Báo cáo khoa học: Production of a recombinant mouse monoclonal antibody in transgenic silkworm cocoons pptx

Production of a recombinant mouse monoclonal antibody in transgenic silkworm cocoons Masashi Iizuka1, Shingo Ogawa2, Atsushi Takeuchi1, Shinichi Nakakita3, Yuhki Kubo4, Yoshitaka Miyawaki4, Jun Hirabayashi3 and Masahiro Tomita1 Neosilk Co., Ltd, Higashihiroshima, Hiroshima, Japan Research Institute, Koken Co., Ltd, Kita-ku, Tokyo, Japan Life Science Research Center, Kagawa University, Kita-gun, Kagawa, Japan Masuda Chemical Industries Co., Ltd, Takamatsu, Japan Keywords IgG; monoclonal antibody; N-glycosylation; recombinant protein; silk gland Correspondence M Tomita, Neosilk Co., Ltd, 3-13-26 Kagamiyama, Higashihiroshima, Hiroshima 739-0046, Japan Fax: +81 82 431 0654 Tel: +81 82 431 0652 E-mail: mtomita@neosilk.co.jp (Received 15 June 2009, revised August 2009, accepted August 2009) doi:10.1111/j.1742-4658.2009.07262.x In the present study, we describe the production of transgenic silkworms expressing a recombinant mouse mAb in their cocoons Two transgenic lines, L- and H-, were generated that carried cDNAs encoding the L- and H-chains of a mouse IgG mAb, respectively, under the control of the enhancer-linked sericin-1 promoter Cocoon protein analysis indicated that the IgG L- or H-chain was secreted into the cocoons of each line We also produced a transgenic line designated L ⁄ H, which carried both cDNAs, by crossing the L- and H-lines This line efficiently produced the recombinant mAb as a fully assembled H2L2 tetramer in its cocoons, with negligible L- or H-chain monomer and H-chain dimer production Thus, the H2L2 tetramer was synthesized in, and secreted from, the middle silk gland cells Crossing of the L ⁄ H-line with a transgenic line expressing a baculovirusderived trans-activator produced a 2.4-fold increase in mAb expression The recombinant mAb was extracted from the cocoons with a buffer containing m urea and purified by protein G affinity column chromatography The antigen-binding affinity of the purified recombinant mAb was identical to that of the native mAb produced by a hybridoma Analysis of the structure of the N-glycans attached to the recombinant mAb revealed that the mAb contained high mannose-, hybrid- and complex-type N-glycans By contrast, insect-specific paucimannose-type glycans were not detected Fucose residues a-1,3- and a-1,6-linked to the core N-acetylglucosamine residue, both of which are found in insect N-glycans, were not observed in the N-glycans of the mAb Introduction mAbs comprise the fastest growing class of therapeutic proteins; thus, there is an increasing need for their costeffective production Current standard procedures for the production of recombinant mAbs rely on mammalian cell lines as hosts [1] because their use meets current regulatory requirements However, enormous investment is required for the construction of the bioreactors used to culture the cells and to run the reactors On the other hand, numerous production systems for mAbs have been developed using non-mammalian hosts, Abbreviations AAL, Aleuria aurantia lectin; CBB, Coomassie brilliant blue; DsRed, red fluorescent protein; GnT, N-acetylglucosaminyltransferase; HRP, horseradish peroxidase; MGFP, monster green fluorescent protein; MSG, middle silk gland; PA-N-glycans, pyridylaminated-N-glycans; PNGaseF, peptide-N-glycosidase F; PSG, posterior silk gland 5806 FEBS Journal 276 (2009) 5806–5820 ª 2009 The Authors Journal compilation ª 2009 FEBS M Iizuka et al including plants [2–4], filamentous fungi [5], chickens [6] and insect cells [7–9] A single IgG molecule is a tetramer consisting of two H- and two L-chains The recombinant mAbs produced by the above non-mammalian production systems are intact H2L2 tetramers with normal antigen-binding ability N-glycans are attached to Asn297 of the H-chain constant region in IgG mAbs Because differences in the structures of these N-glycans can cause allergic reactions [10] or lead to rapid clearance of the mAbs from the human body [11–13], it is important to humanize them when the mAbs produced by non-mammalian hosts are to be used for therapeutic applications Several attempts have been made to produce recombinant mAbs with humanized N-glycans using plants as hosts For example, immunogenic b-1,2xylose and a-1,3-fucose residues have been removed from the glycans by inhibiting b-1,2-xylosyltransferase and a-1,3-fucosyltransferase, respectively, using RNA interference or knockout technology [4,14,15] The silkworm Bombyx mori synthesizes large amounts of silk proteins in its silk glands and spins them into silk fibers to build a cocoon This ability to synthesize silk proteins in large quantities may be useful for the production of recombinant proteins By increasing the number of reared silkworms, the procedure for protein production can be scaled up with ease Therefore, the silkworm might be suited as a host for the mass production of recombinant mAbs compared to mammalian cultured cells and non-mammalian organisms The silk fibers are composed of the proteins fibroin and sericin, which constitute approximately 75% and 25%, respectively, of the fiber weight Fibroin, which constitutes the silk fiber core, is synthesized in the posterior silk gland (PSG) [16] Sericin, which comprises a group of hydrophilic glue proteins that surround the fibroin core, is synthesized in the middle silk gland (MSG) Two sericin genes are known (ser1 and ser2); however, most sericin proteins are encoded by ser1 [17–21] One method for generating germline transgenic silkworms involves the use of piggyBac transposon-derived vectors [22,23] By taking advantage of PSG- and MSG-specific promoters, we developed two recombinant expression systems using transgenic silkworms On the one hand, the recombinant proteins were expressed as fusion proteins with fibroin in the PSG under control of the fibroin promoter [23–25] The silk fibers produced by these silkworms exhibited the properties of both the silk and the recombinant proteins because the recombinant proteins were embedded in the fibroin fibers On the other hand, the ser1 promoter was used to express recombinant proteins in the MSG In this case, the recombinant proteins were secreted into the hydrophilic sericin Production