Biotechnol Lett DOI 10.1007/s10529-014-1686-z
REVIEW
Upstream and downstream processing of recombinant IgA David Reinhart • Renate Kunert
Received: 17 July 2014 / Accepted: 12 September 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Immunoglobulin A (IgA) is the most abundant antibody class in the human body and has a unique role in mediating immunity. The everincreasing knowledge about the potential of IgAs has renewed interest in this antibody class for therapeutic use against a variety of infectious and malignant diseases, and as a preventive agent for mucosal pathogens. Despite the considerable therapeutic potential of IgA the exploration thereof has often been hampered due to difficulties in producing and purifying desired quantities. Large amounts of pure IgA will be required for in vivo studies. This work reviews current achievements and bottlenecks in upstream and downstream processing of recombinant IgA from a biotechnological point of view. We also highlight recent accomplishments with diverse expression systems and presents different affinity techniques for the capture of recombinant IgA to compare their purification potential. Keywords Antibody production Bioaffinity Immunoglobulin A (IgA) Mucosal immunity Protein purification
D. Reinhart (&) R. Kunert Department of Biotechnology, Vienna Institute of BioTechnology, University of Natural Resources and Life Sciences, Muthgasse 11, 1190 Vienna, Austria e-mail: [email protected] R. Kunert e-mail: [email protected]
Introduction To date, immunoglobulin G (IgG) predominates antibody-based therapeutics on the market and in clinical trials. However, the ever-increasing knowledge about the potential of IgA has drawn much attention to its therapeutic use against infectious and malignant diseases as well as for the prevention of mucosal infections (Lamm 1997; Corthe´sy and Spertini 1999; Dechant and Valerius 2001; Woof and Kerr 2006). Despite the fact that IgG is the classical isoform for all available cancer therapies, IgA offers several advantages as a therapeutic agent. For example, monomeric IgA recruits polymorphonuclear neutrophils, the most abundant effector cell population in humans for antibody-dependent cellular cytotoxicity against tumor cells, significantly more effective than IgG (Otten et al. 2005; Dechant et al. 2007). Furthermore, the ability of IgA to dimerize (dimeric IgA; dIgA) enhances its signalling capacity on tumor cells and allows transport into mucosal secretions with a potentially improved targeting of certain carcinomas (Breitz et al. 1992; Terskikh et al. 1994). The secretory form of IgA (secretory IgA; sIgA) represents the predominant antibody class for immune protection at mucosal tissues such as the respiratory, gastrointestinal and genitourinary tracts. With a combined surface area of roughly 400 m2 of mucosal linings, IgA acts as the first line of defence and prevents the attachment and penetration of viruses, bacteria and bacterial toxins in several in vitro and
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in vivo studies (Strugnell and Wijburg 2010). At mucosal surfaces specific sIgA serves as an immunological barrier and is correlated with resistance to in vivo infections (Corthe´sy 2002). The ability of pathogen aggregation, immobilization and neutralization at mucosal tissues is greatly enhanced due to the multivalent nature of sIgA. This multivalency could also be beneficial in an IgA drug formulation specific to the human immunodeficiency virus (HIV) (Ruffin et al. 2012). HIV-specific polyclonal IgAs may aggregate virions during sexual intercourse directly at the site of viral entry and therefore prevent it from breaching through the mucosal linings to infect human individuals. Currently, the need for effective vaccines along with the ongoing discovery of new pathogens and the increasing occurrence of antibiotic resistant microorganisms has renewed interest in sIgA as a prophylactic and therapeutic tool due to its naturally protective role in preventing pathogens from invading through mucosal surfaces into the human body (Corthe´sy 2002). Despite IgA’s sizeable therapeutic potential, the exploration thereof is frequently hampered due to difficulties in producing and purifying the desired amounts. This review summarizes current knowledge about recombinant IgA production in diverse expression systems. Furthermore, different affinity techniques for the capture of recombinant IgA and their purification potential will be discussed.
The molecular forms of IgA Similar to all other immunoglobulins, an IgA molecule comprises two identical heavy (HC) and two light chain (LC) polypeptides that are assembled to form a monomeric unit. While human serum contains mostly monomeric IgA, dimeric and higher polymeric isoforms are predominant in external secretions. Polymerization is a natural ability of IgA molecules facilitated through the presence of an 18 amino acid extension at the C-terminus, termed tail-piece. Thereby two or more IgA monomers (Brunke et al. 2013) can be linked as shown in Fig. 1. The success of this linkage to form dimers is greatly enhanced by the co-expression of joining (J) chain polypeptides (Hendrickson et al. 1995). In fact, some reports showed that recombinant CHO cells (Morton et al.
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1993), plant cells (Ma et al. 1995), and insect cells (Carayannopoulos et al. 1994), which carried the respective genes for LC and HC produced only monomeric IgA, but no covalently linked dimers. The production of the dimeric isoform was then established upon co-transfection of a J chain gene. J chain is a natural product of IgA-secreting plasma cells that helps to connect two IgA monomers via a disulphide linkage of their a chains. The two remaining a chains in the dimer are directly bound via another covalent bond (Sørensen et al. 2000). The incorporation of a J chain into polymeric IgA (pIgA) is a prerequisite for transcytosis through epithelial cells. The J chain binds to the polymeric immunoglobulin receptor (pIgR) and is thereby transported to the apical surface of external secretions (Johansen et al. 2001). Upon transcytosis, the ectoplasmic and transmembrane domain of the pIgR (secretory component, SC) is proteolytically cleaved and remains attached to pIgA that is from then on termed secretory IgA (Fig. 1). The stabilizing and protective function of the highly glycosylated SC makes sIgA ten times more resistant to protease digestion than dIgA (Berdoz et al. 1999) which is especially important in the harsh environments of external secretions. In humans, two different IgA subclasses are known, termed IgA1 and IgA2, which mainly differ in the length of their hinge region (Fig. 1). IgA1 contains a 13 amino acid hinge region that enables it to have a more extended reach (Woof and Kerr 2006). Furthermore, the extended hinge region of IgA1 contains three to six O-linked glycosylation sites (Tarelli et al. 2004). IgA2 occurs in two allotypic variants, IgA2m(1) and IgA2m(2), and a third allotype (IgA2n) probably exists (Chintalacharuvu et al. 1994). Remarkably, unlike other immunoglobulins there are no disulphide bonds linking LC and HC in IgA2m(1), but the LCs are bound to each other instead and interact non-covalently with HC (Woof and Kerr 2006). While there is a IgA1:IgA2 ratio of roughly 9:1 in human serum, the ratio may alternate in secretions but most often is 6:4 (Kerr 1990). The degree of N-glycosylation ranges between 6 and 7 % in IgA1 and between 8 and 10 % in IgA2 with respect to the molecular weight (Woof and Kerr 2006) with high variability in content, composition and number of oligosaccharides.
Biotechnol Lett Fig. 1 Human IgA structures. Molecular models of monomeric IgA1 and IgA2m(1), as well as dIgA1 and sIgA1 are illustrated (PDB accession numbers 1IGA, 1R70, 2QTJ, and 3CHN, resprectively). Note that dIgA most often contains a J chain molecule, as shown for sIgA. The different polypeptides of heavy chain (blue), light chain (red), J chain (orange) and SC (green) are depicted in color
Upstream processing of recombinant IgA IgA antibodies are complex molecules whose production requires elaborate expression systems that are able to perform extensive post-translational modifications. So far, successful IgA production has been demonstrated in mammalian (Wolbank et al. 2003; Reinhart et al. 2012), insect (Zhao et al. 2008; Carayannopoulos et al. 1994) and plant cells (Ma et al. 1995; Juarez et al. 2013). Despite several different mammalian cell lines being available for recombinant protein expression, Chinese hamster ovary (CHO) cells are the predominately applied host for IgA production (Table 1). While IgG antibody production in fed batch processes has been optimized to yield specific productivities of
50–60 pg/cell 9 day (pcd) with cell concentrations far beyond 107 cells/ml and titers up to 5 g/l (Jayapal et al. 2007), recombinant IgA production clearly lags behind. The reported specific productivities typically range between 1–5 pcd, with some reports claiming 16 pcd in serum-free culture or up to 22 pcd with the addition of serum (Table 1). Wolbank et al. (2003) class-switched the anti-HIV1 antibody 2F5 from IgG to IgA and stably expressed LC, HC and J chain genes with the adherent CHO DUKX expression system (Table 1). Upon gene amplification with methotrexate, the serum-containing cultures reached specific productivities of up to 10 pcd in 10 ml T- flasks (personal communication). Another group used the same adherent host cell to initially transfect it with only LC and HC to produce
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Biotechnol Lett Table 1 Recombinant IgA production IgA isotype
Clone designation
Transfected genes
Cell line
Medium
Specific productivity (pg/cell 9 day)
Reference
Chimeric IgA1 (k)/IgA2 (k)
n.a.
