in exon 4 and terminates in exon 9. Targeting deletes exon 4 and 1.4 kb of intron 4, and a BamHI site (labeled B) is inserted. The coding sequence of PrP is entirely within exon 3; targeting deletes this region and two BglI sites (labeled Bg). Arrows indicate translation initiation sites. Black boxes represent exons, hatched boxes represent neo-pA sequence, and open box represents pBlueScript sequence. Location of PCR primers (GGTA1 uses G1/2 and G1/3; PrP uses P1/2 and P1/3) and the 5′ external probes for Southern blot analysis are shown. Scale bar represents 2 kb.
Figure 2. Targeted mutations are retained through development. DNA was isolated from cells before nuclear transfer or from derived fetuses, then analyzed by PCR and Southern blot. Samples with a targeted allele are indicated by an asterisk (*). See Figure 1 for location of primers and probes. (A) GGTA1 PCR. Lanes 1, 2, and 3 show 3C6* cells, and fetuses at day 85* and day 118*. Lanes 4, 5, and 6 show 5E1* cells, and fetuses at day 49* and day 49*. Lanes 7, 8, and 9 show 4H2 cells, and fetuses at day 49 and day 148. (B) PrP PCR. Lane 4 shows nontargeted parental cells. Lane 1 shows YH6* cells. Lanes 2 and 3 show lambs carried to term*. Lane 5 shows the targeted lamb that survived to 12 days*. (C) Southern blot analysis. GGTA1 samples were digested with BamHI. The targeted allele hybridizes with the 5′ and neo probes; lanes 1, 2, and 3 show samples from fetuses at day 118*, day 49*, and day 49*. Lane 4 shows a nontargeted sample. PrP samples were digested with BglI. Lane 5 shows the targeted lamb that survived to 12 days*. Lanes 6 and 7 show samples from fetuses at term*. Lane 8 shows a nontargeted sample.
Figure 3. PrP-/+ lamb photographed at six days postpartum.
Deletion of the ??(1,3)galactosyl transferase (GGTA1) gene and the prion protein (PrP) gene in sheep

July 2001


215 Reads

Nuclear transfer offers a cell-based route for producing precise genetic modifications in a range of animal species. Using sheep, we report reproducible targeted gene deletion at two independent loci in fetal fibro-blasts. Vital regions were deleted from the alpha(1,3)galactosyl transferase (GGTA1) gene, which may account for the hyperacute rejection of xenografted organs, and from the prion protein (PrP) gene, which is directly associated with spongiform encephalopathies in humans and animals. Reconstructed embryos were prepared using cultures of targeted or nontargeted donor cells. Eight pregnancies were maintained to term and four PrP-/+ lambs were born. Although three of these perished soon after birth, one survived for 12 days. These data show that lambs carrying targeted gene deletions can be generated by nuclear transfer.

Overexpression of a cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase in tobacco enhances photosynthesis and growth

November 2001


242 Reads

Transgenic tobacco plants expressing a cyanobacterial fructose-1,6/sedoheptulose-1,7-bisphosphatase targeted to chloroplasts show enhanced photosynthetic efficiency and growth characteristics under atmospheric conditions (360 p.p.m. CO2). Compared with wild-type tobacco, final dry matter and photosynthetic CO2 fixation of the transgenic plants were 1.5-fold and 1.24-fold higher, respectively. Transgenic tobacco also showed a 1.2-fold increase in initial activity of ribulose 1,5 bisphosphate carboxylase/oxygenase (Rubisco) compared with wild-type plants. Levels of intermediates in the Calvin cycle and the accumulation of carbohydrates were also higher than those in wild-type plants. This is the first report in which expression of a single plastid-targeted enzyme has been shown to improve carbon fixation and growth in transgenic plants.

Table 1 Moleculizer modules
Lok, L. & Brent, R. Automatic generation of cellular reaction networks with Moleculizer 1.0. Nature Biotechnol. 23, 131-136

February 2005


77 Reads

Accurate simulation of intracellular biochemical networks is essential to furthering our understanding of biological system behavior. The number of protein complexes and of chemical interactions among them has traditionally posed significant problems for simulation algorithms. Here we describe an approach to the exact stochastic simulation of biochemical networks that emphasizes the contribution of protein complexes to these systems. This simulation approach starts from a description of monomeric proteins and specifications for binding, unbinding and other reactions. This manageable specification is reasonably intuitive for biologists. Rather than requiring the inclusion of all possible complexes and reactions from the outset, our approach incorporates new complexes and reactions only when needed as the simulation proceeds. As a result, the simulation generates much smaller reaction networks, which can be exported to other simulators for further analysis. We apply this approach to the automatic generation of reaction systems for the study of signal transduction networks.

