Plant-based recombinant butyrylcholinesterase production methods
09688970 ยท 2017-06-27
Assignee
Inventors
- Greg Pogue (Austin, TX, US)
- Ernie Hiatt (Maceo, KY, US)
- Romy Kandzia (Halle, DE)
- Stefan Werner (Halle, DE)
- Frank THIEME (Halle, DE)
- Tsafrir Mor (Tempe, AZ, US)
- Steven Hume (Owensboro, KY, US)
Cpc classification
C12Y301/01008
CHEMISTRY; METALLURGY
A61K38/465
HUMAN NECESSITIES
International classification
C12N15/82
CHEMISTRY; METALLURGY
Abstract
A new, reliable, easily scalable and reproducible method for the production of recombinant butyrylcholinesterase (rBuChE) is provided. Through the utilization of a plant transfection procedure, various plant strains have been shown to generate effective and scalable amounts of rBuChE under acceptable manufacturing processes to permit reliable levels of such enzymes for desired nerve agent protection requirements (including tetrameric products). As well, such methods in engineered plant lines have shown suitable production of these enzymes in tetramer form with glycan formation and sialyalation (for terminal groups) to allow for optimal potency against organophosphorus agent exposure as well as proper immunogenic response within the plant sources. The overall production method, including the transfection and production within mammalian cells, as well as the process steps involved for such a reliable sourcing platform from plants is thus encompassed within the invention.
Claims
1. A nucleotide construct providing tetramer formation in a recombinant butyrylcholinesterase product, the construct comprising first and second nucleotide sequences, wherein the first nucleotide sequence encodes a butyrylcholinesterase molecule covalently linked to a signal peptide and the second nucleotide sequence encodes a signal peptide covalently linked to a polyproline adhesion domain permitting butyrylcholinesterase tetramer formation.
2. The construct of claim 1 wherein said construct is suitable for expression in plants or plant cells.
3. The construct of claim 2 which is co-expressed with at least one sialylation vector comprising genes encoding enzymes for sialic acid synthesis and for sialic acid transfer to terminal N-linked glycan structures permitting production of a recombinant butyrylcholinesterase product exhibiting about 50%-73% sialylation.
4. The construct of claim 1 which permits expression of a recombinant butyrylcholinesterase product having between 50%-75% tetramer formation.
5. The construct of claim 3 wherein said recombinant butyrylcholinesterase product exhibits between 60%-70% sialylation.
6. The construct of claim 4 wherein said recombinant butyrylcholinesterase product exhibits between 60%-70% tetramer formation and at least 70% sialylation.
7. The construct of claim 1 wherein said second nucleotide sequence is chosen from SEQ ID NO: 1 and SEQ ID NO: 4.
8. The construct of claim 1 wherein said second nucleotide sequence is chosen from SEQ ID NO: 2 and SEQ ID NO: 3.
9. The construct of claim 4 wherein said second nucleotide sequence is chosen SEQ ID NO: 1 and SEQ ID NO: 4.
10. A method for production of recombinant butyrylcholinesterase from a living cell, said method comprising the following steps: a) providing said living cell with at least one vector capable of expressing said butyrylcholinesterase; b) incubating said living cell in conditions that cause the synthesis of said butyrylcholinesterase and including the generation of sialylated glycans and tetramer formation of said butyrylcholinesterase permitting formation of a butyrylcholinesterase product exhibiting sialylation and tetramer formation; wherein the at least one vector capable of expressing said butyrylcholinesterase comprises first and second nucleotide sequences, the first nucleotide sequence encodes a butyrylcholinesterase molecule covalently linked to a signal peptide and the second nucleotide sequence encodes a signal peptide covalently linked a polyproline adhesion domain permitting butyrylcholinesterase tetramer formation, and c) isolating said butyrylcholinesterase product of step b from said living cell.
11. The method of claim 10 wherein said conditions permitting formation of sialylated glycans on said butyrylcholinesterase include the introduction within said living cell of genes expressing sialic acid synthesis, galactose transfer, and sialic acid transfer, wherein said gene expressions generate butyrylcholinesterase sialylation in vivo.
12. The method of claim 10 wherein said living cell is a plant cell.
13. The method of claim 11 wherein said living cell is a plant cell.
14. The method of claim 10 wherein said second nucleotide sequence is chosen from SEQ ID NO: 1 and SEQ ID NO: 4.
15. The method of claim 10 wherein the second nucleotide sequence is chosen from SEQ ID NO: 2 and SEQ ID NO: 3.
16. The method of claim 15 wherein said living cell is a plant cell.
17. The method of claim 10 wherein said butyrylcholinesterase product exhibits between 50%-73% sialylation and between 50%-75% tetramer formation.
18. The method of claim 17 wherein said butyrylcholinesterase product exhibits between 60%-70% sialylation and between 60%-70% tetramer formation.
19. The method of claim 10 wherein said at least one vector of step a comprises two vectors, one of which expresses peptide tetramerization and one of which also expresses glycoprotein sialylation.
20. The method of claim 16 wherein said butyrylcholinesterase product exhibits between 50%-73% sialylation and between at least 50%-75% tetramer formation.
21. The method of claim 20 wherein said butyrylcholinesterase product exhibits between 60%-70% sialylation and between 60%-70% tetramer formation.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
(20)
(21)
(22)
(23)
(24)
(25)
(26)
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS AND FIGURES
(27) The following descriptions and explanations of the accompanying figures are intended specifically to provide information pertaining to possible embodiments of the present invention. No limitation of the breadth and scope of the overall invention is to be construed by the disclosures provided herein.
(28) As used herein, the following terms are intended to be interpreted as follows:
(29) transfect or transfection or like word is intended to mean the deliberate introduction of nucleic acids within cells (whether native or non-native) in order to allow for expression of genetic material within such cells;
(30) vector or vectors or like word is intended to mean a DNA molecule (such as a plasmid, for example) that serves as a vehicle to transfer foreign genetic material into a cell (whether native or non-native), thus allow for gene expression therein;
(31) expression or gene expression or like word or words is intended to mean the process of transferring information from a gene in order to synthesize a subsequent functional gene product:
(32) endogenous is intended to mean originating from within a cell, tissue, or Organism; and
(33) transgenic or transgenesis or like word is intended to mean a process of introducing a gene into a living organism for transfer of a new property that is then passed to the organism's offspring. All transgenic strategies described herein involved the utilization of a vector to allow for gene expression, as well.
Example 1: Production of Monomeric/Dimeric rBuChE in NbRNAiDXF Plants
(34) The inventive production system employs a transient minimal virus-based system launched by infiltration of plants with Agrobacterium strains containing a transient plant-virus based production system. The technology and its applications have been described in numerous publications. This transient system (
(35) As shown in
(36) Furthermore, the utilized vectors are built from two different plant virus genomes: TMV-related virus turnip vein clearing tobamovirus (TVCV;
(37) For transient expression of the monomeric rBuChE in plants, Nicotiana benthamiana (Nb) plants are infiltrated with Agrobacterium strains containing the virus expression vector-encoded plasmids. Nb plants were grown for 24-26 days in an enclosed growth room at 22-24 C. were used for vacuum infiltration. Overnight-grown Agrobacterium cultures for rBuChE (Vector ID: pBCHEKBP007) were mixed in the infiltration buffer (10 mM MES, 10 mM MgSO.sub.4, pH 5.5). The vector pPBCHKBP007 was diluted 1:1000 (Agrobacterium cells:buffer). Production is conducted in wild-type and a transgenic Nb plant line in which RNAi technology was used to knock down both xylose and fucose transferase activities (NbRNAiXF). Proteins derived from NbRNAiXF plants show highly homogenous mammalian glycoforms such that endogenous and recombinant proteins almost void of any plant-specific fucose or xylose residues. The infiltration solution was transferred into vacuum (infiltration) chambers custom built by Kentucky Bioprocessing (as shown in
(38) As further shown in
(39) Furthermore, a scalable extraction, clarification, and non-affinity purification methodology was developed to purify resultant monomeric/dimeric rBuChE. Enzyme extraction was accomplished using mechanical disintegration of infected biomass in the presence of a phosphate buffer. The initial extract was clarified using pH shifting followed by depth filtration employing a plate/frame filter press and diatomaceous earth filter aid. The rBuChE was captured from the clarified extract using Capto Adhere multimodal resin (GE Healthcare), with elution accomplished using decreasing pH. The eluent from the capture step was then diluted to low conductivity and applied to Ceramic Hydroxyapatite (CHT) Type I multimodal resin (Bio-Rad Laboratories). The rBuChE was eluted from CHT using an increasing sodium chloride gradient, with host proteins stripped from the column using a high concentration of sodium phosphate. The CHT eluent was incubated with 1% v/v Triton X-114, followed by heating to produce a precipitated detergent phase that contains contained the majority of endotoxin. The aqueous phase (supernatant) was then removed from the detergent phase and diluted to low conductivity in preparation for final polishing. Residual detergent was removed by binding the rBuChE onto Capto Q strong anion exchange resin (GE Healthcare), followed by extensive washing with buffer to fully flush the detergent from the column. Elution of rBuChE from Capto Q was accomplished using an increasing sodium chloride gradient. The Capto Q eluent was then difiltered into phosphate-buffered saline containing arginine, followed by concentration to at least 25 mg/mL using tangential flow ultrafiltration. The bulk drug substance was sterilized using 0.2 m filtration and stored at 2-8 C.