LC, HC
COS
DMEM, 10 % FCS
n.a.
Kerr (1990); Morton et al. (1993)
Chimeric IgA1 (k)/IgA2 (k) Chimeric IgA1 (j)
n.a.
LC, HC
CHO-K1
n.a.
K7; K2
LC, HC
CHO-K1
DMEM, 10 % FCS DMEM, 10 % FCS
Chimeric IgA2 (j)
K7; K2
LC, HC
CHO-K1
DMEM, 10 % FCS
5.0; 3.8
Chimeric IgA1 (j) Chimeric IgA2 (j)
K7; K2
LC, HC
CHO-K1
0.8; 1.0
K7; K2
LC, HC
CHO-K1
CD-CHO, serum-free CD-CHO, serum-free
Chimeric IgA1 (j)
K7; K2
LC, HC
CHO-K1
CD-CHO, serum-free
0.34; 0.30
Chimeric IgA2 (j) Chimeric IgA1 (j)
K7; K2
LC, HC
CHO-K1
0.13; 0.26
n.a.
LC, HC
CHO-K1
CD-CHO, serum-free CD-CHO, serum-free
Chimeric IgA2(j)
n.a.
LC, HC
CHO-K1
CD-CHO, serum-free
5
Human IgA1 (j)
2F5
CHO DUKX
Clone 22
DMEM, 10 % FCS AMEM, 10 % FCS
6–10
Chimeric IgA2(j)
LC, HC, J chain LC, HC
Chimeric IgA2(j)
Clone F
LC, HC, J chain
CHO DUKX
AMEM, 10 % FCS
20
Chimeric IgA2(j) Chimeric IgA1 (j)
Clone 6
LC, HC, J chain. SC LC, HC
CHO DUKX
AMEM, 10 % FCS CD-CHO, serum-free
19
Chimeric IgA1(j)
225-IgA1
LC, HC, J chain
CHO-K1
CD-CHO, serum-free
2.3
Human IgA1(j)
3D6//11H7
CHO DUKX
4B3//5D1
ProCHO5, serum-free ProCHO5, serum-free
16
HumanIgA1(k)
LC, HC, J chain LC, HC, J chain
Chimeric IgA1(j)
mAb 93G7
LC, HC, J chain
Insect cells Sf9
0.75
Chimeric IgA1(j) scFv IgA
mAb 93G7
LC, HC, J chain scFv
Insect cells Sf9
Inefficient
Silkworm (5th instar larvae)
n.a.
Zhao (2008)
Human IgA1(k)
HCa1kdelLCk
LC, HC, J chain
Nicotiana benthamiana
n.a.
Juarez et al. (2013)
Human sIgA1(k)
HCa1-LCkJC-Sckdel
LC, HC, J chain, SC
Nicotiana benthamiana
n.a.
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225-IgA1
BmNPV/ 45scFvLCHa
CHO DUKX
CHO-K1
CHO DUKX
1.8; 3.8
Beyer et al. (2009)
0.8; 2.2
3
22
1.2
Dechant et al. (2007)
Wolbank et al. (2003), personal communication Berdoz et al. (1999)
Lohse et al. (2011), personal communication
Reinhart et al. (2012)
0.38 Carayannopoulos et al. (1994)
Biotechnol Lett Table 1 continued IgA isotype
Clone designation
Transfected genes
Cell line
Medium
Hybrid IgAG(j)
CaroRx
LC, HC, J chain, SC
Nicotiana benthamiana
Specific productivity (pg/cell 9 day)
Reference
n.a.
Ma et al. (1995, 1998)
The following table represents an overview of recombinant IgA expression in mammalian, insect and plant cells. Information about the expressed IgA subclass, clone designation, transfected genes, applied expression cell line, medium and achieved specific productivity is shown. DMEM refers to Dulbecco’s Modified Eagle Medium, GMEM refers to Glasgow Minimum Essential Medium and AMEM refers to a-Minimal Essential Medium
monomeric IgA (Berdoz et al. 1999). The best producer was subsequently super-transfected with the J chain gene to obtain pIgA, and then an SC gene to express secretory IgA. The authors reported the highest specific IgA productivities for serum-containing CHO cell cultures with 22 pcd (monomeric IgA), 20 pcd (pIgA) and 19 pcd (secretory IgA), as shown in Table 1. Interestingly, the cultures stopped IgA expression if FCS was omitted. Similar observations were made by Beyer et al. (2009), who initially transfected a serum-dependent CHO-K1 host cell line with LC and HC genes and subsequently adapted the resulting clones to grow in the absence of serum. In this case, the specific productivities dropped by at least 40 % from 3.8 pcd to 2.2 pcd in a three-day batch culture. The ongoing move of cell cultivation towards serum-free, chemically defined media is also observed for recombinant IgA production. In our group, we cotransfected genes for LC, HC and J chain into a serum free adapted CHO DUKX to generate two cell lines, which expressed two different recombinant pIgAs (Reinhart et al. 2012). Upon gene amplification using methotrexate, the specific productivity was boosted 3.6-fold to 16.4 pcd, in one case. However, the second cell line had remarkably low specific productivities of a maximum of 0.17 pcd (Table 1). The low specific productivity was independent of gene copy numbers and mRNA levels (Reinhart et al. 2013) but was ascribed to insufficiencies in protein maturation and/or secretion. Bottlenecks in recombinant IgA expression were determined by visualizing intracellular protein deposits of misassembled and accumulated HC aggregates, which occasionally failed to associate with their free LC partners although they were abundantly present. Despite bottlenecks in protein processing, the cells’ quality checkpoints remained intact, and
predominantly correctly processed IgA was exported into the culture supernatant (Reinhart et al. 2014). Lohse et al. (2011) stably expressed LC and HC genes in the absence or presence of J chain in serumfree CHO-K1 cell lines. Upon gene amplification using the glutamine synthetase system they achieved quite impressive cell concentrations of up to 5 9 107 cells/ml in a seven-day batch process. The specific productivities obtained ranged from 1.2 pcd (monomeric IgA) to 2.3 pcd (dIgA) (see Table 1). In another publication, under similar conditions, the group reported specific productivities of 3 and 5 pcd for recombinantly expressed IgA1 and IgA2 (Dechant et al. 2007). Recombinant IgA production is clearly dominated by the use of CHO cell expression systems. There has been one report in which LC and HC genes were transiently transfected into the fibroblast-like COS cell line derived from monkey kidney tissue (Morton et al. 1993). Although the antibodies were successfully secreted by the respective cell line, data about specific IgA productivities are unfortunately not available. Certainly, a comparison of the COS cell line or other mammalian expression system to the conventional CHO cell line would be highly interesting. Insect cells are a possible alternative to mammalian cells for antibody production. However, insect cells lack some enzymes that are necessary to produce sialylated complex carbohydrates. This fact could be essential since IgA can be highly sialylated (unlike IgG) (Yoo and Morrison 2005). Carayannopoulos et al. (1994) transfected LC and HC genes into Spodoptera frugiperda (Sf9) cells using baculoviral DNA. With the reported system specific productivities of 0.75 pcd were achieved within 3 days after infection (Table 1). The additional expression of J chain has also been attempted but was stated as not being
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efficient. Another group described the feasibility of expressing single chain IgA molecules in silkworms (Zhao et al. 2008). Roughly 20 lg product/ml could be collected from the hemolymph, which showed the desired effectivity in recruiting neutrophils to kill carcinoembryonic antigen expressing tumor cells. Nevertheless, the production of IgA in silkworms will certainly run into capacity and handling limits depending on the desired scale. Plant cells are another alternative for recombinant protein production. As an expression system they offer many benefits such as low production costs, rapid and efficient scale-up, easy storage of transgenic plants as a seed as well as minimal risk of contamination with human pathogens (Fischer et al. 2003). Unlike mammalian cells, plants conduct slightly altered posttranslational modifications in which a1,3-fucose and b1,2-xylose residues are present in complex glycan structures (Cabanes-Macheteau et al. 1999). However, Chargelegue et al. (2000) have shown that despite some differences in glycosylation a plant-derived recombinant IgG antibody did not elicit any immunogenic reaction in a mouse model. Juarez et al. (2013) transiently expressed IgA antibodies in tobacco plant (Nicotiana benthamiana) leaves by agroinfiltration of the respective genes by Agrobacterium tumefaciens. In their work they tested the combinatorial expression of 16 versions of HC (a1 or a2), LC (j or k), J chain, and linking a KDEL retrieval signal to the HC and/or SC. The best performing versions (HCa1kdel-LCk and HCa1LCk-JC-SCkdel) yielded [30 lg IgA per g fresh weight. The most prominent IgA representative is most probably the Guy’s Hospital 13 SIgA/G antibody, also known as CaroRx. It is the first plant-derived antibody (Ma et al. 1995) and is currently undergoing Phase II clinical trials in humans. In a randomized, doubleblind, placebo-controlled trial at Guy’s Hospital, London, subjects treated with either secretory IgA (CaroRx) or the original IgG remained free of Streptococcus mutans, a bacterium causing dental decay, over 4 months of follow-up (Ma et al. 1995). Both the original IgG and the CaroRx IgA preparation had similar dissociation constants; however, the CaroRx had a higher functional affinity (avidity) and a higher half-life (Ma et al. 1998). Such a higher effectiveness at a lower concentration for a longer duration certainly is beneficial since such topical
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passive immunotherapies require relatively large amounts of antibody. For CaroRx, 22.5 mg antibody were administered per course of treatment of six applications, which were purified from approx. 1 kg whole mature plants (about 10–15 plants). In summary, similar to IgG antibodies, the dominating expression system for recombinant IgA production comprises mammalian cell lines and in particular CHO cells. However, upstream processing of recombinant IgA seems to be in its infancy still as the reported cultures are mostly conducted in simple batch mode and the working volumes are far from a litre scale. Although IgA antibodies are complex molecules that are difficult to express, product concentrations in the culture harvest are likely to increase through advances in cell line development and more efficient procedures for clone screening and isolation. Further optimization of the applied medium formulation (comparable to media optimization in high-yielding IgG production processes) and the use of more advanced process strategies such as fed batch or perfusion culture are likely to boost IgA titers and increase the process output to yield the desired amounts.