of mouse mAb by transgenic silkworms layers without being fused to the silk proteins; thus, they were extractable from the cocoons with mild neutral aqueous solutions such as NaCl ⁄ Pi or NaCl ⁄ Tris [26,27] We previously reported an increase in the expression of recombinant proteins in the MSG using the baculovirus-derived enhancer hr3, the trans-activator IE1 [27] and the 5¢-UTR of baculovirus polyhedrin mRNA [28] Recombinant mRNAs were efficiently transcribed from their transgenes in MSG cells by using both the above enhancer and trans-activator; the amounts of the mRNAs observed reached 30–40% of the endogenous ser1 mRNA level On the other hand, the 5¢-UTR enhanced recombinant protein expression in the MSG cells at the level of translation, leading to a 1.5-fold increase in recombinant protein synthesis In the present study, we generated germline transgenic silkworms that synthesize both the L- and H-chains of a mouse IgG mAb in their MSG cells and secrete the mAb as an H2L2 tetramer into the sericin layer of their silk fibers Expression of the mAb was increased by introducing the gene encoding the baculovirus-derived trans-activator The recombinant mAb was extracted and purified from the silk fibers, and the antigen-binding properties of the purified mAb were compared with those of a natural mAb from a hybridoma that had been used as the source of the introduced IgG genes, demonstrating that the binding properties of the recombinant mAb were identical to those of the hybridoma-derived natural mAb The structures of the N-glycans attached to the recombinant mAb were also determined Paucimannose-type N-glycans were not detected, whereas high mannose-, hybrid- and complex-type N-glycans were detected No core fucosylations were found in the N-glycans of the recombinant mAb Major N-glycans in insect cells have paucimannose structures with core fucosylations and high mannose structures, although some variations in the glycan structure are observed depending on the synthesized glycoproteins Further analysis of the N-glycans from silkworm tissues revealed that the above-described N-glycan structures in the recombinant mAb are a result of the tissue specificity of silk glands Results Generation of transgenic silkworms carrying cDNAs encoding a mouse IgG mAb We constructed two vectors, pIgGL ⁄ M1.1MG and pIgGH ⁄ M1.1R, for the generation of transgenic silkworms expressing a mouse IgG mAb (Fig 1) The former vector contained the cDNA for monster green fluorescent protein (MGFP) as a marker under the control of an FEBS Journal 276 (2009) 5806–5820 ª 2009 The Authors Journal compilation ª 2009 FEBS 5807 Production of mouse mAb by transgenic silkworms pIgGL/M1.1MG piggyBac right arm M Iizuka et al P3xP3 MGFP SV40 polyA hr3 Pser1 IgG L-chain piggyBac fibL left arm polyA BmNPVpol 5′-UTR pIgGH/M1.1R piggyBac P right arm 3xP3 DsRed SV40 polyA hr3 Pser1 piggyBac fibL polyA left arm IgG H-chain BmNPVpol 5′-UTR pIE1 piggyBac right arm P3xP3 DsRed SV40 Pser1 polyA ie1 ie1 piggyBac polyA left arm Fig Structures of the transformation vectors Three transformation vectors (pIgGL ⁄ M1.1MG, pIgGH ⁄ M1.1R and pIE1) were constructed, each of which contained expression units for selection markers and the recombinant proteins between the right and left arms of piggyBac In the selection marker units, the gene encoding DsRed (DsRed) or MGFP (MGFP) was placed between the 3xP3 promoter (P3xP3) and SV40 polyA signal sequence (SV40 polyA) The recombinant protein units were designed to express IgG L- and H-chains in pIgGL ⁄ M1.1MG and pIgGH ⁄ M1.1R, respectively; thus, the L-chain (IgG L-chain) or H-chain (IgG H-chain) cDNA was placed between the BmNPV hr3 enhancer, (hr3)-ser1 promoter (Pser1) and fibroin L-chain polyA signal sequence (fibL polyA) The recombinant protein unit in pIE1 was composed of the ser1 promoter (Pser1), IE1 gene (ie1) and ie1 polyA signal sequence (ie1 polyA) eye- and nervous tissue-specific promoter, 3xP3, plus the cDNA for the IgG L-chain under the control of the ser1 promoter The latter vector contained the cDNAs for red fluorescent protein (DsRed) and the IgG H-chain under the control of the 3xP3 and ser1 promoters, respectively (Fig 1) pIgGL ⁄ M1.1MG and pIgGH ⁄ M1.1R were injected into 3154 and 2854 eggs, respectively, and the hatched G0 larvae were allowed to develop to moths G1 embryos from the G0 moths were screened for MGFP or DsRed fluorescence to obtain transgenic silkworms Genomic Southern blot analysis of the transgenic silkworms demonstrated the existence of 13 and 17 independent transgenic lines, respectively, for pIgGL ⁄ M1.1MG- and pIgGH ⁄ M1.1R in relation to the chromosomal insertion positions and copy numbers of the transgenes Transgenic lines with a single-copy transgene were selected, and the cocoon proteins of the lines were analyzed by SDS ⁄ PAGE The lines with the highest levels of IgG L- and H-chain expression were used in the subsequent experiments as the L- and H-lines, respectively To generate transgenic silkworms bearing both the L- and H-chain cDNAs, an L-line worm was crossed with an H-line worm, and the silkworms in the subsequent generation that expressed both MGFP and DsRed in their eyes were selected The silkworms carrying both the L- and H-chain cDNAs were referred to as L ⁄ H-line silkworms Analysis of recombinant mouse IgG in cocoons To analyze secreted proteins in the sericin layer of the silk fibers, all proteins in the layer were dissolved in a 5808 buffer containing m urea, electrophoresed under reducing conditions, and analyzed by western blotting using polyclonal anti-mouse IgG serum Recombinant mouse IgG L- and H-chain was detected in the L- and H-lines, respectively (Fig 2A, lanes and 9) The H-chain was also identified in the H-line proteins by Coomassie brilliant blue (CBB) staining (Fig 2A, lane 4), whereas the L-chain was not, as a result of the presence of endogenous silk proteins with a similar molecular weight (Fig 2A, lane 3) Both L- and H-chains were detected in the cocoon proteins from the L ⁄ H-line by CBB staining and western blotting (Fig 2A, lanes and 10) The amount of L- or H-chain in the L ⁄ H-line appeared to be higher than that in the L- or H-line The intensity of the H-chain on the CBB-stained gels was quantified by densitometry The mean ± SEM amount of H-chain present in 0.1 mg of cocoons from the L ⁄ H- and H-lines was 319 ± ng (n = 3) and 139 ± ng (n = 3), respectively To investigate the assembly of the recombinant L- and H-chains, cocoon proteins were analyzed by electrophoresis under nonreducing conditions No L- or H-chain was detected among the cocoon proteins from the L- and H-lines by CBB staining, respectively (Fig 2B, lanes and 4) Western blotting revealed that the L-chain in the L-line cocoons existed as a monomer (Fig 2B, lane 8) By contrast, the H-chain was detected as a dimer in the H-line cocoon proteins (Fig 2B, lane 9) Intense bands with an apparent molecular weight that exceeded that of the H-chain dimer were also visible on the blot Although the FEBS Journal 276 (2009) 5806–5820 ª 2009 The Authors Journal compilation ª 2009 FEBS M Iizuka et al Production of mouse mAb by transgenic silkworms A Reducing M W L H L/H St W L H L/H St kDa 80 60 50 40 30 H H L L 20 10 11 CBB Western be derived from random aggregates of the chains, were also detected on the blot as the single expression of the H-chain These aggregates appear in much smaller amounts than the H2L2 because the H2L2 was detected as a major product on the CBB-stained gel No L-chain monomer was present among the cocoon proteins (Fig 2B, lane 10) These results suggest that a fully assembled mouse IgG mAb with an H2L2-subunit structure was synthesized in the MSG cells and secreted into the sericin layer of the silk fibers in silkworms carrying both the IgG L- and H-chain cDNAs Quantification of L- and H-chain mRNAs in MSGs Nonreducing B M W L H L/H St kDa 220 120 100 80 60 50 40 30 W L H L/H St H2L2 H2L H2 H2L2 20 L 10 11 CBB Western Fig Analysis of the cocoon proteins in the L-, H- and L ⁄ H-lines The proteins in the cocoons of wild-type (W), L- (L), H- (H) or L ⁄ H-line (L ⁄ H) silkworms were extracted with (A) M urea containing 2% (v ⁄ v) b-mercaptoethanol and 50 mM Tris–HCl, pH 8.0 (i.e reducing conditions) or (B) M urea containing 50 mM Tris–HCl, pH 8.0 (i.e nonreducing conditions) Aliquots of the extracts were subjected to SDS ⁄ PAGE Some of the gels were stained with CBB, whereas others were subjected to western blotting using the rabbit anti(mouse IgG) as a primary antibody (western) ‘H2L2’, ‘H2L’, ‘H2’, ‘H’ and ‘L’ to the right of the gel indicate the H2L2 tetramer, H2L trimer, H2 dimer, H monomer and L monomer, respectively The numbers to the left of the gel are the molecular masses (kDa) as determined by the migration of the markers (M) St, commercially available standard mouse IgG detailed structures of these high molecular weight products were unclear, the products most likely were random aggregates of H-chain connected by interchain disulfide bonds CBB staining of the cocoon proteins from the L ⁄ H-line revealed a band co-migrating with the standard IgG H2L2 tetramer (Fig 2B, lanes and 6) In addition to the H2L2 tetramer, small amounts of H2L and H2 were detected among the proteins from the L ⁄ H-line by western blotting, which were also detectable in the standard mouse IgG (Fig 2B, lanes 10 and 11) Bands with the higher molecular weight than H2L2, which were assumed to As described above, the amounts of L- and H-chain in the cocoons were increased by the co-expression of both chains compared to the expression of either chain We therefore investigated whether these increases arose from an increase in the corresponding mRNAs in the cells Total RNA was extracted from the MSGs of fifth-instar larvae of the L-, H- and L ⁄ H-lines, and the L- and H-chain mRNA levels were measured by quantitative RT-PCR The amount of sericin-1 mRNA was also determined to allow for normalization of the expression of the L- and H-chains As shown in Table 1, the amount of L- or H-chain mRNA in the L ⁄ H-line was lower than that of the corresponding chain in the L- or H-line, most likely as a result of the co-expression of the two genes from the same promoter These results suggest that the increases in IgG L- and H-chain in the L ⁄ H-line cocoons were not caused by the transcriptional regulation of mRNA expression, but by the regulation of protein synthesis and secretion Enhanced transgene expression using trans-activator IE1 We previously demonstrated that the baculovirusderived trans-activator IE1 stimulates the transcripTable Copy numbers of mRNAs of the L-chain, H-chain and sericin-1 in MSG cells Line L-chaina H-chaina Sericin-1a Percentage of L-chain to sercin-1 L H L⁄H 2.4 ± 0.4b 0.0 1.4 ± 0.2 0.0 3.0 ± 0.5 1.6 ± 0.1 58.3 ± 1.8 67.7 ± 4.6 47.0 ± 2.5 4.1 ± 0.6 0.0 3.4 ± 0.3 Percentage of H-chain to sercin-1 0.0 4.4 ± 0.7 2.9 ± 0.1 a Copy numbers of mRNAs of L-chain and H-chains of the recombinant IgG, and sericin-1 per 10 ng of total RNA The indicated values are 10)5 of the actual copy numbers b Data are the mean ± SEM of the results obtained from three MSGs FEBS Journal 276 (2009) 5806–5820 ª 2009 The Authors Journal compilation ª 2009 FEBS 5809 Production of mouse mAb by transgenic silkworms M Iizuka et al tional activity of the ser1 promoter in the presence of the baculovirus-derived enhancer hr3 in MSG cells [27] This mechanism was used to express recombinant proteins in transgenic silkworms [26,27] However, the simultaneous introduction of hr3 and ie1 using a single transformation vector induced the leaky expression of ie1 in tissues other than the MSG because of the selfactivation of ie1 expression through an interaction between IE1 and hr3, resulting in high silkworm mortality In the present study, ie1-bearing silkworms were generated using a transformation vector lacking hr3 but containing ser1 promoter-linked ie1 and crossed with the L ⁄ H-line to obtain silkworms carrying the genes encoding L-chain, H-chain and IE1 The resultant silkworms, which were designated the L ⁄ H ⁄ IE1line, showed no lethality or abnormalities (data not shown), and were therefore used in the subsequent experiments aiming to investigate the increases in L- and H-chain in the cocoons The proteins contained in the cocoons of the L ⁄ Hand L ⁄ H ⁄ IE1-lines were separated by SDS ⁄ PAGE under reducing conditions and stained with CBB (data not shown) L- and H-chains in the L ⁄ H ⁄ IE1-line cocoons were more highly expressed than those in the L ⁄ H-line cocoons The intensities of the