(40) The inventive procedure thus involves the utilization of this plant transient expression technology along with Nb host plants to overexpress rBuChE at levels many-fold higher than published transgenic plant approaches while integrating multigene expression strategies to achieve tetramer formation in rBuChE and host modifications to provide for sialylation of the product in vivo as shown in other examples. This technology system relies on scalable infiltration of Nb plants with Agrobacterium strains containing the DNA expression vectors to launch gene expression. This system has been used to produce more than 1 gram quantity lots of monomeric/dimeric rBuChE at >95% purity. This enzyme (produced at 1.5 gram/lot) shows high enzymatic activity, similar to positive control material purified from transgenic goat sources provided by U.S. Army Medical Research Institute of Chemical Defense, as shown in
Example 2: Generation of Transient Sialylation Vectors
(41) Multigenic constructs were designed and built to contain genes encoding the proteins to synthesize sialic acid and transfer galactose and sialic acid to terminal N-linked glycan structures in the plant Golgi.
(42) Assembly of the construct was done using the Golden Gate cloning technology (as shown in Engler et al., 2008.sup.7) in conjunction with the modular cloning system for multi-gene constructs (as shown in Weber et al., 2011.sup.15, and within Werner et 42011 and 2012.sup.16). First, all basic elements were cloned in level 0 vectors containing BsaI recognition sites generating 4 bp overlaps specific for each type of module (e.g. AATG and GCTT for CDS modules). The level 0 modules were then assembled into level 1 expression cassettes using the Golden Gate restriction/ligation procedure. Level 1 vectors are framed by BpiI recognition sites specific for one of seven positions defining the order of expression cassettes in the final construct. Two additional positions were covered by two ca. 400 bp random sequences at the beginning and the end which will facilitate the analysis of transgene integration sites. The modular cloning system is designed in such a way that one can assemble six level 1 constructs in one reaction. Since seven genes and two random sequences (i.e. nine level 1 vectors) were required for the final construct, the assembly was done in two steps. The BAR gene expression cassette and the random sequences at both ends were introduced into the destination vector prior to assembly of the other genes.
(43) In the first step, level 1 constructs were assembled via BpiI Golden Gate reaction in two level M vectors (
(44) The following basic elements used for pICH88266 are presented in Table 3.
(45) TABLE-US-00003 TABLE 3 Expression elements and genes (CDS - coding regions) for genes comprised in pICH88266 Posi- tion Promoter 5UTR CDS terminator 1 Maize Spm TMV- BAR (ppt acetyl Arabidopsis transferase) GCRP 2 Nos TMV- GNE (GlcNAc Nos epimerase) 3 34S TMV- SAS (sialic acid 35S phosphate synthase) 4 Actin2 TMV- CMAS (CMP sialic Actin2 acid synthase) 5 Arabidopsis TMV- CST (CMP sialic Rbcs1 Rbcs1 acid transporter) 6 Arabidopsis TMV- Gal (Galactosyl Agrocinopine LHB1B1 transferase) synthase (Ags) 7 Potato STLS TMV- ST (Sialyl g7 transferase)
Example 3: Testing of Transient Sialylation Process for Recombinantly Plant Produced Proteins
(46) For transient co-expression of rBuChE (viral expression vector) with sialylation pathway vector pICH88266 normally a 1:10 dilution of the agrobacterium overnight culture that harbors the latter vector is used. Viral expression vectors are based on the above-described technology (Gleba, et al., 2005.sup.9; Gleba et al., 2007.sup.10); binary vectors developed by Icon Genetics using elements from Tobacco Mosaic Virus (TMV) or Potato Virus X (PVX) (Giritch et al., 2006.sup.8). Taking into account that pICH88266 is not a viral vector and unable to spread (short distance movement) from infected cells, for large scale infiltration of plants a large volume of Agrobacterium culture containing pICH88266 would be required. To investigate the possibility to use smaller amounts of Agrobacteria, an experiment was conducted with higher dilutions of Agrobacterium cultures harboring vector pICH88266 which were used for co-expression with the BuChE viral expression vector. Dilutions of 1:10, 1:25, 1:50, 1:100, 1:500 and 1:1000 of overnight cultures (ca. 2 OD.sub.600) were compared in respect to provide for sialylation of rBuChE.
(47) Nicotiana benthamiana wild-type plants were infiltrated with agrobacteria harboring the BuChE viral expression construct (pICH92631) at a 1:1000 dilution and different dilutions of the sialylation pathway construct pICH88266. Prior to purification, crude plant extracts (100 mg plant tissue extracted in 0.3 ml 0.2 M citrate buffer pH6 supplemented with 1 mM EDTA) harvested from 7-days post-infiltration plant tissue were pretested for BuChE activity using an Ellman assay (Ellman, 1961.sup.6). The results of the pretest are shown in
(48) After this pretest, the His-tagged BCHE was purified from the different samples using Ni-NTA chromatography. Comparable amounts of purified BCHE (2 g each) were separated on polyacrylamide gels supplemented with SDS, blotted on PVDF membranes and probed with biotinylated SNA lectin (Sambucus nigra lectin that binds preferentially to sialic acid attached to terminal galactose in -2,6 and to a lesser degree, -2,3 linkage of sialic acid to glycans; Vector laboratories, Peterborough, UK) and streptavidin-HRP conjugate (Life Technologies, Darmstadt, Germany) for detection of sialic acid. To confirm the detection of sialylated rBuChE, the membranes were stripped and reprobed with a goat anti-BCHE polyclonal antibody (Santa Cruz Biotechnology, Heidelberg, Germany) and anti-goat IgG-peroxidase conjugate (Sigma-Aldrich, St. Louis, USA). Sialylation of rBuChE was detected for all tested dilutions, but stronger for dilutions 1:10 up to 1:100. Additionally, a shift in protein size corresponding to rBuChE sialylation was visible for dilutions 1:10 up to 1:100. The results of Western blot analyses are shown in
(49) These results show that higher dilutions up to 1:100 of the agrobacteria containing plCH88266 could be used in principle; however, results were not quantitative. Therefore, MALDI analysis of samples with plCH88266 dilutions of 1:10, 1:25, 1:50 and 1:100 was conducted. Analysis of the free glycans showed for all analyzed dilutions a similar content of sialylation (1:10 dilution: 46%; 1:25 dilution: 49%; 1:50 dilution: 57%; 1:100 dilution: 42%, details are provided more effectively in
(50) Sialylation of rBuChE can be achieved using either (a) transient or (b) transgenic strategies. Transient strategies (a) involve the co-transfection of pBCHEKBP007 alone, or with polyproline adhesion domain (PRAD) peptide expressing vectors (as described above for monomeric or tetrameric product, respectively), along with the pICH88266 plant expression vector in appropriate Agrobacterium stains. PRAD peptides were identified in association with human and equine BuChE (Duysen et al., 2002.sup.5; Li et al., 2008.sup.12; Ilyushina et al., 2013.sup.11). Experiments in mammalian cells suggest that co-expression of PRAD peptides can produce increased levels of tetramerized rBuChE. However, to date, no one has produced an efficiently tetramerized, sialylated rBuChE in mammalian cells (see references above). The method described below demonstrate that PRAD co-expression with rBuChE results in high proportion in excess of 60% rBuChE tetramer formation that can be sialylated by transient and transgenic methodologies efficiency (with about 70% of linkages sialylated).
(51) The pICH88266 plasmid contains expression constructs for the seven genes to allow sialic acid to be synthesized, functionalized and transferred to nascent glycan strains in the plant Golgi apparatus along with the BAR selectable marker gene.
(52) For producing tetramer rBuChE, co-transfection with a transient vector expressing a PRAD or tetramerizing peptide were tested. The following PRAD Sequences (amino acid) were used to produce genetic constructs for expression in planta:
(53) TABLE-US-00004 1. mankhlslslflvllglsaslasgAPSPPLPPPPPPPPPPPPPPP PPPPPLP(SEQIDNO:1) 2. mankhlslslflvllglsaslasgACCLLMPPPPPLFPPPFF (SEQIDNO:2) 3. mankhlslslflvllglsaslasgACCLLMPPPPPLFPPPFFDYK DDDDK(SEQIDNO:3) 4. mankhlslslflvllglsaslasgAQPTFINSVLPISAALPGLDQ KKRGNHKACCLLMPPPPPLFPPPFF(SEQIDNO:4)
(54) All four genes were optimized for Nb codon bias and synthesized. Of these, PRAD peptide 1 (derived from Lamellipodin) and PRAD peptide 4 (derived from collagen-like ColQ) were successfully cloned into PVX transient vectors. Unfortunately, PRAD peptide constructs encoding proteins 2 and 3 were not successfully cloned. Both constructs produced tetramer-like protein, and PRAD peptide 4 was chosen for continued testing due to time constraints.