Secretory IgA production in vitro IgA occurs naturally as secretory molecules in external secretions of the human body. The association of secretory component (SC) with pIgA stabilizes both molecules and mutually protects them in protease-rich environments. The biotechnological expression of secretory IgA is generally considered to be challenging as the ten required polypeptides (4 9 LC, 4 9 HC, 1 9 J chain, 1 9 SC), which are naturally expressed by two different cell lines, need to be expressed and correctly assembled by a single cell. As discussed above, Berdoz et al. (1999) generated a recombinant CHO cell line that expressed secretory IgA at considerably high specific productivities (19 pcd) in serum-containing culture. Most sIgA molecules were correctly expressed, however, a considerable amount of IgA did not associate with SC. In order to test the contribution of SC to the proteolytic resistance of IgA, they exposed purified sIgA and dIgA to proteasecontaining intestinal washes at different dilutions. Despite not all molecules being associated with SC in the sIgA preparation, ten times more proteases were required to digest the sIgA product in comparison to the
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dIgA. In our group, we generated serum-free recombinant CHO cell lines using a similar approach. Cotransfection of LC, HC, J chain and SC plasmids to express sIgA yielded very low titers of sIgA, but the majority of secreted product consisted of various assemblies of the diverse polypeptides (unpublished data). In contrast, co-transfection of only LC, HC and J chain generated transfectants that secreted correctly processed monomeric and pIgA at low-to-high titers (Reinhart et al. 2012). The difficulty of producing sIgA by one cell has led to approaches in which pIgA and SC were expressed separately and subsequently associated in vitro. Crottet and Corthe´sy (1998) recombinantly expressed SC in vaccinia virus-infected HeLa S3 cells and dIgA in mouse hybridoma ZAC33 cells. After purification both molecules were incubated in PBS at equimolar amounts of pIgA and SC. After 1 h at ambient temperature the association of pIgA with SC reached its maximum level with 80 % of SC covalently bound to pIgA. While a 4 h incubation between 15–37 °C yielded similar results, incubation at 4 °C reduced the formation of covalent complexes to 35 %. The authors claimed that the addition of redox reagents such as DTT and glutathione or the addition of PDI (protein disulphide isomerase) did not visibly improve the formation of covalent bonds beyond 80 % at ambient temperature. However, closer evaluation might still be worth carrying out since another group reported that the addition of glutathione/ glutathione disulphide redox buffer to human dIgA and recombinant SC increased sIgA formation from 15–60 to 90 % (Jones et al. 1998). Recently, an approach which combines sIgA in vitro association and protein purification has been published (Moldt et al. 2014). In their report, dIgA2 was initially purified by Protein L chromatography and size exclusion chromatography and subsequently loaded once more onto a Protein L affinity matrix that is specific for kappa LCs. Then, sterile-filtered culture supernatant of recombinant human SC-expressing CHO cells was applied to the column to associate with the IgA bound fraction. Upon pH 3.0 elution, purified sIgA was recovered at an average yield of 30 %.
Downstream processing of IgA Despite having a key role in mediating mucosal immunity, IgA has lacked the enabling bioaffinity
capture method that permitted the commercial success of more than two dozens of IgGs for a long time. Protein A has been documented to bind human VH3 encoded IgAs (Grov 1975, 1976) but, in the 35 years since this discovery, there has been no report of Protein A being seriously considered as a general IgA purification tool. Furthermore, such purification would be limited to IgAs with that particular VH3 heavy chain subfamily. Jacalin, an a-D-galactose-binding lectin derived from jackfruit, is frequently employed for IgA purification. However, the absence of O-glycans containing a-D-galactose in IgA2, renders this affinity method solely useful for the isolation of IgA1. The applicability of immobilized jacalin for IgA1 purification is further limited since similarly glycosylated proteins will co-elute as impurities with the target product (Roque-Barreira and Campos-Neto 1985). In our group, we employed immobilized jacalin chromatography to isolate two recombinant IgA1(j) and IgA1(k) antibodies from concentrated cell culture supernatant (Reinhart et al. 2012). Despite high yields (97–98 %) and appreciable purities (80–90 %), host cell proteins of several different molecular weights were co-eluted during lectin chromatography (Table 2). Even more impurities were co-purified with the target IgA fraction when the antibody was present at lower concentrations in the supernatant. Furthermore, the recoveries dropped in a concentration dependent manner when the supernatant was concentrated [209 (unpublished data) probably due to higher competition with similarly glycosylated proteins for binding sites on the ligand. Another critical point when aiming to employ jacalin affinity chromatography is the requirement of galactose for IgA elution, which is expensive and impractical to remove. Protein L is an alternative for IgA purification. Protein L is a bacterial-derived protein that binds to the framework region 1 of certain kappa LCs. However, it does not recognize human kappa LCs of subtype 2 and antibodies with lambda LCs (Nilson et al. 1992) and thus is not valid as a generically applicable IgA capture step. Another drawback of Protein L is that free LC, which can leave a cell even in the absence of HC (unlike free HC), will probably coelute with the target protein fraction (Boes et al. 2011). To enable the purification of a mouse/human chimeric IgA2 antibody by Protein L, Boes et al. (2011) replaced selected framework region 1 residues in the
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Biotechnol Lett Table 2 Recombinant IgA purification IgA isotype
Original source
Step
Method
Yield (%)
Purity (%)
Reference
Human IgA1(j); IgA1(k)
CHO DUKX supernatant (serum-free; concentrate)
Lectin chromatography
Immobilized jacalin (Thermo Fisher Scientific)
97; 98
[90; [80
Reinhart et al. (2012)
Mouse/human chimeric IgA2(j)
Nicotiana tabacum leaves
Protein L
Cbind-L (SigmaAldrich)
54.7
high
Boes et al. (2011)
Human IgA2m(2)
CHO supernatant (5 % FCS) spiked with purified IgA or sIgA
Ligand peptide chromatography
Hexamer peptide ligand (HWRGWV)
96.0; 94.3
90.3; 91.7
Liu et al. (2013)
Human IgA2m(2)
1009 concentrated CHO supernatant
Ligand peptide chromatography
Hexamer peptide ligand (HWRGWV)
54.8–77.2
2.3–4.6
Liu et al. (2013)
Chimeric IgA1(j); IgA2(j)
CHO-K1 supernatant (serumfree)
VHH chromatography
Capture Select Fab Kappa (Life Technologies)
n.a.
97; 97
Beyer et al. (2009)
Human IgA1(j)/ IgA1(k)
CHO DUKX concentrated supernatant (serum-free)
VHH chromatography
Capture Select human IgA (Life Technologies)
97; 96
[95; \95
Reinhart et al. (2012)
The following table represents an overview of affinity methods used to purify recombinantly expressed IgA antibodies. Data is shown about the purified IgA antibody, source material and applied purification techniques as well as results of achieved purities and yields
variable domain of a murine LC(j) that do not interact with Protein L with corresponding residues from a LC(j) subtype that are able to bind. The best performing variant was isolated at high purity with a recovery yield of roughly 55 % IgA (Table 2). The generally low yields were suggested to be due to inefficient elution from the affinity matrix or possible denaturation of some variants during the acidic elution step. A hexameric pepide ligand, HWRGWV, has been described to have potential for IgA purification by affinity chromatography (Liu et al. 2013). The synthetic peptide ligand was tested in different scenarios. In one case, CHO cell culture supernatants were spiked with previously purified IgA and sIgA and subsequently loaded to the affinity resin. In both cases, high antibody recoveries could be achieved (96 % IgA; 94.3 % sIgA) with purities over 90 % (Table 2). The purities improved when additional washing steps were introduced, however, the yields decreased dramatically. In another experiment, low antibody-containing CHO cell culture supernatants were 1009 concentrated and then applied onto the resin. Depending on the number of washing steps the recoveries ranged between 55–77 % with purities of \5 %.