H-chain bands on the gels were quantified by densitometry The mean ± SEM amount of H-chain per 0.1 mg of cocoon in the L ⁄ H- and L ⁄ H ⁄ IE1-lines was 319 ± ng (n = 3) and 754 ± 36 ng (n = 3), respectively Thus, the expression of IE1 induced an approximate 2.4-fold increase in the expression of the IgG mAb in the silkworms The mAb content in the cocoons of the L ⁄ H ⁄ IE1-line was estimated to be 1.1% Extraction and purification of recombinant mouse mAb from cocoons Recombinant mAb was extracted from L ⁄ H ⁄ IE1-line cocoons at °C with NaCl ⁄ Pi or a buffered solution containing urea at a variety of concentrations (in the range 2–8 m), and the resultant extracts were analyzed by SDS ⁄ PAGE (Fig 3A, lanes 4–10) All the proteins in the sericin layers were solubilized using m urea and 2% b-mercaptoethanol with heating and then subjected to SDS ⁄ PAGE (Fig 3A, lane 3) The ratios of the amount of mAb extracted with NaCl ⁄ Pi or the ureacontaining solutions to the total amount of mAb in the sericin layers were calculated by quantifying the band intensities of the CBB-stained H-chains When the extracted proteins with NaCl ⁄ Pi were analyzed, faint bands of the H- and L-chains were detected The ratio of the extracted H-chains to all H-chains in the sericin layers was estimated to be 8% (Fig 3A, lane 10) In 5810 the case of the extraction with a buffered solution containing urea at a concentration of m or less, the amount of the mAb increased as the urea concentration increased; the extraction efficiencies were 18%, 25% and 40% at 2, and m urea, respectively (Fig 3A, lanes 4–6) In the previous studies, recombinant enhanced green fluorescent protein and human serum albumin were efficiently extracted with NaCl ⁄ Pi or NaCl ⁄ Tris saline from cocoons of transgenic silkworms [26,27] In the present study, however, the addition of urea in the saline was required for the efficient extraction of the mAb This difference in the protein extraction may be a result of the difference in the structure of recombinant proteins or their affinities for sericin Endogenous sericin variants were hardly solubilized by urea at concentrations of less than m (Fig 3A, lanes 4, and 10) More than 80% of the mAb was recoverable using a solution containing more than m urea (Fig 3A, lanes 7–9) Under these conditions, however, a large proportion of the sericin variants were solubilized Thus, in our subsequent purification experiment, L ⁄ H ⁄ IE1-line cocoons were treated with a buffered solution containing m urea to reduce the level of contamination in the extract by sericin variants An L ⁄ H ⁄ IE1-line cocoon extract prepared with m urea and 50 mm Tris-HCl (pH 7.4) was dialyzed against 20 mm phosphate buffer (pH 7.0) and subjected to protein G affinity column chromatography As shown in Fig 3B, this process was sufficient to purify the mAb to apparent homogeneity (Fig 3B, lane 5) SDS ⁄ PAGE under nonreducing conditions revealed that the purified mAb was fully assembled H2L2 (Fig 3B, lane 6) As shown in Table 2, we were able to obtain 1.2 mg of purified mAb from 500 mg of L ⁄ H ⁄ IE1-line cocoons Antigen-binding properties of recombinant mAb The IgG L- and H-chain cDNAs used in the present study were cloned from a mouse hybridoma that produces an IgG mAb against human IgG Therefore, binding of the recombinant mAb to human IgG was analyzed by ELISA to compare the antigen-binding properties of the recombinant mAb with those of the hybridoma-derived one As depicted in Fig 4, both mAbs produced almost identical binding curves to the antigen with similar EC50 values The mouse mAb to human surfactant protein D that was used as a negative control did not bind human IgG at all We also surveyed the binding of the recombinant and hybridoma-derived mAbs to human IgM as a negative control; no binding was detected in either case (data not shown) FEBS Journal 276 (2009) 5806–5820 ª 2009 The Authors Journal compilation ª 2009 FEBS B M W Urea conc.(M) NaCl/Pi Heating L/H/IE1 A sA 120 100 80 60 50 40 30 Heating MW sM kDa 220 sP H L Purification Production of mouse mAb by transgenic silkworms L/H/IE1 Extraction M Iizuka et al kDa 220 H2L2 120 100 80 60 50 40 30 H L 20 20 10 Extraction Purification Fig Extraction and purification of recombinant mAb from cocoons (A) Extraction of recombinant mAb from L ⁄ H ⁄ IE1-line cocoons The proteins in the sericin layer of the silk fibers from wild-type (lane 2) or L ⁄ H ⁄ IE1-line (lane 3) silkworms were extracted by maintaining the cocoons at 80 °C for in M urea, 2% (v ⁄ v) b-mercaptoethanol and 50 mM Tris–HCl (pH 8.0), at 10 mg dry weightỈmL)1 The proteins from the L ⁄ H ⁄ IE1-line cocoons were also extracted with 50 mM Tris–HCl (pH 7.4) containing 2, 3, 4, 5, or M urea (lanes 4–9) or NaCl ⁄ Pi (lane 10) at °C for 24 h The extracted proteins were separated by SDS ⁄ PAGE and stained with CBB (B) Purification of the recombinant mAb from the L ⁄ H ⁄ IE1-line cocoons Recombinant mAb extracted with 50 mM Tris–HCl (pH 7.4) containing M urea was purified using a protein G column The extract (lane 4) and purified mAb (lane 5) were electrophoresed under reducing conditions The purified mAb was also subjected to SDS ⁄ PAGE under nonreducing conditions (lane 6) The electrophoresed proteins were stained with CBB The numbers to the left of the gel indicate the molecular masses (kDa) as determined by the migration of the markers (M) ‘sM’, ‘sA’, and ‘sP’ to the right of the gel represent sericin M, A and P, respectively ‘H2L2’, ‘H’ and ‘L’ represent the H2L2 tetramer, H-chain monomer and L-chain monomer of the IgG, respectively Table Purification of the recombinant mAb from 500 mg of cocoons Purification step Amount of mAb (mg) Recovery (%) Cocoons Extract Eluate from protein G column 5.5 1.4 1.2 100 25 22 Structures of N-glycans attached to recombinant mAb The N-glycan profiles of the recombinant mAb produced by the silkworms were determined The purified mAb with a protein G column was used for this determination Protein G, as well as protein A, binds the CH2 and CH3 domain interface region distal to the glycosylation site in the CH2 domain of IgG [29], and the affinity of the IgG-binding proteins for IgG is unchanged by deglycosylation of IgG [30] Thus, it is unlikely that any specific mAb glycoform is preferentially