(55) For transient expression of the Tetramer-sialylated rBuChE in plants, the transfection procedure described above was used with minor modifications. Plants grown for 24-26 days in an enclosed growth room at 22-24 C. were used for vacuum infiltration. Overnight-grown Agrobacterium cultures for rBuChE (Vector ID: pBCHEKBP007), the tetramerizing peptide from ColQ (Vector ID: Tetra 4; [both ColQ and Lamellipodin vectors where shown to work similarly in other studies]) and the sialylation pathway (Vector ID: pICH88266) were mixed in the infiltration buffer (10 mM MES, 10 mM MgSO4, pH 5.5). The vector pPBCHKBP007 was diluted 1:1000 (Agrobacterium cells:buffer), Tetra 4 was diluted 1:200 (Agrobacterium cells:buffer) and pICH88266 was diluted 1:10 (Agrobacterium cells:buffer). The infiltration solution was transferred into custom built (Kentucky Bioprocessing, Owensboro, Ky.) vacuum chambers. The aerial parts of entire plants were submerged upside down into the bacterial/buffer solution and a vacuum of 24 of mercury was applied for 2 min and released. For 80 kg of harvested plant biomass 280 L of infiltration solution was made requiring 280 mL of vector pBCHEKBP007, 1.4 L of vector Tetra 4 and 28 L of vector pICH88266. Post infiltration, plants were returned to the growth room under standard growing conditions. Harvest of the aerial parts of the entire plants occurred at 7 dpi (days post infiltration).
(56) A scalable extraction, clarification, and non-affinity purification methodology was developed to purify Tetramer rBuChE. Enzyme extraction was accomplished using mechanical disintegration of infected biomass in the presence of a phosphate buffer. The initial extract was clarified using pH shifting followed by depth filtration employing a plate/frame filter press and diatomaceous earth filter aid. The rBuChE was captured from the clarified extract using Capto Adhere multimodal resin (GE Healthcare), with elution accomplished using decreasing pH. The eluent from the capture step was then diluted to low conductivity and applied to Ceramic Hydroxyapaptite (CHT) Type I multimodal resin (Bio-Rad Laboratories). The rBuChE was eluted from CHT using an increasing sodium chloride gradient, with host proteins stripped from the column using a high concentration of sodium phosphate. The CHT eluent was incubated with 1% v/v Triton X-114, followed by heating to produce a precipitated detergent phase that contained the majority of endotoxin. The aqueous phase (supernatant) was then removed from the detergent phase and diluted to low conductivity in preparation for final polishing. Residual detergent was removed by binding the rBuChE onto Capto Q strong anion exchange resin (GE Healthcare), followed by extensive washing with buffer to fully flush the detergent from the column. Elution of rBuChE from Capto Q was accomplished using an increasing sodium chloride gradient. The Capto Q eluent was then diafiltered into phosphate-buffered saline containing arginine, followed by concentration to at least 25 mg/mL using tangential flow ultrafiltration. The bulk drug substance was sterilized using 0.2 m filtration and stored at 2-8 C. Final product properties are shown in
(57) In this situation, however, it was determined that the plant-based methodologies described herein involving transient rBuChE expression with PRAD peptides with the transient sialylation system or in transgenic plants (such as within Example 5 below, as one non-limiting example) produce highly sialylated products that accumulate throughout transfected plant cells and can be readily, not just selectively, isolated. This inventive technological breakthrough is thus not only highly unexpected in terms of viability, scalability, and reliability, but is also more efficient for producing material sufficient for detailed animal studies for OPs treatment analyses, and further is also highly cost effective (reducing overall production costs by potentially 10-100 fold compared with serum-derived enzyme BuChE products).
Example 5: Production of Transiently Sialylated Monomer Form of rBuChE in XTFT N. benthamiana (80 kg Plant Biomass) Using Transient Sialyalation Methods
(58) For transient expression of the monomer-sialylated rBuChE in plants, the transfection procedure as described above was used with minor modifications. Plants grown for 24-26 days in an enclosed growth room at 22-24 C. were used for vacuum infiltration. Overnight-grown Agrobacterium cultures for rBuChE (Vector ID: pBCHEKBP007) and the sialylation pathway (Vector ID: pICH88266) were mixed in the infiltration buffer (10 mM MES, 10 mM MgSO.sub.4, pH 5.5). The vector pPBCHKBP007 was diluted 1:1000 (Agrobacterium cells:buffer and pICH88266 was diluted 1:10 (Agrobacterium cells:buffer). The infiltration solution was transferred into custom built (Kentucky Bioprocessing, Owensboro, Ky.) vacuum chambers. The aerial parts of entire plants were submerged upside down into the bacterial/buffer solution and a vacuum of 24 of mercury was applied for 2 min and released. For 80 kg of harvested plant biomass 280 L of infiltration solution was made requiring 280 mL of vector pBCHEKBP007 and 28 L of vector pICH88266. Post infiltration, plants were returned to the growth room under standard growing conditions. Harvest of the aerial parts of the entire plants occurred at 7 dpi (days post infiltration).
(59) A scalable extraction, clarification, and non-affinity purification methodology was developed to purify Monomer-sialylated rBuChE. Enzyme extraction was accomplished using mechanical disintegration of infected biomass in the presence of a phosphate buffer. The initial extract was clarified using pH shifting followed by depth filtration employing a plate/frame filter press and diatomaceous earth filter aid. The rBuChE was captured from the clarified extract using Capto Adhere multimodal resin (GE Healthcare), with elution accomplished using decreasing pH. The eluent from the capture step was then diluted to low conductivity and applied to Ceramic Hydroxyapaptite (CHT) Type I multimodal resin (Bio-Rad Laboratories). The rBuChE was eluted from CHT using an increasing sodium chloride gradient, with host proteins stripped from the column using a high concentration of sodium phosphate. The CHT eluent was incubated with 1% v/v Triton X-114, followed by heating to produce a precipitated detergent phase that contained the majority of endotoxin. The aqueous phase (supernatant) was then removed from the detergent phase and diluted to low conductivity in preparation for final polishing. Residual detergent was removed by binding the rBuChE onto Capto Q strong anion exchange resin (GE Healthcare), followed by extensive washing with buffer to fully flush the detergent from the column. Elution of rBuChE from Capto Q was accomplished using an increasing sodium chloride gradient. The Capto Q eluent was then diafiltered into phosphate-buffered saline containing arginine, followed by concentration to at least 25 mg/mL using tangential flow ultrafiltration. The bulk drug substance was sterilized using 0.2 m filtration and stored at 2-8 C.
(60) The SNA reactivity to final purified product is similar for both monomeric and tetrameric protein products. These data are encouraging for the potential efficacy of the transiently sialylated product as well as the potential for increased sialylation from the new transgenic plant strains. These data suggest that significant in vivo sialylation is possible using the transgenic plant production strategy (such as in
(61) The native serum-derived BuChE is shown in
Example 6: Glycan Analysis of Transiently Sialylated Monomer/Dimer rBuChE
(62) Glycan analysis of dialylated monomer protein was carried out as described: the LC/MS setup for separation of the enzyme digest consisted of a capillary HPLC using a 1 mm150 cm C18 reverse phase column with formic acid (FA) mobile phase and acetonitrile gradient elution. The detection of peptide and glycopeptide ions is by mass detection in a quadrupole time of flight (QTOF) mass spectrometer. The tryptic maps of the resulting glycopeptide ion spectra are used to identify specific glycan structures by comparison of the observed ion mass to the predicted mass. The expected peptides resulting from trypsin digest were analyzed for N-link glycosylation. The Base Peak tryptic map of Lot 13B003, Lot 13B005, monomeric sialylated rBuChE, and CHT Elution were determined. The tryptic maps have notable differences. Monomeric, sialylated rBuChE, and CHT Elution were analyzed separately from Lot 13B003 and 13B005. For the 13B003 and 13B005 lots were analyzed using an optimized gradient to capture the early eluting glycopeptides. The samples were comparable when they were analyzed using the same gradient.
(63) BuChE has nine potential N-Link sites, the present study focused on the identity and quantitation of glycan species on five of the N-Link sites presented in
(64) The native serum-derived BuChE is shown in
(65) Glycan analysis of dialyated monomer protein was carried out as described: the LC/MS setup for separation of the enzyme digest consisted of a capillary HPLC using a 1 mm150 cm C18 reverse phase column with formic acid (FA) mobile phase and acetonitrile gradient elution. Detection of peptide and glycopeptide ions is by mass detection in a quadrupole time of flight (QTOF) mass spectrometer. The tryptic maps of the resulting glycopeptide ion spectra are used to identify specific glycan structures by comparison of the observed ion mass to the predicted mass. The expected peptides resulting from trypsin digest were analyzed for N-link glycosylation. The Base Peak tryptic map of Lot 13B003, Lot 13B005, monomeric sialylated rBuChE, and CHT Elution were determined. The tryptic maps have notable differences. Monomeric, sialylated rBuChE and CHT Elution were analyzed separately from Lot 13B003 and 13B005. For the 13B003 and 13B005 lots were analyzed using an optimized gradient to capture the early eluting glycopeptides. The samples were comparable when they were analyzed using the same gradient.
(66) BuChE has nine potential N-Link sites, the present study focused on the identity and quantitation of glycan species on five of the N-Link sites presented in
(67) TABLE-US-00005 TABLE 6 Monomeric rBuChE Transiently Expressed Glycan Forms Tryptic Glycopeptides Msial Formulated Sum of all N-Link Sites Glycan Total Vol Area % 0000 9,890,025 8.3 1000 9,436,467 7.9 1100 552,364 0.5 2002 2,483,922 2.1 2010 484,716 0.4 2011 1,659,161 1.4 2020 112,697 0.1 2021 2,645,798 2.2 2022 77,606,591 64.9 2110 191,087 0.2 2120 179,291 0.1 2121 1,198,000 1.0 2122 1,050,067 0.9 Agly 12,110,846 10.1 119,601,032 100.0 Sum of all N-Link Sites Sialylated 70.4% Non-Sialylated 19.5% Aglycosylated 10.1%
(68) The native serum-derived BuChE is shown in
(69) Further analysis was undertaken of both mono- and oligo-saccharide constituents on transiently sialylated tetramer products generated through this general method. The glycan structures were basically analyzed for content and whether the transient production scheme accorded effective and high occupancy measurements for optimal OPs effectiveness.