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IgA-specific camelid VHH ligands constitute a recent advancement in IgA purification. VHH ligands are 12–15 kDa single-domain ligands that lack any LC and the CH1 domain of conventional antibodies. Their unique stability and specificity has successfully been demonstrated for the purification of several different molecules (Detmers et al. 2010). Beyer et al. (2009) described the use of a human kappa light chain specific VHH ligand (Capture Select Fab Kappa; Life Technologies) as having the obvious disadvantage of not binding IgAs with lambda LCs. By using the respective VHH-ligand, recombinant IgA1(j) and IgA2(j) antibodies were isolated at high purities with recovery yields of 96.7 % (Table 2). In our group, we used a human alpha-chain specific VHH ligand (Capture Select Human IgA; Life Technologies) to purify recombinant IgA1(j) and IgA1(k) (Reinhart et al. 2012). Unlike the kappa LC-specific variant, the IgAspecific VHH ligand can theoretically be used to purify any IgA, irrespective of the HC or LC subtype. Therefore, the respective ligand truly provides the potential of a generally applicable purification tool for any IgA. In our case, both antibodies were isolated at high purity ([95 %) and recovery (96–97 %) in a single chromatographic step (Table 2).
Biotechnol Lett
Product quality
Concluding remarks
The transfection of LC and HC genes into a cellular expression system typically results in the secretion of monomeric IgA. Co-transfection of an additional J chain gene may yield recombinant CHO cell lines that secrete simultaneously monomers, dimers, trimers and even higher oligomers at different distributions (Reinhart et al. 2012). Regarding polymerization, CHO cells secrete IgA monomers similar to most IgA-secreting hybridomas, whereas 20–80 % of IgA can be expressed as dimers (Berdoz et al. 1999). Comparable to recombinant expression, IgA monomers are predominantly found in human serum, despite dIgA enhancing the signalling capacity on tumor cells and allowing their transport into mucosal secretions with a potentially improved targeting of certain carcinomas (Breitz et al. 1992; Terskikh et al. 1994). For passive immunotherapy, e.g. against HIV, virus envelope protein-specific IgA dimers block transcytosis and intracellularly neutralize primary isolates (Bomsel et al. 1998). IgA dimers are promising; however, it remains to be determined if higher IgA oligomers are similarly potent or if one isoform should be preferred for specific applications. Evaluating IgA glycosylation, considerable heterogeneity with respect to number, sites of attachment, composition and primary structure of their glycan side-chains exists among human polyclonal sIgA and monoclonal IgA1 and IgA2 (Mestecky and Russell 2009). The heterogeneous glycosylation of IgAs is considered beneficial as it allows the antibodies to interact with diverse glycan receptors on epithelial cells and thereby prevent different microorganisms from binding. The different glycosylation patterns of various expression systems may be beneficial to be used to generate a recombinant IgA with the desired glycan profile against a specific pathogen. Nevertheless, glycosylation has to be tightly monitored since reduced galactosylation of O-linked N-acetylgalactosamine residues with or without changes in the terminal sialylation of the Olinked sugars in IgA1 has been associated with IgA nephropathy (Barratt et al. 2007). Furthermore, IgA glycosylation may significantly affect the antibody’s in vivo pharmacokinetics as demonstrated by alternating interactions with the asialoglycoprotein receptor (Rifai et al. 2000).
To date, antibody-based therapies on the market and in clinical trials are still dominated by IgGs. Nevertheless, the unique role of IgA antibodies in mediating immunity will foster the treatment or prevention of a variety of diseases and IgA-based therapies will take their place along with IgGs in future. For a long time, exploration of the in vivo potential has been hampered for the IgA antibody class due to relatively low product yields and the absence of bioaffinity capture methods for downstream processing. Regarding upstream processing, 10-fold higher specific productivities are typically achieved in optimized IgG bioprocesses. Despite IgAs being very complex molecules, advances in cell line development and especially more efficient procedures for clone screening and isolation will lead to more efficient expression systems. Furthermore, moving on from simple batch processes to fed batch or perfusion cultures and applying more optimized media formulations will also boost the process output of IgA. Regarding downstream processing, several different affinity methods have been applied. Most techniques still have one or more downsides regarding purity, recovery or general applicability. The consequent development of chromatographic ligands similar to Protein A for IgGs, permits IgA isolation at high purity and yield in a single chromatographic step. Whether the VHH bioaffinity capture method is applied on an industrial scale or other methods are developed will have to be demonstrated in future. There is still space for improvement, however, with the available techniques and suggestions contained herein more efficient upstream and downstream processing is certainly feasible that will help to accumulate enough pure material to explore the clinical potential of recombinant IgA antibodies further.
References Barratt J, Smith AC, Feehally J (2007) The pathogenic role of IgA1 O-linked glycosylation in the pathogenesis of IgA nephropathy. Nephrology 12:275–284 Berdoz J, Blanc CT, Reinhardt M, Kraehenbuhl JP, Corthe´sy B (1999) In vitro comparison of the antigen-binding and stability properties of the various molecular forms of IgA
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Grov A (1975) Human IgM interacting with staphylococcal protein A. Acta Pathol Microbiol Scand Section Immunol C 83:173–176 Grov A (1976) Human colostral IgA interacting with staphylococcal protein A. Acta Pathol Microbiol Scand Section Immunol C 84:71–72 Hendrickson BA, Conner DA, Ladd DJ, Kendall D, Casanova JE, Corthesy B, Max EE, Neutra MR, Seidman CE, Seidman JG (1995) Altered hepatic transport of immunoglobulin A in mice lacking the J chain. J Exp Med 182:1905–1911 Jayapal KP, Wlaschin KF, Yap MGS, Hu WS (2007) Recombinant protein therapeutics from CHO cells—20 years and counting. Chem Eng Prog 103:40–47 Johansen FE, Braathen R, Brandtzaeg P (2001) The J chain is essential for polymeric Ig receptor-mediated epithelial transport of IgA. J Immunol 167:5185–5192 Jones RM, Schweikart F, Frutiger S, Jaton JC, Hughes GJ (1998) Thiol-disulfide redox buffers maintain a structure of immunoglobulin A that is essential for optimal in vitro binding to secretory component. Biochim Biophys Acta 1429:265–274 Juarez P, Huet-Trujillo E, Sarrion-Perdigones A, Falconi EE, Granell A, Orzaez D (2013) Combinatorial analysis of secretory immunoglobulin A (sIgA) expression in plants. Int J Mol Sci 14:6205–6222 Kerr MA (1990) The structure and function of human IgA. Biochem J 271:285–296 Lamm ME (1997) Interaction of antigens and antibodies at mucosal surfaces. Annu Rev Microbiol 51:311–340 Liu Z, Gurgel PV, Carbonell RG (2013) Affinity chromatographic purification of human immunoglobulin a from Chinese hamster ovary cell culture supernatant. Biotechnol Prog 29:91–98 Lohse S, Derer S, Beyer T, Klausz K, Peipp M, Leusen JH, van de Winkel JG, Dechant M, Valerius T (2011) Recombinant dimeric IgA antibodies against the epidermal growth factor receptor mediate effective tumor cell killing. J Immunol 186:3770–3778 Ma JK, Hiatt A, Hein M, Vine ND, Wang F, Stabila P, van Dolleweerd C, Mostov K, Lehner T (1995) Generation and assembly of secretory antibodies in plants. Science 268:716–719 Ma JK, Hikmat BY, Wycoff K, Vine ND, Chargelegue D, Yu L, Hein MB, Lehner T (1998) Characterization of a recombinant plant monoclonal secretory antibody and preventive immunotherapy in humans. Nat Med 4:601–606 Mestecky J, Russell MW (2009) Specific antibody activity, glycan heterogeneity and polyreactivity contribute to the protective activity of S-IgA at mucosal surfaces. Immunol Lett 124:57–62 Moldt B, Saye-Francisco K, Schultz N, Burton DR, Hessell AJ (2014) Simplifying the synthesis of SIgA: combination of dIgA and rhSC using affinity chromatography. Methods 65:127–132 Morton HC, Atkin JD, Owens RJ, Woof JM (1993) Purification and characterization of chimeric human IgA1 and IgA2 expressed in COS and Chinese hamster ovary cells. J Immunol 151:4743–4752 Nilson BH, Solomon A, Bjo¨rck L, Akerstro¨m B (1992) Protein L from Peptostreptococcus magnus binds to the kappa light chain variable domain. J Biol Chem 267:2234–2239