selected by purification using protein G When the pyridylaminated-N-glycans (PA-N-glycans) prepared from the mAb were separated by anion exchange chromatography, the glycans were detected only in a flow-through fraction (data not shown), suggesting the absence of negatively-charged saccharides such as sialic acids Subsequently, the PA-N-glycans in the fraction were separated by size fractionation and RP-HPLC Six major PA-N-glycan fractions were obtained, and their structures were analyzed by MALDI-TOF-MS The results obtained are summarized in Table The six PA-N-glycan fractions were identified as GlcNAcMan3GlcNAc2-PA (GNb), Man2Man3GlcNAc2-PA (M5), GlcNAc2Man3 GlcNAc2-PA (GN2), Man3Man3GlcNAc2-PA (M6), Man4Man3GlcNAc2-PA (M7) and Man5Man3GlcNAc2-PA (M8) Most of the major N-glycans were high mannose-types such as M5 (51.1%) and M6 (11.9%) On the other hand, significant amounts of hybrid-type (GNb) and complex-type N-glycans (GN2) having one and two GlcNAc residues at their nonreducing termini, respectively, were detected at ratios of 18.1% and 11.7%, respectively Pauci- FEBS Journal 276 (2009) 5806–5820 ª 2009 The Authors Journal compilation ª 2009 FEBS 5811 Production of mouse mAb by transgenic silkworms 1.25 Absorbance at 490 nm 1.00 M Iizuka et al Recombinant EC50: 73.9 ng·mL–1 Hybridoma EC50: 99.4 ng·mL–1 Negative control 0.75 0.50 0.25 0.00 10–1 100 101 102 103 Concentration (ng·mL–1) 104 Fig Antigen binding of recombinant mouse mAb The binding of recombinant mouse mAb to human IgG was analyzed by ELISA Recombinant mAb (closed triangles), hybridoma-derived natural mAb (closed squares) and negative control (anti-human surfactant protein D mouse IgG1; closed circles) at various concentrations (3333.33, 1111.11, 370.37, 123.46, 41.15, 13.72, 4.57, 1.52, 0.51 and 0.17 ngỈmL)1) were reacted against human IgG The EC50 values for the binding of the mAbs to the antigen were determined from binding curves mannose-type N-glycans such as Man3GlcNAc2-PA (M3), which are typically found in insects, were not observed [31] It is also noteworthy that fucose residues linked to the core GlcNAc residue were not detected To confirm the absence of fucose residues in the N-glycans attached to the mAb, SDS ⁄ PAGE with lectin blotting using Aleuria aurantia lectin (AAL) or concanavalin A was performed on the purified recombinant mAb and standard human IgG from human serum treated with or without peptide-N-glycosidase F (PNGaseF) The AAL lectin used in this analysis recognizes fucose residues a-1,3- and a-1,6-linked to the GlcNAc residue [32] Concanavalin A lectin recognizing mannose was also used as a control CBB staining showed that PNGaseF-treated H-chains in both the recombinant mAb and standard human IgG were slightly lower in molecular mass than the corresponding untreated chains (Fig 5, lanes 1–4) This indicates that PNGaseF actually removed N-glycans from the H-chains Concanavalin A reacted with both the recombinant and standard H-chains (Fig 5, lanes and 7); however, this reaction was not observed after PNGaseF treatment (Fig 5, lanes and 8), confirming the presence of mannose residues in the N-glycans of both H-chains On the other hand, AAL did not react Table N-glycan structures of the recombinant mouse mAb produced by silkworms The proposed structure is illustrated using symbols: closed square, open circle and closed circle indicate N-acetylglucosamine, mannose and aminopyridine, respectively ODS, octadecyl silane Abbreviation of N-glycan structure Theoretical m ⁄ z (mass + H+)a Observed m ⁄ z (mass + H+)b % Peak area obtained from HPLC (ODS)c GNb 1192.469 1192.576 18.1 M5 1313.495 1313.591 51.1 GN2 1395.548 1395.444 11.7 M6 1475.548 1475.654 11.9 M7 1637.601 1637.597 2.7 M8 1799.654 1799.650 4.5 Proposed structureb Theoretical m ⁄ z of PA-N-linked glycans was calculated as the monoisotopic mass of (mass + H+) b Observed m ⁄ z (mass + H)) were obtained from reflector mode MALDI-TOF mass spectra of the labeled N-glycans c % Peak area calculated from the result of RP-HPLC a 5812 FEBS Journal 276 (2009) 5806–5820 ª 2009 The Authors Journal compilation ª 2009 FEBS M Iizuka et al Production of mouse mAb by transgenic silkworms R PNGase KDa 50 35 – St + – R + – St + – R + – St + – + H L 25 CBB Con A 10 11 12 AAL Fig Analysis of N-glycans in the recombinant mAb by lectin blotting Recombinant mAb was subjected to lectin blotting with AAL and concanavalin A The purified recombinant mAb (R) and standard human IgG (St) treated with (+) or without ()) PNGaseF were electrophoresed on polyacrylamide gradient gels One gel was stained with CBB (lanes 1–4), whereas the others were subjected to concanavalin A (lanes 5–8) or AAL blotting (lanes 9–12) ‘H’ and ‘L’ to the right of the gel represent the H- and L-chains of IgG, respectively The numbers to the left of the gel correspond to the molecular masses (kDa) as determined by the migration of the markers with the recombinant H-chain, whereas the standard H-chain was clearly stained with this lectin (Fig 5, lanes and 11) On the basis of these results, together with our structural data, we conclude that the N-glycans attached to the recombinant mouse mAb contained no detectable a-1,3-linked or a-1,6-linked fucose residues N-Glycan structures of endogenous proteins in cocoons and larval tissues As described above, the recombinant mAb contained high mannose, hybrid and complex N-glycans On the other hand, major N-glycans synthesized in insect cell lines have paucimannose structures with a-1,3- and ⁄ or a-1,6-fucose residues and high mannose structures, although some variations in the N-glycan structure are observed depending on the synthesized glycoproteins [33] To investigate the reason for this difference in N-glycan structure, we analyzed the N-glycans contained in the cocoons and two larval tissues (MSGs and fat bodies) of wild-type pnd-w1 silkworms The results obtained are shown in Table The major N-glycans in the cocoons were M5 (48.5%) and GN2 (36.2%), with small amounts of M3 (1.2%) Fucosylated glycans were not detected in the cocoons, as in the case of the recombinant mAb Thus, the N-glycans attached to the endogenous cocoon proteins were similar to those attached to the mAb Similar N-glycan structures were noted in the MSG N-glycans, suggesting that the structural