(70) The collected samples were dialyzed against running deionized water for about 24 hours through a 4-kDa membrane. After dialysis, the samples were then lyophilized in preparation for monosaccharide composition analysis. Subsequently, an aliquot of each sample was allocated for neutral and amino sugars analysis (200 g), and for sialic acids analysis (200 g). The aliquots for neutral and amino sugars were hydrolyzed with 2.0 N trifluoroacetic acid (TFA), whereas the aliquots for sialic acids were hydrolyzed with 2 M acetic acid. After hydrolysis, the digests were dried under a stream of nitrogen gas, dissolved with H.sub.2O, sonicated for 5 min on ice and transferred to injection vials.
(71) A mix of neutral and amino sugar standards, and sialic acid standards with known number of moles were hydrolyzed in the same manner and at the same time as the samples. Four concentrations of standard mixtures (neutral and amino sugars, and sialic acids) were prepared to establish a calibration equation. The number of moles of each monosaccharide in the sample was quantified by linear interpolation of residue area units into the calibration equation.
(72) The monosaccharides were analyzed by HPAEC using a Dionex ICS3000 system equipped with a gradient pump, an electrochemical detector, and an autosampler. The individual neutral and amino sugars, and sialic acids were separated by a Dionex CarboPac PA20 (3150 mm) analytical column with an amino trap. The gradient program used the following mobile phase eluents: for neutral and amino sugars, degassed nanopure water and 200 mM NaOH; For sialic acids, 100 mM NaOH and 1 M sodium acetate in 100 mM NaOH. Injection was made every 40 min for neutral and amino sugars and every 35 min for sialic acids.
(73) Two samples were then analyzed for certain glycan residues with results presented in Table 7.
(74) TABLE-US-00006 TABLE 7 Glycosyl Residue Identifications and Measurements on Transient Sialylated Products Total Residues in Sam- Sample ple Hydrolyzed %, by Mole ID Glycosyl Residue nanomoles g 1 Fucose (Fuc) 2.98 18.14 3.44 N-acetyl galactosamine (GalNAc) nd N-acetyl glucosamine (GlcNAc) 36.61 165.52 42.26 Galactose (Gal) 12.39 68.77 14.30 Glucose (Glc) nd Mannose (Man) 18.46 102.46 21.31 N-acetyl neuraminic acid (NANA) 16.19 52.35 18.69 N-glycolyl neuraminic nd acid(NGNA) 2 Fucose (Fuc) 0.40 2.45 3.66 N-acetyl galactosamine (GalNAc) nd N-acetyl glucosamine (GlcNAc) 4.25 19.21 38.80 Galactose (Gal) 1.36 7.55 12.42 Glucose (Glc) nd Mannose (Man) 2.89 16.03 26.36 N-acetyl neuraminic acid (NANA) 2.05 6.64 18.76 N-glycolyl neuraminic nd acid(NGNA) .sup.1Dialyzed aliquots hydrolyzed from each sample was ~200 g for neutral&amino sugars and ~200 g for sialic acids.
(75) Fucose, N-acetylglucosamine, galactose and mannose were detected in all four samples. Among the sialic acids, NANA was detected in all four glycoproteins.
Example 7: Production of Sialylated Tetramer Form of rBuChE
(76) Sialylation of rBuChE can be achieved using either (a) transient or (b) transgenic strategies. Transient strategies (a) involve the co-transfection of pBCHEKBP007 alone, or with proline rich adhesion domain (PRAD) peptide expressing vectors (as described above for monomeric or tetrameric product, respectively), along with the pICH88266 plant expression vector in appropriate Agrobacterium stains. The pICH88266 plasmid contains expression constructs for the seven genes to allow sialic acid to be synthesized, functionalized and transferred to nascent glycan strains in the plant Golgi apparatus along with the BAR selectable marker gene.
(77) For transient expression of the Tetramer-sialylated rBuChE in plants, the magnifection (Icon Genetics GmbH, Halle/Saale, DE) procedure was used with minor modifications. Plants grown for 24-26 days in an enclosed growth room at 22-24 C. were used for vacuum infiltration. Overnight-grown Agrobacterium cultures for rBuChE (Vector ID: pBCHEKBP007), the tetramerizing peptide from ColQ (Vector ID: Tetra 4; [both ColQ and Lamellipodin vectors where shown to work similarly in other studies]) and the sialylation pathway (Vector ID: pICH88266) were mixed in the infiltration buffer (10 mM MES, 10 mM MgSO4, pH 5.5). The vector pPBCHKBP007 was diluted 1:1000 (Agrobacterium cells:buffer), Tetra 4 was diluted 1:200 (Agrobacterium cells:buffer) and pICH88266 was diluted 1:10 (Agrobacterium cells: buffer). The infiltration solution was transferred into custom built (Kentucky Bioprocessing, Owensboro, Ky.) vacuum chambers. The aerial parts of entire plants were submerged upside down into the bacterial/buffer solution and a vacuum of 24 of mercury was applied for 2 min and released. For 80 kg of harvested plant biomass 280 L of infiltration solution was made requiring 280 mL of vector pBCHEKBP007, 1.4 L of vector Tetra 4 and 28 L of vector pICH88266. Post infiltration, plants were returned to the growth room under standard growing conditions. Harvest of the aerial parts of the entire plants occurred at 7 dpi (days post infiltration).
(78) A scalable extraction, clarification, and non-affinity purification methodology was developed to purify the inventive tetramer rBuChE. Enzyme extraction was accomplished using mechanical disintegration of infected biomass in the presence of a phosphate buffer. The initial extract was clarified using pH shifting followed by depth filtration employing a plate/frame filter press and diatomaceous earth filter aid. The rBuChE was captured from the clarified extract using Capto Adhere multimodal resin (GE Healthcare), with elution accomplished using decreasing pH. The eluent from the capture step was then diluted to low conductivity and applied to Ceramic Hydroxyapatite (CHT) Type I multimodal resin (Bio-Rad Laboratories). The rBuChE was eluted from CHT using an increasing sodium chloride gradient, with host proteins stripped from the column using a high concentration of sodium phosphate. The CHT eluent was incubated with 1% v/v Triton X-114, followed by heating to produce a precipitated detergent phase that contained the majority of endotoxin. The aqueous phase (supernatant) was then removed from the detergent phase and diluted to low conductivity in preparation for final polishing. Residual detergent was removed by binding the rBuChE onto Capto Q strong anion exchange resin (GE Healthcare), followed by extensive washing with buffer to fully flush the detergent from the column. Elution of rBuChE from Capto Q was accomplished using an increasing sodium chloride gradient. The Capto Q eluent was then diafiltered into phosphate-buffered saline containing arginine, followed by concentration to at least 25 mg/mL using tangential flow ultrafiltration. The bulk drug substance was sterilized using 0.2 m filtration and stored at 2-8 C. Final product properties are shown in
Example 8: Production of Transgenic Sialylating SIAL-NbRNAiXF Plant Line from XTFT N. benthamiana Plants
(79) Transgenic strategies have been developed for exogenous gene sialylation by transforming the genes from the pICH88266 vector into the NbRNAiXF plant strain to produce SIAL-NbRNAiXF plant lines. The strategy used is detailed in
(80) Nb SIAL-NbRNAiXF seed was mixed for production due to small amount of seed from each lines and the extensive production required. The amounts were equal upon weight, but each line could have different efficiencieshigher or lower of sialylation activity. The pooled plant from seed and Nb SIAL-NbRNAiXF-88266#11 in particular were infiltrated with Agrobacteria harboring the BuChE viral expression construct (pICH92631) at a 1:1000 dilution. Prior purification, crude plant extracts (100 mg plant tissue extracted in 0.3 ml 0.2 M citrate buffer pH6 supplemented with 1 mM EDTA) from harvested 7 dpi plant tissue were pretested for BuChE activity using an Ellman assay. After this pretest, the His-tagged BCHE was purified from the different samples using Ni-NTA chromatography. Comparable amounts of purified BCHE (2 g each) were separated on polyacrylamide gels supplemented with SDS, blotted on PVDF membranes and probed with biotinylated SNA lectin (Sambucus nigra lectin; Vector laboratories, Peterborough, UK) and streptavidin-HRP conjugate (Life Technologies, Darmstadt, Germany) for detection of sialic acid. To confirm the detection of sialylated rBuChE, the membranes were stripped and re-probed with a goat anti-BCHE polyclonal antibody (Santa Cruz Biotechnology, Heidelberg, Germany) and anti-goat IgG-peroxidase conjugate (Sigma-Aldrich, St. Louis, USA).
(81) Sialylation of rBuChE was detected within many plants from the NbRNAiXF-88266#11 line (
(82) Nb SIAL-NbRNAiXF seed was planted and tested for sialylation capability using the SNA Western blot methodology.