Biotechnol Lett Otten MA, Rudolph E, Dechant M, Tuk CW, Reijmers RM, Beelen RH, van de Winkel JG, van Egmond M (2005) Immature neutrophils mediate tumor cell killing via IgA but not IgG Fc receptors. J Immunol 174:5472–5480 Reinhart D, Weik R, Kunert R (2012) Recombinant IgA production: single step affinity purification using camelid ligands and product characterization. J Immunol Method 378:95–101 Reinhart D, Sommeregger W, Debreczeny M, Gludovacz E, Kunert R (2013) Characterization of recombinant IgA producing CHO cell lines by qPCR. BMC Proc 7(Suppl 6):P114 Reinhart D, Sommeregger W, Debreczeny M, Gludovacz E, Kunert R (2014) In search of expression bottlenecks in recombinant CHO cell lines-a case study. Appl Microbiol Biotechnol 98:5959–5965 Rifai A, Fadden K, Morrison SL, Chintalacharuvu KR (2000) The N-glycans determine the differential blood clearance and hepatic uptake of human immunoglobulin (Ig)A1 and IgA2 isotypes. J Exp Med 191:2171–2182 Roque-Barreira MC, Campos-Neto A (1985) Jacalin: an IgAbinding lectin. J Immunol 134:1740–1743 Ruffin N, Borggren M, Euler Z, Fiorino F, Grupping K, Hallenga¨rd D, Javed A, Mendonca K, Pollard C, Reinhart D, Saba E, Sheik-Khalil E, Sko¨ld A, Ziglio S, Scarlatti G, Gotch F, Wahren B, Shattock RJ (2012) Rational design of HIV vaccines and microbicides: report of the EUROPRISE annual conference 2011. J Transl Med 10:144
Sørensen V, Rasmussen IB, Sundvold V, Michaelsen TE, Sandlie I (2000) Structural requirements for incorporation of J chain into human IgM and IgA. Int Immunol 12:19–27 Strugnell RA, Wijburg OL (2010) The role of secretory antibodies in infection immunity. Nat Rev Microbiol 8:656–667 Tarelli E, Smith AC, Hendry BM, Challacombe SJ, Pouria S (2004) Human serum IgA1 is substituted with up to six O-glycans as shown by matrix assisted laser desorption ionisation time-of-flight mass spectrometry. Carbohydr Res 339:2329–2335 Terskikh A, Couty S, Pe`legrin A, Hardman N, Hunziker W, Mach JP (1994) Dimeric recombinant IgA directed against carcino-embryonic antigen, a novel tool for carcinoma localization. Mol Immunol 31:1313–1319 Wolbank S, Kunert R, Stiegler G, Katinger H (2003) Characterization of human class-switched polymeric (immunoglobulin M [IgM] and IgA) anti-human immunodeficiency virus type 1 antibodies 2F5 and 2G12. J Virol 77:4095–4103 Woof JM, Kerr MA (2006) The function of immunoglobulin A in immunity. J Pathol 208:270–282 Yoo EM, Morrison SL (2005) IgA: an immune glycoprotein. Clin Immunol 116:3–10 Zhao J, Kuroki M, Shibaguchi H, Wang L, Huo Q, Takami N, Tanaka T, Kinugasa T (2008) Recombinant human monoclonal igA antibody against CEA to recruit neutrophils to CEA-expressing cells. Oncol Res 17:217–222
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REVIEW
Upstream and downstream processing of recombinant IgA David Reinhart • Renate Kunert
Received: 17 July 2014 / Accepted: 12 September 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Immunoglobulin A (IgA) is the most abundant antibody class in the human body and has a unique role in mediating immunity. The everincreasing knowledge about the potential of IgAs has renewed interest in this antibody class for therapeutic use against a variety of infectious and malignant diseases, and as a preventive agent for mucosal pathogens. Despite the considerable therapeutic potential of IgA the exploration thereof has often been hampered due to difficulties in producing and purifying desired quantities. Large amounts of pure IgA will be required for in vivo studies. This work reviews current achievements and bottlenecks in upstream and downstream processing of recombinant IgA from a biotechnological point of view. We also highlight recent accomplishments with diverse expression systems and presents different affinity techniques for the capture of recombinant IgA to compare their purification potential. Keywords Antibody production Bioaffinity Immunoglobulin A (IgA) Mucosal immunity Protein purification
D. Reinhart (&) R. Kunert Department of Biotechnology, Vienna Institute of BioTechnology, University of Natural Resources and Life Sciences, Muthgasse 11, 1190 Vienna, Austria e-mail: [email protected] R. Kunert e-mail: [email protected]
Introduction To date, immunoglobulin G (IgG) predominates antibody-based therapeutics on the market and in clinical trials. However, the ever-increasing knowledge about the potential of IgA has drawn much attention to its therapeutic use against infectious and malignant diseases as well as for the prevention of mucosal infections (Lamm 1997; Corthe´sy and Spertini 1999; Dechant and Valerius 2001; Woof and Kerr 2006). Despite the fact that IgG is the classical isoform for all available cancer therapies, IgA offers several advantages as a therapeutic agent. For example, monomeric IgA recruits polymorphonuclear neutrophils, the most abundant effector cell population in humans for antibody-dependent cellular cytotoxicity against tumor cells, significantly more effective than IgG (Otten et al. 2005; Dechant et al. 2007). Furthermore, the ability of IgA to dimerize (dimeric IgA; dIgA) enhances its signalling capacity on tumor cells and allows transport into mucosal secretions with a potentially improved targeting of certain carcinomas (Breitz et al. 1992; Terskikh et al. 1994). The secretory form of IgA (secretory IgA; sIgA) represents the predominant antibody class for immune protection at mucosal tissues such as the respiratory, gastrointestinal and genitourinary tracts. With a combined surface area of roughly 400 m2 of mucosal linings, IgA acts as the first line of defence and prevents the attachment and penetration of viruses, bacteria and bacterial toxins in several in vitro and
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in vivo studies (Strugnell and Wijburg 2010). At mucosal surfaces specific sIgA serves as an immunological barrier and is correlated with resistance to in vivo infections (Corthe´sy 2002). The ability of pathogen aggregation, immobilization and neutralization at mucosal tissues is greatly enhanced due to the multivalent nature of sIgA. This multivalency could also be beneficial in an IgA drug formulation specific to the human immunodeficiency virus (HIV) (Ruffin et al. 2012). HIV-specific polyclonal IgAs may aggregate virions during sexual intercourse directly at the site of viral entry and therefore prevent it from breaching through the mucosal linings to infect human individuals. Currently, the need for effective vaccines along with the ongoing discovery of new pathogens and the increasing occurrence of antibiotic resistant microorganisms has renewed interest in sIgA as a prophylactic and therapeutic tool due to its naturally protective role in preventing pathogens from invading through mucosal surfaces into the human body (Corthe´sy 2002). Despite IgA’s sizeable therapeutic potential, the exploration thereof is frequently hampered due to difficulties in producing and purifying the desired amounts. This review summarizes current knowledge about recombinant IgA production in diverse expression systems. Furthermore, different affinity techniques for the capture of recombinant IgA and their purification potential will be discussed.
The molecular forms of IgA Similar to all other immunoglobulins, an IgA molecule comprises two identical heavy (HC) and two light chain (LC) polypeptides that are assembled to form a monomeric unit. While human serum contains mostly monomeric IgA, dimeric and higher polymeric isoforms are predominant in external secretions. Polymerization is a natural ability of IgA molecules facilitated through the presence of an 18 amino acid extension at the C-terminus, termed tail-piece. Thereby two or more IgA monomers (Brunke et al. 2013) can be linked as shown in Fig. 1. The success of this linkage to form dimers is greatly enhanced by the co-expression of joining (J) chain polypeptides (Hendrickson et al. 1995). In fact, some reports showed that recombinant CHO cells (Morton et al.