features of the N-glycans in the cellular glycoproteins of the MSG cells are comparable to those in the secreted cocoon glycoproteins The N-glycan structures from the fat bodies were different from the MSG The major fat body glycans had a fucosylated paucimannose structure (Man2[Fuc1]GlcNAc2-PA [FM2; 37.4%]) with high mannose structures having more than six mannoses Because FM2 was identified only by MS, it was not possible to determine whether the fucose residues were a-1,3- or a-1,6-linked to the GlcNAc residues The M5 observed in the cocoons and MSGs as a major high mannose-type glycan was not present in the fat bodies We also analyzed the N-glycan structures in the tissues of another silkworm strain, Kinshu, and found that they were essentially the same as those in the pnd-w1 silkworms (data not shown) From these results, we conclude that the structural features of the N-glycans in the recombinant mAb are attributed to the tissue specificity of the silk glands Discussion In the present study, we generated three transgenic lines, L-, H- and L ⁄ H-, that synthesized mouse IgG L-chain and H-chain, or both L- and H-chains, respectively The L-line silkworms secreted L-chain as a monomer into their cocoons, whereas the H-line silkworms secreted H-chain as a dimer and higher molecular aggregates In the case of the L ⁄ H-line, the co-expressed L- and H-chains formed H2L2 tetramers that were secreted as a major product into the cocoons L-chain monomers and H-chain dimers were hardly detected in the L ⁄ H-line cocoons The amount of H-chain in the L ⁄ H-line cocoons was approximately 2.3-fold higher than that in the H-cocoons Quantitative analysis of the H-chain mRNA in the MSG cells revealed that the increase in H-chain in the L ⁄ H-line cocoons was not the result of a rise in the mRNA level Thus, H2L2 tetramers were preferentially synthesized and secreted through post-transcriptional regulation In vertebrate antibody-producing cells, H-chain dimers synthesized in the absence of L-chain expression are not secreted, but are retained within the cells This inhibition of secretion is caused by the stable association of an endoplasmic reticulum-resident stress protein, BiP, with the H-chain dimer [34–36] This mechanism could be present in the MSG cells of silkworms However, the regulation of IgG secretion may be insufficient in MSG cells because the L-chain monomers or H-chain dimers were secreted from the cells in the case of the single expression of each chain Similar observations were reported in Drosophila cells transfected with the genes encoding humanized IgG [37] When the H-chain gene was expressed in these cells, H-chain was efficiently secreted as a dimer into the culture medium Furthermore, a Drosophila BiP homolog, hsc72, transiently interacts with the H-chain FEBS Journal 276 (2009) 5806–5820 ª 2009 The Authors Journal compilation ª 2009 FEBS 5813 Production of mouse mAb by transgenic silkworms M Iizuka et al Table Structures of N-glycans from cocoons, MSGs and fat bodies The proposed structure is illustrated using symbols: closed square, open circle, open diamond, closed triangle and closed circle indicate N-acetylglucosamine, mannose, galactose, fucose and aminopyridine, respectively ODS, octadecyl silane % Peak area obtained from HPLC (ODS)a Abbreviation of N-glycan structure Proposed structure Cocoons MSGs Fat bodies FM2 0.0 0.0 37.4 M3 1.2 5.7 7.9 GNb 4.5 10.9 0.0 GNa 1.7 7.2 0.0 M5 48.5 42.3 0.0 GN2 36.2 24.0 0.0 M6 3.8 4.9 7.2 M7 2.5 3.1 13.7 M8 1.6 0.0 16.5 M9 0.0 1.9 17.3 GAa ⁄ b 0.0 0.0 0.0 GA2 0.0 0.0 0.0 a % Peak area calculated from the result of RP-HPLC during its secretion [37] Unlike vertebrate BIP, Drosophila hsc72 dissociates from the H-chain independently of the L-chain association, allowing the secretion of the H-chain as the dimer The silkworm genome contains a homolog of BiP (NCBI accession number AB016836), and this gene is expressed in MSG and PSG cells [38] It is also suggested that endoplasmic reticulum-resident chaperone proteins such as BiP are involved in the synthesis and secretion of fibroin in PSG cells [16] Therefore, it is reasonable to assume that the silkworm BiP homolog and ⁄ or other chaperone proteins involved in the secretion of silk proteins might also function in that of recombinant IgG Although the function of these factors in IgG-secretion is not sufficient in MSG cells, as observed in the Drosophila cells, the factors might have selectively enhanced the secretion of the IgG H2L2 tetramers from the cells into the cocoons 5814 Accordingly, we were able to collect the mAb as fully assembled H2L2 tetramers from the cocoons Previous studies have shown that the major N-glycans in insects have paucimannose- and high mannosestructures [9,33,39] Paucimannose-type N-glycans such as M3 are characteristic of insects and are not found in mammals On the other hand, in the present study, paucimannose-type N-glycans were detected at very low levels in the cocoons and MSGs, whereas N-glycans of this type were present at high levels in the fat bodies Paucimannose-type N-glycans arise from GNb by the removal of a GlcNAc residue by the Golgi membraneassociated enzyme b-N-acetylglucosaminidase [40] In the MSG cells of silkworms, b-N-acetylglucosaminidase activity might be absent or very low, resulting in the nondetection of paucimannose structures in the N-glycans of the cocoons On the other hand, it is likely that N-acetylglucosaminyltransferase (GnT)-I and II activity FEBS Journal 276 (2009) 5806–5820 ª 2009 The Authors Journal compilation ª 2009 FEBS M Iizuka et al is present in the MSG cells of silkworms as in other insect cells or tissues [41–43] Therefore, it is reasonable to find that significant amounts of N-glycans having GlcNAc residues at their nonreducing termini were detected among the MSG-synthesized glycoproteins We also detected large amounts of M5, which is a possible substrate for GnT-I [44] The accumulation of M5 suggests relatively low GnT-I activity in the MSG cells No b-1,4-galactose-containing N-glycans were detected among the MSG-synthesized glycoproteins, implying little or no b-1,4-galactosyltransferase activity in the cells This is consistent with previous observations in other insect cells and tissues [45–47] One surprising finding