(83) 1. Negative Control Benz
(84) 2. Negative Control Transgenic
(85) 3. NBG 41 with pBCHEKBP007 and Tetra Peptide 4
(86) 4. NBG 42 with pBCHEKBP007 and Tetra Peptide 4
(87) 5. NBG 43 with pBCHEKBP007 and Tetra Peptide 4
(88) 6. NBG 45 with pBCHEKBP007 and Tetra Peptide 4
(89) 7. NBG 46 with pBCHEKBP007 and Tetra Peptide 4
(90) 8. NBG 47 with pBCHEKBP007 and Tetra Peptide 4
(91) Extractions were performed using a 2:1 buffer to biomass ratio. 45 mL of green juice was immediately centrifuged to produce a S1 sample. S1 pellets were produced at 10000g for 10 at 4 C. 1000 L of S1 was centrifuged at 16000g for 2 to produce an S2. Reduced NuPAGE samples were prepared and gels ran at 200V, 50. The results and loading order are provided in
(92) These data demonstrate that six sialylation transgenic seed lines grown in 104 cell trays show strong expression of rBuChE and the rBuChE and other protein bands show strong reactivity with the SNA lectin indicating that the sialylation system is intact and function in all T2 lines (
(93) Further breeding and selection continues for T3 lines expected to show more stability with regards to sialylation pathway. Seed stock designation Nbg-45 and Nbg-43 (both from initial sialic acid transgenic plant Nb88266-11) were surface sterilized using ethanol and sodium hypochlorite then plated onto Murashige and Skoog media plus Gamborg's vitamins plates supplemented with the herbicide Phosphinothricin. Phosphinothricin resistance serves as a marker gene in the transgenic system. Plants that germinate and remain green contain the marker gene and suggest the sialylation gene cassette is intact. Germination and Phosphinothricin results were recorded ten days post sowing (dps) with all plants thriving and green. Plants were transplanted from the media plates to soilless plant media in five gallon pots for seed production. Thirteen pots were established for plants from seed stock Nbg-45 and 12 pots for Nbg-43.
(94) Seed pod collection on plants commenced 61 days post-transplant. Per the standard seed production protocol, seed pods from individual plants are collected over a period of one to two weeks then moved to light carts to complete the pod drying process. Seeds were then cleaned and sized through a sieving process. Seed too small or too big was understood to be possibly retained for research purposes, but the seed retained by the proper sized sieve was that which was qualified. This harvest, drying and cleaning step was repeated over the entire harvesting time period. The cleaned seed was bulked together by individual plants and is kept separate until sufficient testing concludes that seed from individual plants can be bulked together. Typical N. benthamiana XF seed lot testing includes germination, vigor, morphology, and nptII tests (indicative of kanamycin resistance marker gene). Specific testing for sialic acid transgenic seed lines was undertaken in relation to the following protocol: 1. Germination on Phosphinothricin media plates. If segregation for Phosphinothricin was recorded the seed from segregating plants was not bulked with seed from non-segregating plants. 2. If Phosphinothricin media results are 100% positive plantlets were transferred to pots containing soilless media and grown until large enough to syringe IF with a his-tagged BuChE vector. Extracts of IF spots were performed, followed by BuChE enrichment using magnetic beads (Dynabeads His-Tag Isolation and Pulldown). Anti-BuChE Westerns and SNA blots were performed for determinations of the presence of BuChE and sialylation. If segregation for sialylation was recorded, seed from a segregating plant was not bulked with seed from non-segregating plants.
(95) Seed lots that tested 100% positive for the Phosphinothricin herbicide selection and the BuChE/SNA Western (along with germination, vigor, morphology and nptII) tests were bulked together and approved for use in future BCHE production runs. Prior to large scale use seed (all or a portion) was pelleted to facilitate ease of use with an automated seeding system.
(96) Approximately 200 g of seed was collected with approximately 75 g cleaned and sieved (3 g per plant25 plants=75 g). Seed collection continued for another six to eight weeks until it was shown that the predicted estimate of approximately 600-800 grams of seed was produced within a final bulked seed lot (with all 25 plants combined). Such a seed quantity can potentially produce 7.2-9.6 million plants demonstrating the scalability and robustness of the transgenic strategy for production.
Example 9: Production of Tetrameric rBuChE in Transgenic Sialylating SIAL-NbRNAiXF Plant Lines
(97) Tetramer formation for the rBuChE plant-based products was undertaken through the utilization of the following Vectors: rBuChE: pBCHEKBP007 (1:1000 dilution), PRAD peptide 4: Tetra 4 (1:200 dilution)
(98) Nb SIAL-NbRNAiXF seed was mixed for production due to small amount of seed from each lines and the extensive production required. The amounts were equal in terms of weight, but each line could have different efficiencies in terms of higher or lower sialylation activity. For transient expression of the tetramer-sialylated rBuChE in plants, the magnifection procedure was again used with minor modifications. Plants grown for 24-26 days in an enclosed growth room at 22-24 C. were used for vacuum infiltration. Overnight-grown Agrobacterium cultures for rBuChE (Vector ID: pBCHEKBP007), the tetramerizing peptide from ColQ (Vector ID: Tetra 4) and the sialylation pathway (Vector ID: pICH88266) were mixed in the infiltration buffer (10 mM MES, 10 mM MgSO4, pH 5.5). The vector pPBCHKBP007 was diluted 1:1000 (Agrobacterium cells:buffer), Tetra 4 was diluted 1:200 (Agrobacterium cells:buffer) and pICH88266 was diluted 1:10 (Agrobacterium cells:buffer). The infiltration solution was transferred into vacuum chambers. The aerial parts of entire plants from Nicotiana benthamiana SIAL-NbRNAiXF pooled seed were submerged upside down into the bacterial/buffer solution and a vacuum of 24 of mercury was applied for 2 minutes and released. For 80 kg of harvested plant biomass, 280 L of infiltration solution was made requiring 280 mL of vector pBCHEKBP007, 1.4 L of vector Tetra 4, and 28 L of vector pICH88266. Post infiltration, plants were returned to the growth room under standard growing conditions. Harvest of the aerial parts of the entire plants occurred at 7 dpi.
(99) A scalable extraction, clarification, and non-affinity purification methodology was developed to purify the tetramer-sialylated rBuChE products. Enzyme extraction was accomplished using mechanical disintegration of infected biomass in the presence of a phosphate buffer. The initial extract was clarified using acidic pH shifting followed by depth filtration employing a plate/frame filter press and diatomaceous earth filter aid. The rBuChE was captured from the clarified extract using Capto Adhere multimodal resin (GE Healthcare), with elution accomplished using decreasing pH. The eluent from the capture step was then diluted to lower conductivity and applied to Ceramic Hydroxyapaptite (CHT) Type I multimodal resin (Bio-Rad Laboratories). The rBuChE was eluted from CHT using an increasing sodium chloride gradient, with host proteins stripped from the column using a high concentration of sodium phosphate. The CHT eluent was incubated with 1% v/v Triton X-114, followed by heating to produce a precipitated detergent phase that contained the majority of endotoxin. The aqueous phase (supernatant) was then removed from the detergent phase and diluted to lower conductivity in preparation for final polishing. Residual detergent was removed by binding the rBuChE onto Capto Q strong anion exchange resin (GE Healthcare), followed by extensive washing with buffer to fully flush the detergent from the column. Elution of rBuChE from Capto Q was accomplished using an increasing sodium chloride gradient. The Capto Q eluent was then diafiltered into phosphate-buffered saline containing arginine, followed by concentration to at least 25 mg/mL using tangential flow ultrafiltration. The bulk drug substance was sterilized using 0.2 m filtration and stored at 2-8 C.
(100) The binding of OPs agents was compared between the transgenically sialylated rBuChE and plasma-derived BuChE. A continuous method of assessing inhibition constants (with butyrylthiocholine in a modified Ellman assay) was used to determine k.sub.i values of OPs nerve agents with KBP BuChE, as compared to human plasma-derived BuChE. As shown in
(101) Also shown in
Example 10: Glycan Analysis of Transgenically Sialylated rBuChE
(102) Further analysis was undertaken of the glycan constituents on the tetramerized sialylated rBuChE plant-derived product. The glycan structures were basically analyzed for content and whether the transient production scheme accorded effective and high occupancy measurements for optimal OPs effectiveness.
(103) An intact aliquot of each sample was allocated for neutral and amino sugars analysis (200 g), and for sialic acids analysis (200 g). The aliquots for neutral and amino sugars were hydrolyzed with 2.0 N trifluoroacetic acid (TFA), whereas the aliquots for sialic acids were hydrolyzed with 2 M acetic acid. After hydrolysis, the digests were dried under a stream of nitrogen gas, dissolved with H.sub.2O, sonicated for 5 min on ice and transferred to injection vials.
(104) A mix of neutral and amino sugar standards, and sialic acid standards with known number of moles were hydrolyzed in the same manner and at the same time as the samples. Four concentrations of standard mixtures (neutral and amino sugars, and sialic acids) were prepared to establish a calibration equation. The number of moles of each monosaccharide in the sample was quantified by linear interpolation of residue area units into the calibration equation.