Figure 4. Adenovirus-based β-galactosidase expression in mouse liver before and after hepatocellular regeneration. C57Bl/6-scid mice were injected into the tail vein with 1 × 10 9 TU each of HD-TLacZ and HD-SB-Flp (A, E), HD-FRT-TLacZ alone (B, F), HD-FRT-TLacZ and HD-mSB-Flp (C, G), or HD-FRT-TLacZ and HD-SB-Flp (D, H). Transposase expression was induced in a stepwise manner during the five weeks immediately following vector administration by addition of increasing concentrations of ZnSO 4 to the animal drinking water. Representative sections are shown from liver removed during a surgical two-thirds partial hepatectomy (PH) 5 weeks postinjection (p.i.) (A–D; n = 5 mice per group) and again after an additional 16 weeks (E–I; n = 3 mice per group). All animals received intraperitoneal injections of CCl 4 at 8, 10, 12, 15, 17, and 19 weeks after vector administration to further promote hepatocellular cell cycling. Arrows denote the presence of β-galactosidase + foci, examples of which are shown in more detail in the six lower (I) panels. Magnifications: A–D, original ×100; E–H, original ×40; I, originals ×200. Bars, 500uM. Bar in D applies to A-D and bar in H applies to E-H.  
Figure 5. In vivo human Factor IX persistence in actively dividing mouse livers via the adeno-transposon system. C57Bl/6 mice (n = 5 mice per group) were injected into the tail vein with 5 × 10 8 TU of HD-FRT-ThFIX together with 5 × 10 8 TU of either HD-SB-Flp () or HD-mSB-Flp () as a control. Transposase expression was induced in treated animals during the first three weeks immediately following vector administration by addition of drinking water containing ZnSO 4 (25 mM final). Approximately three weeks postinjection, the zinc was removed from the water and a surgical two-thirds partial hepatectomy (PH) was done to stimulate hepatocellular regeneration and loss of episomal vectors. Arrows indicate when animals received intraperitoneal injections of CCl 4 to further promote hepatic cell cycling.
Figure 6. Transposition in vivo from a single Ad vector encoding transposon and Flp-activated transposase activities. (A) Experimental strategy for controlled transposase expression from a single Ad-based transposition vector. A linear adenoviral vector (HD-FRT-TNC-SB) containing the entire Sleeping Beauty transposon system flanked by a pair of FRT sites forms a circular intermediate in the presence of the Flp recombinase. Circularization of the transposition cassette results in cis-activation of transposase expression and genomic transposition from the adenoviral circle. In this configuration, the episome-encoded transposase gene is either degraded after transposition or lost over time during cell division. CMV, Minimal cytomegalovirus core promoter. (B) Transposon insertion site sequences from mice injected with a single integrating adenovirus vector. C57Bl/6-scid mice (n = 3) were injected with 2.4 × 10 9 TU of HD-FRT-TNC-SB through the tail vein. One week later, animals were injected with 25 µg pCMV-Flpe to promote Flp-mediated vector rearrangement, and seven weeks later, total liver DNA was isolated and used to recover integrated transposons through a plasmid recovery strategy. Target site duplications are shown in bold uppercase, novel flanking sequences are in lowercase, and transposon sequences are denoted by the central shaded box.
Yant, SR, Ehrhardt, A, Mikkelsen, JG, Meuse, L, Pham, T and Kay, MA. Transposition from a gutless adeno-transposon vector stabilizes transgene expression in vivo. Nat Biotechnol 20: 999-1005

November 2002


199 Reads

A major limitation of adenovirus-mediated gene therapy for inherited diseases is the instability of transgene expression in vivo, which originates at least in part from the loss of the linear, extrachromosomal vector genomes. Herein we describe the production of a gene-deleted adenovirus-transposon vector that stably maintains virus-encoded transgenes in vivo through integration into host cell chromosomes. This system utilizes a donor transposon vector that undergoes Flp-mediated recombination and excision of its therapeutic payload in the presence of the Flp and Sleeping Beauty recombinases. Systemic in vivo delivery of this system resulted in efficient generation of transposon circles and stable transposase-mediated integration in mouse liver. Somatic integration was sufficient to maintain therapeutic levels of human coagulation Factor IX for more than six months in mice undergoing extensive liver proliferation. These vectors combine the versatility of adenoviral vectors with the integration capabilities of a eukaryotic DNA transposon and should prove useful in the treatment of genetic diseases.

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