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1993), plant cells (Ma et al. 1995), and insect cells (Carayannopoulos et al. 1994), which carried the respective genes for LC and HC produced only monomeric IgA, but no covalently linked dimers. The production of the dimeric isoform was then established upon co-transfection of a J chain gene. J chain is a natural product of IgA-secreting plasma cells that helps to connect two IgA monomers via a disulphide linkage of their a chains. The two remaining a chains in the dimer are directly bound via another covalent bond (Sørensen et al. 2000). The incorporation of a J chain into polymeric IgA (pIgA) is a prerequisite for transcytosis through epithelial cells. The J chain binds to the polymeric immunoglobulin receptor (pIgR) and is thereby transported to the apical surface of external secretions (Johansen et al. 2001). Upon transcytosis, the ectoplasmic and transmembrane domain of the pIgR (secretory component, SC) is proteolytically cleaved and remains attached to pIgA that is from then on termed secretory IgA (Fig. 1). The stabilizing and protective function of the highly glycosylated SC makes sIgA ten times more resistant to protease digestion than dIgA (Berdoz et al. 1999) which is especially important in the harsh environments of external secretions. In humans, two different IgA subclasses are known, termed IgA1 and IgA2, which mainly differ in the length of their hinge region (Fig. 1). IgA1 contains a 13 amino acid hinge region that enables it to have a more extended reach (Woof and Kerr 2006). Furthermore, the extended hinge region of IgA1 contains three to six O-linked glycosylation sites (Tarelli et al. 2004). IgA2 occurs in two allotypic variants, IgA2m(1) and IgA2m(2), and a third allotype (IgA2n) probably exists (Chintalacharuvu et al. 1994). Remarkably, unlike other immunoglobulins there are no disulphide bonds linking LC and HC in IgA2m(1), but the LCs are bound to each other instead and interact non-covalently with HC (Woof and Kerr 2006). While there is a IgA1:IgA2 ratio of roughly 9:1 in human serum, the ratio may alternate in secretions but most often is 6:4 (Kerr 1990). The degree of N-glycosylation ranges between 6 and 7 % in IgA1 and between 8 and 10 % in IgA2 with respect to the molecular weight (Woof and Kerr 2006) with high variability in content, composition and number of oligosaccharides.
Biotechnol Lett Fig. 1 Human IgA structures. Molecular models of monomeric IgA1 and IgA2m(1), as well as dIgA1 and sIgA1 are illustrated (PDB accession numbers 1IGA, 1R70, 2QTJ, and 3CHN, resprectively). Note that dIgA most often contains a J chain molecule, as shown for sIgA. The different polypeptides of heavy chain (blue), light chain (red), J chain (orange) and SC (green) are depicted in color
Upstream processing of recombinant IgA IgA antibodies are complex molecules whose production requires elaborate expression systems that are able to perform extensive post-translational modifications. So far, successful IgA production has been demonstrated in mammalian (Wolbank et al. 2003; Reinhart et al. 2012), insect (Zhao et al. 2008; Carayannopoulos et al. 1994) and plant cells (Ma et al. 1995; Juarez et al. 2013). Despite several different mammalian cell lines being available for recombinant protein expression, Chinese hamster ovary (CHO) cells are the predominately applied host for IgA production (Table 1). While IgG antibody production in fed batch processes has been optimized to yield specific productivities of
50–60 pg/cell 9 day (pcd) with cell concentrations far beyond 107 cells/ml and titers up to 5 g/l (Jayapal et al. 2007), recombinant IgA production clearly lags behind. The reported specific productivities typically range between 1–5 pcd, with some reports claiming 16 pcd in serum-free culture or up to 22 pcd with the addition of serum (Table 1). Wolbank et al. (2003) class-switched the anti-HIV1 antibody 2F5 from IgG to IgA and stably expressed LC, HC and J chain genes with the adherent CHO DUKX expression system (Table 1). Upon gene amplification with methotrexate, the serum-containing cultures reached specific productivities of up to 10 pcd in 10 ml T- flasks (personal communication). Another group used the same adherent host cell to initially transfect it with only LC and HC to produce
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Biotechnol Lett Table 1 Recombinant IgA production IgA isotype
Clone designation
Transfected genes
Cell line
Medium
Specific productivity (pg/cell 9 day)
Reference
Chimeric IgA1 (k)/IgA2 (k)
n.a.
LC, HC
COS
DMEM, 10 % FCS
n.a.
Kerr (1990); Morton et al. (1993)
Chimeric IgA1 (k)/IgA2 (k) Chimeric IgA1 (j)
n.a.
LC, HC
CHO-K1
n.a.
K7; K2
LC, HC
CHO-K1
DMEM, 10 % FCS DMEM, 10 % FCS
Chimeric IgA2 (j)
K7; K2
LC, HC
CHO-K1
DMEM, 10 % FCS
5.0; 3.8
Chimeric IgA1 (j) Chimeric IgA2 (j)
K7; K2
LC, HC
CHO-K1
0.8; 1.0
K7; K2
LC, HC
CHO-K1
CD-CHO, serum-free CD-CHO, serum-free
Chimeric IgA1 (j)
K7; K2
LC, HC
CHO-K1
CD-CHO, serum-free
0.34; 0.30
Chimeric IgA2 (j) Chimeric IgA1 (j)
K7; K2
LC, HC
CHO-K1
0.13; 0.26
n.a.
LC, HC
CHO-K1
CD-CHO, serum-free CD-CHO, serum-free
Chimeric IgA2(j)
n.a.
LC, HC
CHO-K1
CD-CHO, serum-free
5
Human IgA1 (j)
2F5
CHO DUKX
Clone 22
DMEM, 10 % FCS AMEM, 10 % FCS
6–10
Chimeric IgA2(j)
LC, HC, J chain LC, HC
Chimeric IgA2(j)
Clone F
LC, HC, J chain
CHO DUKX
AMEM, 10 % FCS
20
Chimeric IgA2(j) Chimeric IgA1 (j)
Clone 6
LC, HC, J chain. SC LC, HC
CHO DUKX
AMEM, 10 % FCS CD-CHO, serum-free
19
Chimeric IgA1(j)
225-IgA1
LC, HC, J chain
CHO-K1
CD-CHO, serum-free
2.3
Human IgA1(j)
3D6//11H7
CHO DUKX
4B3//5D1
ProCHO5, serum-free ProCHO5, serum-free
16
HumanIgA1(k)
LC, HC, J chain LC, HC, J chain
Chimeric IgA1(j)
mAb 93G7
LC, HC, J chain
Insect cells Sf9
0.75
Chimeric IgA1(j) scFv IgA
mAb 93G7
LC, HC, J chain scFv
Insect cells Sf9
Inefficient
Silkworm (5th instar larvae)
n.a.
Zhao (2008)
Human IgA1(k)
HCa1kdelLCk
LC, HC, J chain
Nicotiana benthamiana
n.a.
Juarez et al. (2013)
Human sIgA1(k)
HCa1-LCkJC-Sckdel
LC, HC, J chain, SC
Nicotiana benthamiana
n.a.
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225-IgA1
BmNPV/ 45scFvLCHa
CHO DUKX
CHO-K1
CHO DUKX
1.8; 3.8
Beyer et al. (2009)
0.8; 2.2
3
22
1.2
Dechant et al. (2007)
Wolbank et al. (2003), personal communication Berdoz et al. (1999)
Lohse et al. (2011), personal communication
Reinhart et al. (2012)
0.38 Carayannopoulos et al. (1994)
Biotechnol Lett Table 1 continued IgA isotype
Clone designation
Transfected genes
Cell line
Medium
Hybrid IgAG(j)
CaroRx
LC, HC, J chain, SC
Nicotiana benthamiana
Specific productivity (pg/cell 9 day)
Reference
n.a.