(105) The monosaccharides were analyzed by HPAEC using a Dionex ICS3000 system equipped with a gradient pump, an electrochemical detector, and an autosampler. The individual neutral and amino sugars, and sialic acids were separated by a Dionex CarboPac PA20 (3150 mm) analytical column with an amino trap. The gradient program used the following mobile phase eluents: for neutral and amino sugars, degassed nanopure water and 200 mM NaOH; for sialic acids, 100 mM NaOH and 1 M sodium acetate in 100 mM NaOH. Injection was made every 40 min for neutral and amino sugars and every 35 minutes for sialic acids. Table 11, below, provides the analytical results.
(106) TABLE-US-00007 TABLE 11 Monosaccharide composition analysis of intact QC # 7953-4, QC # 8046-1, QC # 8048-1, and QC # 8147-27 glycoproteins by HPAEC-PAD Total Residues in Sam- Sample ple Hydrolized %, by mole ID Glycosyl Residue nanomoles g QC # Fucose (Fuc) nd 8046-1 N-acetyl galactosamine nd (GalNAc) N-acetyl glucosamine 61.98 280.17 47.98 (GlcNAc) Galactose (Gal) 26.78 148.63 20.73 Glucose (Glc) nd Mannose (Man) 40.42 224.28 31.29 N-acetyl neuraminic acid nd (NANA) N-glycolyl neuraminic nd acid(NGNA) QC # Fucose (Fuc) nd 8147-27 N-acetyl galactosamine nd (GalNAc) N-acetyl glucosamine 44.40 200.70 51.45 (GlcNAc) Galactose (Gal) 17.52 97.23 20.31 Glucose (Glc) nd Mannose (Man) 24.36 135.21 28.24 N-acetyl neuraminic acid nd (NANA) N-glycolyl neuraminic nd acid(NGNA) .sup.1Intact aliquots hydrolyzed from each sample was ~200 g for neutral&amino sugars and ~200 g for sialic acids.
(107) The N-glycans were further analyzed subsequent to cleavage from the tetramer products. Such N-glycans cleavage was generated through by enzymatic activity with a solution of PNGase A for 24 hours at 37 C. then PNGase F for another 24 hours at 37 C. The released N-glycans were then purified with a C18 SPE cartridge. The carbohydrate fraction was eluted with 5% acetic acid and dried by lyophilization. The N-linked oligosaccharides were further permethylated (based on a method described by Anumula and Taylor, 1992). The glycans were dried with nitrogen gas and profiled, as described above, by NSI-FTMS.
(108) As above, such analysis was determined using a LTQ Orbitrap XL mass spectrometer (ThermoFisher) equipped with a nanospray ion source. Permethylated N-linked glycans were dissolved in 1 mM NaOH in 50% methanol then infused directly into the instrument at a constant flow rate of 0.5 L/min. A full FTMS spectrum was collected at 30 000 resolution with 3 microscans. The capillary temperature was set at 210 C. and MS analysis was performed in the positive ion mode. For total ion mapping (automated MS/MS analysis), m/z range, 200 to 2000 was scanned with ITMS mode in successive two mass unit windows.
(109) Numerous N-glycans were found in the sample. These primarily were biantennary complex-type glycans with smaller amounts of high mannose glycosylation and hybrid-type glycosylation.
(110) Truncated or damaged glycans found in the sample are denoted in
(111) Further tests were undertaken for enhanced glycan analysis of the transgenically sialylated rBuChE products. Initially, such activities involved hydrolysis and analysis of the samples for monosaccharide identification and measurement determinations. To that end, aliquots of each sample were allocated for neutral and amino sugars and for sialic acids analyses, dried and weighed to determine the actual amounts of samples being used for analysis. The aliquots for neutral and amino sugars were hydrolyzed with 2.0 N trifluoroacetic acid (TFA), whereas the aliquots for sialic acids were hydrolyzed with 2 M acetic acid. After hydrolysis, the digests were dried under a stream of nitrogen gas, dissolved with H.sub.2O, sonicated for 5 min on ice and transferred to injection vials.
(112) A mix of neutral and amino sugars standards, and sialic acids standards with known amounts were hydrolyzed in the same manner and at the same time as the samples. Four concentrations of standard mixtures (neutral and amino sugars, and sialic acids) were prepared to establish a calibration equation. The amount and number of moles of each monosaccharide in the sample was quantified by linear interpolation of residue area units into the calibration equation.
(113) The monosaccharides were analyzed by HPAEC using a Dionex ICS3000 system equipped with a gradient pump, an electrochemical detector, and an autosampler. The individual neutral and amino sugars, and sialic acids were separated by a Dionex CarboPac PA20 (3150 mm) analytical column with an amino trap. The gradient program used the following mobile phase eluents: for neutral and amino sugars, degassed nanopure water and 200 mM NaOH; for sialic acids, 100 mM NaOH and 1 M sodium acetate in 100 mM NaOH. Injection was made every 40 min for neutral and amino sugars and every 35 min for sialic acids. The results are provided in Table 13 for two samples (Lots 4 and 5).
(114) TABLE-US-00008 TABLE 13 Monosaccharide composition analysis of rBuChE glycoproteins by HPAEC-PAD. Total Residues in Sample Hydrolyzed %, by mole rBuChE Glycosyl Residue g nanomoles Lot 4 Fucose (Fuc) 1.30 7.95 5.60 N-acetyl galactosamine nd (GalNAc) N-acetyl glucosamine 9.93 44.91 31.67 (GlcNAc) Galactose (Gal) 4.12 22.86 16.12 Glucose (Glc) nd Mannose (Man) 9.03 50.09 35.32 N-acetyl neuraminic acid 4.95 16.00 11.29 (NANA) N-glycolyl neuraminic nd acid(NGNA) Lot 5 Fucose (Fuc) 1.06 6.48 3.40 N-acetyl galactosamine nd (GalNAc) N-acetyl glucosamine 14.13 63.89 33.57 (GlcNAc) Galactose (Gal) 6.56 36.38 19.11 Glucose (Glc) nd Mannose (Man) 11.77 65.30 34.31 N-acetyl neuraminic acid 5.66 18.29 9.61 (NANA) N-glycolyl neuraminic nd acid(NGNA) .sup.1Intact aliquots hydrolyzed from Lot: 14B002 was ~245 g for neutral&amino sugars and ~245 g for sialic acids, and from Lot: 14B003 was ~370 g for neutral&amino sugars and ~370 g for sialic acids
(115) From these results, it is evident that of the residues analyzed, N-acetylglucosamine, galactose and mannose were detected within both samples (lots). Also, the sialic acid, NANA, was detected in both samples.
(116) From such overall data for the transiently and transgenically sialylated rBuChE products (tetramerized or not), the following observations have been made: From four transgenic production rBuChE batches analyzed, three show 70-73% terminal sialic acid occupancy and one shows 50%. All sugars are present at reasonable molar ratios. It was not determined how a low occupancy level was present within a sample. The transient sample showed 100% sialic acid occupancy with unnatural molar ratios of sugars, possibly due to sugar cleavage and instability (previous data showed 70% occupancy). Pooled transgenic T2 seed lot sialylates were found to be at a level similar to the transient system. Transient strategies show occupancy in 6 of 9 potential sites while the transgenic approach shows glycan occupancy on 8 of 9 sites with the last site problematic. Thus, the transgenic approach appears to be superior and more complete for glycosylation.
(117) Thus, overall, it is evident not only our plant-based rBuChE production methods suitable to provide highly effective OPs protections in mammals (whether delivered intravenously or intramuscularly), but the ability to produce the tetramer form thereof as well as highly sialylated structures reliably and to a necessary scalable level, but such products may also be provided through seed lines for a continuous reliable supply, as well.
Example 11: Pilot and Definitive Pharmacokinetic Studies of rBuChE Produced in Plant System
(118) Two PK studies were performed in male Hartley guinea pigs to evaluate the characteristics of rBuChE. Complete details of these studies are presented in the final reports for SRI Study Nos. B616-13 and B618-13 which are provided in Appendices IV and V. First, a pilot study was conducted that compared three different forms of rBuChE: the neat monomer (Lot 13B001), a sialylated monomer dimer (Lot 13B002), and a sialylated tetramer (Lot 13B003) administered by the intravenous (IV) route. Table 14 presents the design for the pilot study (SRI Study No. B616-13) (with the expectation that a suitable pilot study result would lead to a more definitive test strategy).
(119) TABLE-US-00009 TABLE 14 Overall PK Testing Strategy Dose Dose Dosing rBuChE Activity (mg/kg; Conc. Volume Dose No. of Plasma Collection Group Variant (U/mg) U/kg) (mg/mL) (mL/kg) Route .sup.a Animals Time Points .sup.b,c 1 A 285 25 17.6 1.43 IV 3 Pre-dose, 5, 10, 20, 30, (Neat (7125) 60 min, 2, 4, 8, 24, 36, Monomer) 48, 72, 120 and 168 hr 2 B 338 25 30.8 0.82 IV 3 Pre-dose, 5, 10, 20, 30, (Monomer (8450) 60 min, 2, 4, 8, 24, 36, Dimer 48, 72, 120 and 168 hr Sialylated) 3 C 426 25 28.7 0.88 IV 3 Pre-dose, 5, 10, 20, 30, (Tetramer (10650) 60 min, 2, 4, 8, 24, 36, Sialylated) 48, 72, 120 and 168 hr .sup.a Dose administration was via a jugular vein catheter (JVC). .sup.b Blood was collected from the JVC port not used for dose administration. Due to loss of catheter patency in some of the animals, blood was also collected from methods approved on SRI's IACUC protocol 10006. .sup.cPlasma samples were analyzed by the Ellman assay.