obtained from our N-glycan analysis was the absence of fucose residues among the MSG-synthesized glycoproteins Previously, the N-glycans of insects such as silkworms were reported to contain considerable amounts of fucose residues a-1,3and ⁄ or a-1,6-linked to the core GlcNAc residue [33,48] For example, the ratios of N-glycans with a-1,3-fucose and a-1,6-fucose, and both a-1,3- and a-1,6-fucoses, to the total amount of N-glycans among the membrane glycoproteins in Sf-21 cells were found to be 1.8%, 15.1% and 8.8%, respectively [33] The absence of fucose residues is not attributable to the silkworm strain used in the present study, but to the tissue specificity of the silk glands The Drosophila genome contains genes for a-1,3-fucosyltransferase (FucTA) and a-1,6- fucosyltransferase (FucT6) [49,50] These Drosophila enzymes preferred N-glycans with nonreducing terminal GlcNAc residues as substrates [49,50] Gene homologs of the Drosophila fucosyltransferases were also identified from the silkworm genome (FucTA homolog, NCBI accession number CK537398; FucT6 homolog, NCBI accession number BB987128) Our preliminary analysis demonstrated that the fucosyltransferase mRNAs were expressed in the MSG cells (data not shown), suggesting that the absence of the core fucosylation is not a result of the absence of the fucosyltransferase expression The shortage of GDP-fucose in the cells might lead to the prevention of fucosylation The present study highlights several advantages of using transgenic silkworms as hosts for the production of recombinant mAbs Cocoons of transgenic silkworms contained fully formed H2L2 tetramers with appropriate antigen-binding ability The expression of the mAb was increased up to 1.1% by the introduction of ie1 into the mAb-expressing silkworms The mAb was easily extracted and purified from the cocoons Thus, the present study demonstrated the feasibility of using transgenic silkworms for the mass production of recombinant mAbs Silkworms have been used for the Production of mouse mAb by transgenic silkworms manufacture of silk in the sericultural industry Therefore, the industrial production of recombinant mAbs could be achieved by taking advantage of technologies employed in the sericultural industry, although quality control of the product must be taken into consideration The observed structures of the N-glycans further highlight the potential use of transgenic silkworms for mAb production Although the presence of oligomannose N-glycans such as M5 is not always favorable for the therapeutic use of the mAb, the absence of the core fucosylation is beneficial Fucose residues a-1,3-linked to GlcNAc show high antigenicity when administrated to humans [10] Therefore, the presence of a-1,3-fucose in the recombinant glycoproteins produced by insect cells has been recognized as an important issue for their use in therapeutic applications In our system, this issue may be solved because no a-1,3-fucose residues were detected among the N-glycans in the recombinant proteins The absence of a-1,6-fucose comprises yet another reason supporting the use of this system in the production of mAbs The absence of a-1,6-fucose enhances the activity of antibody-dependent cellular cytotoxicity of IgG [51,52] Thus, the present system might be particularly beneficial for the production of therapeutic mAbs, whose main mechanism of action is antibody-dependent cellular cytotoxicity activity Experimental procedures Experimental animals B mori strain pnd-w1 was obtained from the National Institute of Agrobiological Science (Tsukuba, Japan) The larvae were reared at 25 °C on an artificial diet (Silk Mate PM, Nosan Corp, Kanagawa, Japan) Construction of the vectors used to generate the transgenic silkworms A mouse hybridoma that produces a mouse IgG1 mAb to human IgG was kindly provided by Dr S Usuda (Institute of Immunology, Tokyo, Japan) Total RNA prepared from the hybridoma using an RNeasy kit (Qiagen, Valencia, CA, USA) was reverse-transcribed to produce cDNA fragments Two cDNA fragments encoding the IgG L- and H-chain variable regions were obtained from the hybridoma cDNAs using a SMART RACE cDNA Amplification Kit (Clontech, Palo Alto, CA, USA) in accordance with the manufacturer’s instructions, and the obtained fragments were sequenced The sequences were used to design PCR primers, and the cDNAs encoding the full-length L- and H-chain ORFs were cloned by PCR from the hybridoma cDNAs The 5¢-UTR sequence of BmNPV polyhedrin [53] FEBS Journal 276 (2009) 5806–5820 ª 2009 The Authors Journal compilation ª 2009 FEBS 5815 Production of mouse mAb by transgenic silkworms M Iizuka et al was inserted just upstream of the cDNAs for the L- and H-chain ORFs, as described previously [28] The H-chain ORF cDNA with the 5¢-UTR was then inserted downstream of the hr3-linked ser1 promoter in pMSG1.1R [28] to generate pIgGH ⁄ M1.1R (Fig 1) Similarly, the L-chain ORF cDNA with the 5¢-UTR was introduced into pMSG1.1MG, which was created from pMSG1.1R by replacing the DsRed cDNA with the cDNA for MGFP (Promega, San Luis Obispo, CA, USA) The resulting vector was dubbed pIgGL ⁄ M1.1MG (Fig 1) Quantification of mRNA in MSG cells Total RNA was extracted from the MSGs of the transgenic silkworms using Isogen (Nippon Gene, Tokyo, Japan) cDNAs were synthesized from the RNAs using PowerScript Reverse Transcriptase (BD Bioscience, Rockville, MD, USA) The mRNAs of the IgG L- and H-chains and sericin-1 were quantified using an ABI PRISM 7700 Sequence Detector (Applied Biosystems, Foster City, CA, USA), as described previously [25] Generation of transgenic silkworms Extraction and purification of recombinant mAb pIgGL ⁄ M1.1MG and pIgGH ⁄ M1.1R were each injected with the helper vector pHA3PIG [23] into eggs, as described previously [23] The hatched G0 larvae were reared at 25 °C to moths G1 embryos obtained by mating among siblings or with pnd-w1 were screened for MGFP and DsRed expression in the eyes to obtain transgenic silkworms bearing the IgG L- and H-chain genes, respectively To generate silkworms bearing both the L- and H-chain genes, an L-chain silkworm was crossed with an H-chain silkworm pBac[Ser1 IE1 ⁄ 3xP3-DsRed] [27], which was renamed pIE1 (Fig 1), was used to produce IE1 gene (ie1)-carrying transgenic silkworms pIE1 was injected