Ma et al. (1995, 1998)
The following table represents an overview of recombinant IgA expression in mammalian, insect and plant cells. Information about the expressed IgA subclass, clone designation, transfected genes, applied expression cell line, medium and achieved specific productivity is shown. DMEM refers to Dulbecco’s Modified Eagle Medium, GMEM refers to Glasgow Minimum Essential Medium and AMEM refers to a-Minimal Essential Medium
monomeric IgA (Berdoz et al. 1999). The best producer was subsequently super-transfected with the J chain gene to obtain pIgA, and then an SC gene to express secretory IgA. The authors reported the highest specific IgA productivities for serum-containing CHO cell cultures with 22 pcd (monomeric IgA), 20 pcd (pIgA) and 19 pcd (secretory IgA), as shown in Table 1. Interestingly, the cultures stopped IgA expression if FCS was omitted. Similar observations were made by Beyer et al. (2009), who initially transfected a serum-dependent CHO-K1 host cell line with LC and HC genes and subsequently adapted the resulting clones to grow in the absence of serum. In this case, the specific productivities dropped by at least 40 % from 3.8 pcd to 2.2 pcd in a three-day batch culture. The ongoing move of cell cultivation towards serum-free, chemically defined media is also observed for recombinant IgA production. In our group, we cotransfected genes for LC, HC and J chain into a serum free adapted CHO DUKX to generate two cell lines, which expressed two different recombinant pIgAs (Reinhart et al. 2012). Upon gene amplification using methotrexate, the specific productivity was boosted 3.6-fold to 16.4 pcd, in one case. However, the second cell line had remarkably low specific productivities of a maximum of 0.17 pcd (Table 1). The low specific productivity was independent of gene copy numbers and mRNA levels (Reinhart et al. 2013) but was ascribed to insufficiencies in protein maturation and/or secretion. Bottlenecks in recombinant IgA expression were determined by visualizing intracellular protein deposits of misassembled and accumulated HC aggregates, which occasionally failed to associate with their free LC partners although they were abundantly present. Despite bottlenecks in protein processing, the cells’ quality checkpoints remained intact, and
predominantly correctly processed IgA was exported into the culture supernatant (Reinhart et al. 2014). Lohse et al. (2011) stably expressed LC and HC genes in the absence or presence of J chain in serumfree CHO-K1 cell lines. Upon gene amplification using the glutamine synthetase system they achieved quite impressive cell concentrations of up to 5 9 107 cells/ml in a seven-day batch process. The specific productivities obtained ranged from 1.2 pcd (monomeric IgA) to 2.3 pcd (dIgA) (see Table 1). In another publication, under similar conditions, the group reported specific productivities of 3 and 5 pcd for recombinantly expressed IgA1 and IgA2 (Dechant et al. 2007). Recombinant IgA production is clearly dominated by the use of CHO cell expression systems. There has been one report in which LC and HC genes were transiently transfected into the fibroblast-like COS cell line derived from monkey kidney tissue (Morton et al. 1993). Although the antibodies were successfully secreted by the respective cell line, data about specific IgA productivities are unfortunately not available. Certainly, a comparison of the COS cell line or other mammalian expression system to the conventional CHO cell line would be highly interesting. Insect cells are a possible alternative to mammalian cells for antibody production. However, insect cells lack some enzymes that are necessary to produce sialylated complex carbohydrates. This fact could be essential since IgA can be highly sialylated (unlike IgG) (Yoo and Morrison 2005). Carayannopoulos et al. (1994) transfected LC and HC genes into Spodoptera frugiperda (Sf9) cells using baculoviral DNA. With the reported system specific productivities of 0.75 pcd were achieved within 3 days after infection (Table 1). The additional expression of J chain has also been attempted but was stated as not being
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efficient. Another group described the feasibility of expressing single chain IgA molecules in silkworms (Zhao et al. 2008). Roughly 20 lg product/ml could be collected from the hemolymph, which showed the desired effectivity in recruiting neutrophils to kill carcinoembryonic antigen expressing tumor cells. Nevertheless, the production of IgA in silkworms will certainly run into capacity and handling limits depending on the desired scale. Plant cells are another alternative for recombinant protein production. As an expression system they offer many benefits such as low production costs, rapid and efficient scale-up, easy storage of transgenic plants as a seed as well as minimal risk of contamination with human pathogens (Fischer et al. 2003). Unlike mammalian cells, plants conduct slightly altered posttranslational modifications in which a1,3-fucose and b1,2-xylose residues are present in complex glycan structures (Cabanes-Macheteau et al. 1999). However, Chargelegue et al. (2000) have shown that despite some differences in glycosylation a plant-derived recombinant IgG antibody did not elicit any immunogenic reaction in a mouse model. Juarez et al. (2013) transiently expressed IgA antibodies in tobacco plant (Nicotiana benthamiana) leaves by agroinfiltration of the respective genes by Agrobacterium tumefaciens. In their work they tested the combinatorial expression of 16 versions of HC (a1 or a2), LC (j or k), J chain, and linking a KDEL retrieval signal to the HC and/or SC. The best performing versions (HCa1kdel-LCk and HCa1LCk-JC-SCkdel) yielded [30 lg IgA per g fresh weight. The most prominent IgA representative is most probably the Guy’s Hospital 13 SIgA/G antibody, also known as CaroRx. It is the first plant-derived antibody (Ma et al. 1995) and is currently undergoing Phase II clinical trials in humans. In a randomized, doubleblind, placebo-controlled trial at Guy’s Hospital, London, subjects treated with either secretory IgA (CaroRx) or the original IgG remained free of Streptococcus mutans, a bacterium causing dental decay, over 4 months of follow-up (Ma et al. 1995). Both the original IgG and the CaroRx IgA preparation had similar dissociation constants; however, the CaroRx had a higher functional affinity (avidity) and a higher half-life (Ma et al. 1998). Such a higher effectiveness at a lower concentration for a longer duration certainly is beneficial since such topical
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passive immunotherapies require relatively large amounts of antibody. For CaroRx, 22.5 mg antibody were administered per course of treatment of six applications, which were purified from approx. 1 kg whole mature plants (about 10–15 plants). In summary, similar to IgG antibodies, the dominating expression system for recombinant IgA production comprises mammalian cell lines and in particular CHO cells. However, upstream processing of recombinant IgA seems to be in its infancy still as the reported cultures are mostly conducted in simple batch mode and the working volumes are far from a litre scale. Although IgA antibodies are complex molecules that are difficult to express, product concentrations in the culture harvest are likely to increase through advances in cell line development and more efficient procedures for clone screening and isolation. Further optimization of the applied medium formulation (comparable to media optimization in high-yielding IgG production processes) and the use of more advanced process strategies such as fed batch or perfusion culture are likely to boost IgA titers and increase the process output to yield the desired amounts.
Secretory IgA production in vitro IgA occurs naturally as secretory molecules in external secretions of the human body. The association of secretory component (SC) with pIgA stabilizes both molecules and mutually protects them in protease-rich environments. The biotechnological expression of secretory IgA is generally considered to be challenging as the ten required polypeptides (4 9 LC, 4 9 HC, 1 9 J chain, 1 9 SC), which are naturally expressed by two different cell lines, need to be expressed and correctly assembled by a single cell. As discussed above, Berdoz et al. (1999) generated a recombinant CHO cell line that expressed secretory IgA at considerably high specific productivities (19 pcd) in serum-containing culture. Most sIgA molecules were correctly expressed, however, a considerable amount of IgA did not associate with SC. In order to test the contribution of SC to the proteolytic resistance of IgA, they exposed purified sIgA and dIgA to proteasecontaining intestinal washes at different dilutions. Despite not all molecules being associated with SC in the sIgA preparation, ten times more proteases were required to digest the sIgA product in comparison to the
Biotechnol Lett
dIgA. In our group, we generated serum-free recombinant CHO cell lines using a similar approach. Cotransfection of LC, HC, J chain and SC plasmids to express sIgA yielded very low titers of sIgA, but the majority of secreted product consisted of various assemblies of the diverse polypeptides (unpublished data). In contrast, co-transfection of only LC, HC and J chain generated transfectants that secreted correctly processed monomeric and pIgA at low-to-high titers (Reinhart et al. 2012). The difficulty of producing sIgA by one cell has led to approaches in which pIgA and SC were expressed separately and subsequently associated in vitro. Crottet and Corthe´sy (1998) recombinantly expressed SC in vaccinia virus-infected HeLa S3 cells and dIgA in mouse hybridoma ZAC33 cells. After purification both molecules were incubated in PBS at equimolar amounts of pIgA and SC. After 1 h at ambient temperature the association of pIgA with SC reached its maximum level with 80 % of SC covalently bound to pIgA. While a 4 h incubation between 15–37 °C yielded similar results, incubation at 4 °C reduced the formation of covalent complexes to 35 %. The authors claimed that the addition of redox reagents such as DTT and glutathione or the addition of PDI (protein disulphide isomerase) did not visibly improve the formation of covalent bonds beyond 80 % at ambient temperature. However, closer evaluation might still be worth carrying out since another group reported that the addition of glutathione/ glutathione disulphide redox buffer to human dIgA and recombinant SC increased sIgA formation from 15–60 to 90 % (Jones et al. 1998). Recently, an approach which combines sIgA in vitro association and protein purification has been published (Moldt et al. 2014). In their report, dIgA2 was initially purified by Protein L chromatography and size exclusion chromatography and subsequently loaded once more onto a Protein L affinity matrix that is specific for kappa LCs. Then, sterile-filtered culture supernatant of recombinant human SC-expressing CHO cells was applied to the column to associate with the IgA bound fraction. Upon pH 3.0 elution, purified sIgA was recovered at an average yield of 30 %.