(120) Next, the definitive PK study was performed using the sialylated tetramer (Lot 13B005) administered by both the IV and intramuscular (IM) routes. The decision to evaluate the sialylated tetramer in the definitive study was based on evaluation of PK characteristics determined in the pilot study as well as physicochemical properties analyses conducted at KBP throughout the continuous process development work. Table 15 presents the design for the definitive study (SRI Study No. B618-13).
(121) TABLE-US-00010 TABLE 15 Study Design for Definitive Pharmacokinetic Study of Sialylated Tetramer rBuChE in Guinea Pigs Dose Level Dose Dosing Test Activity (mg/kg; Conc. Volume Dose No. of Plasma Collection Group Article (U/mg) U/kg) (mg/mL) (mL/kg) Route Animals Time Points .sup.a, b 1 rBuChE 525 25 28.0 0.89 IV 4 Pre-dose, 5, 10, 20, (tetramer (13125) 30, 60 min, 2, 4, 8, sialylated) 24, 36, 48, 72, 120 and 168 hr 2 rBuChE 525 25 28.0 0.89 IM 4 Pre-dose, 5, 10, 20, (tetramer (13125) 30, 60 min, 2, 4, 8, sialylated) 24, 36, 48, 72, 120 and 168 hr .sup.a In Group 1, blood was collected from the JVC port not used for dose administration. .sup.b Plasma samples were analyzed by the Ellman assay.
The basic test system undertaken was as follows:
(122) Male Hartley guinea pigs were purchased from Charles River (Raleigh, N.C.), with either a single jugular vein catheter (JVC) or dual JVC by the vendor prior to shipment. The animals were five to six weeks and 320-409 grams in weight. General procedures for animal care and housing were in accordance with the National Research Council (NRC) Guide for the Care and Use of Laboratory Animals, 8.sup.th edition (2011) and the Animal Welfare Standards incorporated in 9 CFR Part 3, 1991. The animals were housed one per cage in hanging polycarbonate cages with hardwood chip bedding, using a 12 hr light/12 hr dark schedule, at 72-73 F., and at 33-46% humidity. The animal room had at least ten room volumes per hour ventilation, with no recirculation of air. Harlan Teklad Certified Guinea Pig chow (#2040C) was provided ad libitum. Feed was analyzed periodically to ensure that contaminants known to be capable of interfering with the study and reasonably expected to be present in such feed were not present at levels that would affect the study. Documentation of feed analyses is maintained in the study records. Water (purified, reverse osmosis) was provided ad libitum. Based on previous reports, no contaminants that could interfere with and affect the results of the study are expected to have been present in the water. Copies of annual analysis reports are maintained at SRI for reference. Animals were individually identified by an ear punch.
(123) Study Procedures and Endpoints:
(124) Dose administration was by the IV route (SRI Study Nos. B616-13 and B618-13) or IM route (SRI Study No. B618-13). Mortality and morbidity were checked at least once daily and clinical observations were recorded immediately post-dose on the day of dose administration, once daily thereafter, or more often as clinical signs warranted. Animals were examined for any altered clinical signs, including gross motor and behavioral activity, and observable changes in appearance. Body weights were determined one day before the start of the study for randomization and on Day 1 for dose administration calculations only. Blood was collected from the JVC port or other site approved by SRI's IACUC and ACURO, into a tube containing K.sub.3EDTA, processed to plasma, and then stored frozen at 7010 C. Approximately 100 L total whole blood (50 L of plasma) was collected from each guinea pig pre-dose, and at 5, 10, 20, 30, 60 min, 2, 4, 8, 24, 36, 48, 72, 120 and 168 hr post-dose.
(125) Pharmacokinetic Analysis Procedures:
(126) Data were subjected to non-compartmental analysis using WinNonlin Model 200 (for extravascular administration) or Model 201 (for IV bolus administration); a uniform weighting factor was applied to each data set. T.sub.max and C.sub.max values were determined directly from the data. AUC.sub.last values were calculated using the log/linear trapezoidal (IV dose) or linear up/log down trapezoidal (IM dose). Values were calculated for each individual guinea pig. The dose administered was input to the program as U/kg, and as a result no additional corrections for individual body weights of the animals were necessary. The background levels of BuChE, determined for each animal from a sample collected prior to dose administration, were not subtracted from the measured plasma concentrations. The actual times recorded for sample collection were used through the first four hours for the calculations. The following parameters and constants were determined for the IV and IM groups: observed maximum plasma concentration (C.sub.max), time to maximum plasma concentration (T.sub.max), area under the plasma concentration-time curve to the last time point (AUC.sub.last), area under the plasma concentration-time curve extrapolated to infinity (AUC.sub.inf), terminal phase elimination half-life (t.sub.1/2), mean residence time to extrapolated infinity (MRT.sub.inf). The volume of distribution at steady state (V.sub.ss) and clearance (Cl) were determined for the IV group only. Bioavailability (F) after IM administration was calculated using the AUC.sub.last values for both the IV and IM groups.
(127) Clinical Observations:
(128) Clinical observations showed that one animal from the tetramer sialylated rBuChE dose group was slightly hypoactive on Day 1. All other animals appeared normal throughout the study.
(129) Plasma Drug Levels:
(130)
(131) Pharmacokinetics Analysis Results:
(132) The results of the PK analysis are presented in Table 16. The elimination half-life values (t.sub.1/2) varied markedly among the three forms of rBuChE. The neat monomer was eliminated from plasma with a t.sub.1/2 of less than one hr (0.37 hr) while the monomer dimer sialylated had a t.sub.1/2 about 20 fold longer, 7.5 hr. The longest t.sub.1/2 was observed for the tetramer sialylated form, 60 hr. The MRT also varied with the form administered from 0.59 hr (Variant A) to 73 hr (Variant C). Cl was highest for Variant A (76.6 ml/hr/kg) and lowest for Variant C (6.27 mL/hr/kg). The highest concentrations of rBuChE were observed immediately after dose administration and were about 80 to 100 fold higher than the background. The neat monomer and monomer dimer sialylated variants had similar C.sub.max values, 98.82.8 U/mL and 91.316.2 U/mL, respectively. The tetramer sialylated variant had a lower mean C.sub.max, 78.73.0 U/mL, despite the administration of a dose with higher activity than the other two variants. This difference was likely due to higher distribution of the tetramer sialylated as shown by the highest Vss of the three rBuChE forms. The more favorable parameters exhibited by the tetramer sialylated resulted in the highest exposure as shown by the AUC, 1704 hr.Math.U/mL, about 18 and 7 fold higher than the AUC.sub.inf for the neat monomer and monomer dimer sialylated, respectively.
(133) TABLE-US-00011 TABLE 16 Pilot Pharmacokinetic Parameters for rBuChE in Guinea Pigs .sup.a Guinea C.sub.max t.sub.1/2 AUC.sub.last AUC.sub.inf Cl Vss MRT.sub.inf Pig (U/mL) (hr) (hr .Math. U/mL) (hr .Math. U/mL) (mL/hr/kg) (mL/kg) (hr) Neat Monomer, 25 mg/kg (7125 U/kg) 1 97.2 0.36 94.2 95.2 74.8 46.1 0.62 2 97.2 0.39 84.3 85.9 82.9 51.2 0.62 3 102 0.36 97.5 98.7 72.2 39.0 0.54 Mean 98.8 0.37 92.0 93.3 76.6 45.4 0.59 SD 2.8 0.02 6.9 6.6 5.6 6.1 0.05 Monomer Dimer Sialylated, 25 mg/kg (8450 U/kg) 4 110 6.6 263 276 30.6 161 5.2 5 82.8 8.3 205 224 37.8 262 6.9 6 81.2 7.5 185 198 42.6 266 6.2 Mean 91.3 7.5 275 233 37.0 230 6.1 SD 16.2 0.9 41 40 6.0 60 0.9 Tetramer Sialylated, 25 mg/kg (10650 U/kg) 7 80.4 45 1598 1701 6.30 354 57 8 80.4 74 1477 1799 5.90 529 89 9 75.2 62 1399 1611 6.60 490 74 Mean 78.7 60 1491 1704 6.27 458 73 SD 3.0 15 100 94 0.35 92 16 .sup.a Pharmacokinetic parameters were determined using plasma concentrations of BuChE that were not corrected for the background enzyme level. Mean BuChE activity in plasma was 1.05 0.185 U/mL.
Pilot Study Conclusions:
(134) Three variants of rBuChE were administered by the IV route to male guinea pigs. The dose of 25 mg/kg was well tolerated. The plasma profile and pharmacokinetic parameters varied markedly among the three proteins. Although the highest initial plasma concentration was observed for Variant A, the neat monomer, this form of rBuChE was rapidly eliminated from the plasma with a t.sub.1/2 and MRT of less than 1 hr and very rapid Cl, resulting in low plasma exposure. The pharmacokinetic characteristics most closely approximating those of human serum derived BuChE were observed for the tetramer sialylated form, which had a biphasic plasma profile, the highest V.sub.ss, suggesting greater distribution of the protein, the lowest clearance (Cl), t.sub.1/2 of 60 hr, and the highest exposure based on the AUC values for the three rBuChE variants.