into eggs, and transgenic silkworms were created as described above The resulting ie1 silkworms were crossed with silkworms carrying both the IgG L- and H-chain genes to obtain silkworms bearing ie1 and the L- and H-chain genes Pieces of cocoons were suspended in 50 mm Tris–HCl (pH 7.4) containing urea at various concentrations and stirred at °C for 24 h The extracted proteins were then analyzed by SDS ⁄ PAGE to determine the optimal conditions for extraction of the recombinant mAb For purification of the mAb, 50 mm Tris–HCl (pH 7.4) containing m urea was used for extraction of the cocoon proteins The extract was centrifuged at 20 000 g for 15 min, and the obtained supernatant was dialyzed against 20 mm phosphate buffer (pH 7.0) Next, the recombinant mAb was purified by protein G affinity column chromatography according to the manufacturer’s instructions (GE Healthcare) The purified mAb was quantified using a mouse IgG1 ELISA quantitation kit (Bethyl Laboratories, Montgomery, TX, USA) Binding affinity assay Analysis of recombinant proteins in cocoons Cocoon fragments were suspended in an extraction buffer comprised of m urea, 2% (v ⁄ v) b-mercaptoethanol and 50 mm Tris-HCl (pH 8.0) at 10 mg dry weightỈmL)1, and maintained at 80 °C for The extracted proteins were then electrophoresed under reducing conditions on 0.1% (w ⁄ v) SDS ⁄ 5–20% (w ⁄ v) polyacrylamide gradient gels (Atto, Tokyo, Japan) For the analysis of subunit assembly, cocoon proteins were extracted with b-mercaptoethanol-free extraction buffer and electrophoresed under nonreducing conditions The gels for protein staining were treated with CBB R250 In some cases, gel images were captured after CBB staining and analyzed using imagej (http://rsb.info nih.gov/ij/) For western blotting, the proteins on the gels were transferred to nitrocellulose membranes (BA85; Schleicher and Schuell, Dassell, Germany) and reacted with AffiniPure Rabbit Anti-Mouse IgG (H + L; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) and then with horseradish peroxidase (HRP)-linked anti-(rabbit IgG) sera (Cell Signaling Technology, Danvers, MA, USA) The antigen–antibody complexes were visualized using the ECL Western Blotting Detection System (GE Healthcare, Little Chalfont, UK) 5816 Commercially available purified human IgG from human serum (Cappel, Irvine, CA, USA) was diluted to a final concentration of lgỈmL)1 with 150 mm NaCl containing 0.1% (w ⁄ v) NaN3, and aliquots were dispensed into the wells of 96-well polystyrene microplates (Greiner Bio-One, Frickenhausen, Germany) The plates were then incubated for 16 h at room temperature, washed with 150 mm NaCl containing 0.05% (w ⁄ v) Tween 20, and blocked with 2% (v ⁄ v) fetal calf serum in NaCl ⁄ Tris for 30 After washing, mAb at various concentrations was added to the wells and incubated for 80 at room temperature The wells were then washed with 150 mm NaCl containing 0.05% (w ⁄ v) Tween 20 and incubated with peroxidase-labeled rabbit anti-(mouse IgG) serum (Jackson ImmunoResearch Laboratories, Inc.) O-phenylenediamine dihydrochloride was used as a substrate for peroxidase The reaction was stopped by adding m H2SO4, and A490 was measured using a microplate reader The effector concentration for half-maximum response (EC50) values for the mAb bound to human IgG was determined from binding curves using graphpad prism 4.0 (GraphPad Software, Avenida de La Jolla, CA, USA) FEBS Journal 276 (2009) 5806–5820 ª 2009 The Authors Journal compilation ª 2009 FEBS M Iizuka et al Preparation of PA-N-glycans The purified mAb and cocoons or larval tissues were hydrazinolyzed at 100 °C for 10 h The liberated N-glycans were then N-acetylated and pyridylaminated, as described previously [54] All excess reagents were removed by phenol ⁄ chloroform extraction [55] and subsequent solid-phase extraction using a Sep-PAK Plus C18 cartridge (Waters, Milford, MA, USA) [56] HPLC A Waters 2694 separation module liquid chromatograph was used to conduct HPLC analyses PA-N-glycans were separated by anion exchange chromatography using a Mono Q ⁄ HR column (0.5 · cm; GE Healthcare) Subsequently, size fractionation and RP-HPLC chromatography were performed on a Shodex Asahipak NH2P-50 column (0.2 · 15 cm; Showa Denko, Tokyo, Japan) and Cosmosil 5C18-P column (0.2 · 25 cm; Nacalai Tesque, Kyoto, Japan), respectively, as described previously [57] The fractionated PA-N-glycans were quantified from the peak areas in comparison with those of standard PA-N-glycans MS For MALDI-TOF-MS, the PA-N-glycans were co-crystallized in a matrix of 2,5-dihydroxybenzoic acid and analyzed with an Autoflex II mass spectrometer (Bruker Daltonics, Billerica, MA, USA) operated in the reflector mode, as described previously [58] Peptide standards were used to achieve a six-point external calibration for mass assignment of the ions Digestion with PNGaseF The purified recombinant mouse mAb and standard human IgG (Cappel) were digested with PNGaseF (Takara Bio Inc., Shiga, Japan) at 37 °C for 17 h under denaturing conditions The reaction was terminated by boiling in SDS ⁄ PAGE sample buffer Lectin blotting mAb treated with or without PNGaseF was electrophoresed under reducing conditions as described above and transferred to ImmobilonÒ poly(vinylidene difluoride) membranes (Millipore Corp., Billerica, MA, USA) The membranes were then treated with TBST buffer [NaCl ⁄ Tris containing 0.05% (w ⁄ v) Tween 20] and reacted with biotinylated AAL (Seikagakukogyo, Tokyo, Japan) at a concentration of lgỈmL)1 or concanavalin A (Seikagakukogyo) at a concentration of 0.3 lgỈmL)1 at room temperature for h After three washes with TBST, the membranes were Production of mouse mAb by transgenic silkworms incubated with lgỈmL)1 HRP-linked streptavidin (Jackson ImmunoResearch Laboratories, Inc.) in TBST at room temperature for h After washing with TBST, the HRP was detected with 3,3¢-diaminobenzidine (Wako Chemicals, Osaka, Japan) at a concentration of 0.2 mgỈmL)1 in NaCl ⁄ Pi References Chadd HE & Chamow SM (2001) Therapeutic 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Concanavalin A lectin recognizing mannose was also used as a control CBB staining showed that PNGaseF-treated H-chains in both the recombinant mAb and standard human IgG were slightly lower in molecular