Downstream processing of IgA Despite having a key role in mediating mucosal immunity, IgA has lacked the enabling bioaffinity
capture method that permitted the commercial success of more than two dozens of IgGs for a long time. Protein A has been documented to bind human VH3 encoded IgAs (Grov 1975, 1976) but, in the 35 years since this discovery, there has been no report of Protein A being seriously considered as a general IgA purification tool. Furthermore, such purification would be limited to IgAs with that particular VH3 heavy chain subfamily. Jacalin, an a-D-galactose-binding lectin derived from jackfruit, is frequently employed for IgA purification. However, the absence of O-glycans containing a-D-galactose in IgA2, renders this affinity method solely useful for the isolation of IgA1. The applicability of immobilized jacalin for IgA1 purification is further limited since similarly glycosylated proteins will co-elute as impurities with the target product (Roque-Barreira and Campos-Neto 1985). In our group, we employed immobilized jacalin chromatography to isolate two recombinant IgA1(j) and IgA1(k) antibodies from concentrated cell culture supernatant (Reinhart et al. 2012). Despite high yields (97–98 %) and appreciable purities (80–90 %), host cell proteins of several different molecular weights were co-eluted during lectin chromatography (Table 2). Even more impurities were co-purified with the target IgA fraction when the antibody was present at lower concentrations in the supernatant. Furthermore, the recoveries dropped in a concentration dependent manner when the supernatant was concentrated [209 (unpublished data) probably due to higher competition with similarly glycosylated proteins for binding sites on the ligand. Another critical point when aiming to employ jacalin affinity chromatography is the requirement of galactose for IgA elution, which is expensive and impractical to remove. Protein L is an alternative for IgA purification. Protein L is a bacterial-derived protein that binds to the framework region 1 of certain kappa LCs. However, it does not recognize human kappa LCs of subtype 2 and antibodies with lambda LCs (Nilson et al. 1992) and thus is not valid as a generically applicable IgA capture step. Another drawback of Protein L is that free LC, which can leave a cell even in the absence of HC (unlike free HC), will probably coelute with the target protein fraction (Boes et al. 2011). To enable the purification of a mouse/human chimeric IgA2 antibody by Protein L, Boes et al. (2011) replaced selected framework region 1 residues in the
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Biotechnol Lett Table 2 Recombinant IgA purification IgA isotype
Original source
Step
Method
Yield (%)
Purity (%)
Reference
Human IgA1(j); IgA1(k)
CHO DUKX supernatant (serum-free; concentrate)
Lectin chromatography
Immobilized jacalin (Thermo Fisher Scientific)
97; 98
[90; [80
Reinhart et al. (2012)
Mouse/human chimeric IgA2(j)
Nicotiana tabacum leaves
Protein L
Cbind-L (SigmaAldrich)
54.7
high
Boes et al. (2011)
Human IgA2m(2)
CHO supernatant (5 % FCS) spiked with purified IgA or sIgA
Ligand peptide chromatography
Hexamer peptide ligand (HWRGWV)
96.0; 94.3
90.3; 91.7
Liu et al. (2013)
Human IgA2m(2)
1009 concentrated CHO supernatant
Ligand peptide chromatography
Hexamer peptide ligand (HWRGWV)
54.8–77.2
2.3–4.6
Liu et al. (2013)
Chimeric IgA1(j); IgA2(j)
CHO-K1 supernatant (serumfree)
VHH chromatography
Capture Select Fab Kappa (Life Technologies)
n.a.
97; 97
Beyer et al. (2009)
Human IgA1(j)/ IgA1(k)
CHO DUKX concentrated supernatant (serum-free)
VHH chromatography
Capture Select human IgA (Life Technologies)
97; 96
[95; \95
Reinhart et al. (2012)
The following table represents an overview of affinity methods used to purify recombinantly expressed IgA antibodies. Data is shown about the purified IgA antibody, source material and applied purification techniques as well as results of achieved purities and yields
variable domain of a murine LC(j) that do not interact with Protein L with corresponding residues from a LC(j) subtype that are able to bind. The best performing variant was isolated at high purity with a recovery yield of roughly 55 % IgA (Table 2). The generally low yields were suggested to be due to inefficient elution from the affinity matrix or possible denaturation of some variants during the acidic elution step. A hexameric pepide ligand, HWRGWV, has been described to have potential for IgA purification by affinity chromatography (Liu et al. 2013). The synthetic peptide ligand was tested in different scenarios. In one case, CHO cell culture supernatants were spiked with previously purified IgA and sIgA and subsequently loaded to the affinity resin. In both cases, high antibody recoveries could be achieved (96 % IgA; 94.3 % sIgA) with purities over 90 % (Table 2). The purities improved when additional washing steps were introduced, however, the yields decreased dramatically. In another experiment, low antibody-containing CHO cell culture supernatants were 1009 concentrated and then applied onto the resin. Depending on the number of washing steps the recoveries ranged between 55–77 % with purities of \5 %.
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IgA-specific camelid VHH ligands constitute a recent advancement in IgA purification. VHH ligands are 12–15 kDa single-domain ligands that lack any LC and the CH1 domain of conventional antibodies. Their unique stability and specificity has successfully been demonstrated for the purification of several different molecules (Detmers et al. 2010). Beyer et al. (2009) described the use of a human kappa light chain specific VHH ligand (Capture Select Fab Kappa; Life Technologies) as having the obvious disadvantage of not binding IgAs with lambda LCs. By using the respective VHH-ligand, recombinant IgA1(j) and IgA2(j) antibodies were isolated at high purities with recovery yields of 96.7 % (Table 2). In our group, we used a human alpha-chain specific VHH ligand (Capture Select Human IgA; Life Technologies) to purify recombinant IgA1(j) and IgA1(k) (Reinhart et al. 2012). Unlike the kappa LC-specific variant, the IgAspecific VHH ligand can theoretically be used to purify any IgA, irrespective of the HC or LC subtype. Therefore, the respective ligand truly provides the potential of a generally applicable purification tool for any IgA. In our case, both antibodies were isolated at high purity ([95 %) and recovery (96–97 %) in a single chromatographic step (Table 2).
Biotechnol Lett
Product quality
Concluding remarks
The transfection of LC and HC genes into a cellular expression system typically results in the secretion of monomeric IgA. Co-transfection of an additional J chain gene may yield recombinant CHO cell lines that secrete simultaneously monomers, dimers, trimers and even higher oligomers at different distributions (Reinhart et al. 2012). Regarding polymerization, CHO cells secrete IgA monomers similar to most IgA-secreting hybridomas, whereas 20–80 % of IgA can be expressed as dimers (Berdoz et al. 1999). Comparable to recombinant expression, IgA monomers are predominantly found in human serum, despite dIgA enhancing the signalling capacity on tumor cells and allowing their transport into mucosal secretions with a potentially improved targeting of certain carcinomas (Breitz et al. 1992; Terskikh et al. 1994). For passive immunotherapy, e.g. against HIV, virus envelope protein-specific IgA dimers block transcytosis and intracellularly neutralize primary isolates (Bomsel et al. 1998). IgA dimers are promising; however, it remains to be determined if higher IgA oligomers are similarly potent or if one isoform should be preferred for specific applications. Evaluating IgA glycosylation, considerable heterogeneity with respect to number, sites of attachment, composition and primary structure of their glycan side-chains exists among human polyclonal sIgA and monoclonal IgA1 and IgA2 (Mestecky and Russell 2009). The heterogeneous glycosylation of IgAs is considered beneficial as it allows the antibodies to interact with diverse glycan receptors on epithelial cells and thereby prevent different microorganisms from binding. The different glycosylation patterns of various expression systems may be beneficial to be used to generate a recombinant IgA with the desired glycan profile against a specific pathogen. Nevertheless, glycosylation has to be tightly monitored since reduced galactosylation of O-linked N-acetylgalactosamine residues with or without changes in the terminal sialylation of the Olinked sugars in IgA1 has been associated with IgA nephropathy (Barratt et al. 2007). Furthermore, IgA glycosylation may significantly affect the antibody’s in vivo pharmacokinetics as demonstrated by alternating interactions with the asialoglycoprotein receptor (Rifai et al. 2000).
To date, antibody-based therapies on the market and in clinical trials are still dominated by IgGs. Nevertheless, the unique role of IgA antibodies in mediating immunity will foster the treatment or prevention of a variety of diseases and IgA-based therapies will take their place along with IgGs in future. For a long time, exploration of the in vivo potential has been hampered for the IgA antibody class due to relatively low product yields and the absence of bioaffinity capture methods for downstream processing. Regarding upstream processing, 10-fold higher specific productivities are typically achieved in optimized IgG bioprocesses. Despite IgAs being very complex molecules, advances in cell line development and especially more efficient procedures for clone screening and isolation will lead to more efficient expression systems. Furthermore, moving on from simple batch processes to fed batch or perfusion cultures and applying more optimized media formulations will also boost the process output of IgA. Regarding downstream processing, several different affinity methods have been applied. Most techniques still have one or more downsides regarding purity, recovery or general applicability. The consequent development of chromatographic ligands similar to Protein A for IgGs, permits IgA isolation at high purity and yield in a single chromatographic step. Whether the VHH bioaffinity capture method is applied on an industrial scale or other methods are developed will have to be demonstrated in future. There is still space for improvement, however, with the available techniques and suggestions contained herein more efficient upstream and downstream processing is certainly feasible that will help to accumulate enough pure material to explore the clinical potential of recombinant IgA antibodies further.
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