(135) From these promising results, it was then determined that a more definitive study was merited to analyze the potential benefits of the plant-derived rBuChE products.
(136) Definitive Study Undertaking:
(137) The same type of guinea pigs were utilized, kept, and tested as above, with a greater amount of rBuChE administered and actual treated subjects exposed to various OPs agents.
(138) Mortality/Morbidity and Clinical Observations:
(139) Clinical observations were collected and all animals appeared normal throughout the study.
(140) Plasma Drug Levels:
(141)
(142) Pharmacokinetics Analysis:
(143) The results of the pharmacokinetics analysis are presented in Table 17. In the IV group, the observed C.sub.max value was 63.5 U/mL, or about 70 fold higher than the background level. rBuChE was eliminated slowly from the plasma with a t.sub.1/2 of 63.4 hr and MRT.sub.inf of 83.5 hr for the IV group; this corresponded to a Cl of 9.8 mL/hr/kg. V.sub.ss was moderate, 836 mL/kg, suggesting extensive extracellular distribution. The AUC.sub.last and AUC.sub.inf were 1124 hr.Math.U/mL and 1368 hr.Math.U/mL, respectively. In the IM group, the observed C.sub.max value was 63.5 U/ml, 8 fold greater than the background, and was observed at the T.sub.max of 36 hr. Both the t.sub.1/2 and MRT.sub.inf were longer than in the IV group, 86.5 hr and 142 hr, respectively. Exposure as shown by AUC.sub.last and AUC.sub.inf values was 689 hr.Math.U/mL and 1005 hr.Math.U/mL, respectively. The bioavailability (F) determined using AUC.sub.last was calculated to be 61.61.8%.
(144) TABLE-US-00012 TABLE 17 Definitive Pharmacokinetic Parameters in Guinea Pigs of rBuChE Administered by IV and IM Routes Guinea C.sub.max T.sub.max t.sub.1/2 AUC.sub.last AUC.sub.inf Cl V.sub.ss MRT.sub.inf F Pig (U/mL) (hr) (hr) (hr .Math. U/mL) (hr .Math. U/mL) (mL/hr/kg) (mL/kg) (hr) (%).sup.a Intravenous group, 25 mg/kg (13125 U/kg) 1 60.7 .sup.NA.sup.b 61.3 978 1129 11.6 874 75.3 NA 2 68.0 NA 64.2 1181 1413 9.3 774 83.4 NA 3 62.5 NA 64.7 1097 1359 9.7 887 91.8 NA 4 62.7 NA 74.1 1241 1569 8.4 818 97.8 NA Mean 63.5 63.4 1124 1368 9.8 838 83.5 SD 3.1 1.8 114 182 1.3 52 8.3 Intramuscular group, 25 mg/kg (13125 U/kg) 5 7.7 36.0 82.6 694 997 .sup.NC.sup.c NC 136 61.7 6 7.4 36.0 90.0 671 1036 NC NC 151 59.7 7 7.5 36.0 87.0 711 1037 NC NC 139 63.3 8 8.0 36.0 77.1 681 950 NC NC 129 60.6 Mean 7.7 36.0 86.5 689 1005 142 61.6 SD 0.3 0.0 3.7 17 41 7.9 1.8 .sup.aCalculated using AUC.sub.last values .sup.bNA, not applicable .sup.cNC, not calculated
Conclusions of the Definitive Study:
(145) A single dose of a sialylated tetramer variant of rBuChE, 25 mg/kg (13125 U/mL), was administered to male Hartley guinea pigs by the IV and IM routes. The treatment was well tolerated and all animals appeared normal for the entire study, 168 hr after dosing. The enzyme exhibited PK parameters more closely comparable to those for human serum derived BuChE when compared to other recombinant moieties developed in this project. After IM administration, rBuChE was detected at levels slightly above the background at the first blood collection time point, with concentrations steadily increasing to a peak at 36 hr after injection. The t.sub.1/2 values were 63.4 hr (IV) and 86.5 hr (IM), corresponding to a slow Cl, 9.8 mL/hr/kg, and V.sub.ss, 836 ml/kg, that is consistent with extracellular distribution. The bioavailability of rBuChE after IM administration was about 60%.
Example 12: Efficacy Testing of Sialylated rBuChE Tetramer
(146) Efficacy studies were conducted using three different nerve agents using a short time point IV model for protection. GD and VX Nerve Agents
(147) For these agents, there was administered an inventive plant-derived tetramer sialylated BuChE to male Hartley guinea pigs (300-350 grams) via an IV carotid catheter at 26.15 mg/kg for each subject.
(148) After 15 minutes, animals administered 26.15 mg/kg were exposed to 3LD50 of GD or VX via s.c. injection (n=6 for each). GB Nerve Agent
(149) For this agent, there was administered a plant-derived tetramer sialylated BuChE to male Hartley guinea pigs (300-350 grams) via an IV carotid catheter at 52.3 mg/kg.
(150) After 15 minutes, animals administered 52.3 mg/kg were exposed to 3LD50 of GB via s.c. injection (n=6).
(151) In each situation, all of the test subject animals survived to 24 hours without any sign of OP intoxication.
(152) These data demonstrate the efficacy potential of the sialylated tetrameric rBuChE to protect mammals from lethal nerve agent exposure. The attractive PK data suggest that this protection can be optimized by improvements in sialylation and tetramerizing efficiencies leading to a very competitively functioning product.
(153) As alluded to above, such tetramerized and sialylated rBuChE products may also be utilized for other mammalian treatments, including neurological conditions (Alzheimer's), addiction therapies (for cocaine addictions treatments, for instance), and even enzyme replacement therapies to overcome BuChE deficiencies due to any number of genetic or other disorders. The viability of production in reliable fashion, particularly with high levels of sialylated glycans and tetramer formation, may further translate into effective mammalian (including human) treatment potential, as well.
(154) Thus, the overall effectiveness of this newly discovered tetramer sialylated rBuChE product accords significant improvements within this industry. The further capability to produce such a new product through transgenic means, transfection processes, and other types of gene expression methodologies, thus opens up a notable area for not only OPs protections, but also other treatments for humans and other mammals that have heretofore been rather limited.
(155) It should be understood that various modifications within the scope of this invention can be made by one of ordinary skill in the art without departing from the spirit thereof. It is therefore wished that this invention be defined by the scope of the appended claims as broadly as the prior art will permit, and in view of the specification if need be.
(156) References cited hereinabove, are as follows:
(157) 1. Castilho et al., 2008. Plant Physiol. 147:331-9; 2. Castilho et al., 2010. J. Biol. Chem. 285:15923-30; 3. Castilho et al., 2011. Glycobiol. 21:813-82; 4. Castilho et al., 2011. Glycoconjugate J. 28:240 5. Diaz et al., Sensitive and specific detection of the non-human sialic Acid N-glycolylneuraminic acid in human tissues and biotherapeutic products. PLoS ONE 2009, 4:e4241 6. U.S. Pat. No. 8,729,245 7. Duysen et al., Wild-type and A328W mutant human butyrylcholinesterase tetramers expressed in Chinese hamster ovary cells have a 16-hour half-life in the circulation and protect mice from cocaine toxicity. Pharmacol Exp Ther. 2002 302:751-8. 8. Ellman, G. L. et al. [1961], Biochem. Pharmacol. 7:88-95 9. Engler et al., 2008, PLoS One, 3, e36472008 10. Giritch et al., 2006, Proc. Natl. Acad. Sci. USA, 103:14701-14706 11. Geyer et al., PNAS, Nov. 23, 2010, vol. 107, no. 47, 20251-20256; 12. Gleba, Y., Klimyuk, V. & Marillonnet, S., 2005, Vaccine 23:2042-2048 13. Gleba, Y., Klimyuk, V. & Marillonnet, S. 2007, Curr. Opin. Biotechnol. 18:134-141 14. Ilyushina et al., Chemical polysialylation of human recombinant butyrylcholinesterase delivers a long-acting bioscavenger for nerve agents in vivo. Proc Natl Acad Sci USA. 2013. 110:1243-8 15. Li et al., Lamellipodin proline rich peptides associated with native plasma butyrylcholinesterase tetramers. Biochem. J. (2008) 411, 425-432. 16. Schneider et al., 2014. Plant Biotechnol. J. March 11. doi: 10.1111/pbi.12184; 17. Schneider et al., Biotechnol J. 2014 April; 9(4):501-10 18. Weber et al., 2011, PLoS One, 6, e16765 19. Werner et al., 2012, Bioeng Bugs 3, 38-43 20. Zheng et al., 2014. A highly efficient cocaine-detoxifying enzyme obtained by computational design. NATURE COMMUNICATIONS |5:3457|DOI: 10.1038/ncomms4457
APPENDIXSEQUENCE LISTINGS
(158) SEQ ID NO: 1
(159) mankhlslslflvllglsaslasgAPSPPLPPPPPPPPPPPPPPPPPPPPLP
(160) SEQ ID NO: 2
(161) mankhlslslflvllglsaslasgACCLLMPPPPPLFPPPFF
(162) SEQ ID NO: 3
(163) mankhlslslflvllglsaslasgACCLLMPPPPPLFPPPFFDYKDDDDK
(164) SEQ ID NO: 4
(165) mankhlslslflvllglsaslasgAQPTFINSVLPISAALPGLDQKKRGNHKACCLLMPPPPPLFPPPFF