Abstract:
A sugar chain synthesizer comprising one or more reaction columns packed with immobilized glycosyltransferase and/or glycosidase; one or more separation means, arranged downstream from the reaction columns, for separating reaction products, unreaction products and byproducts contained in the solution eluted from the reaction columns; a first pump for feeding a primer of water soluble polymer and buffer solution to the reaction columns through a first selector valve; a second pump for feeding a buffer solution and sugar nucleotide solution to any one of the reaction columns through a second selector valve; one or more circulation flow paths connecting between a flow path downstream from the separation means and a flow path upstream from each of the reaction columns; and a third selector valve, arranged between the separation means and one or more circulation flow paths, for selective connection between the separation means and a desired circulation flow path. This synthesizer ensures continuous and automatic synthesis of sugar chains even if complicated.

Description:
FIELD OF THE INVENTION 
     The present invention relates to a sugar chain synthesizer for automating synthesis and separation of a sugar chain. 
     BACKGROUND OF THE INVENTION 
     A glycoconjugate in a cell plays an important role in the signal transduction and identification of cells, for example, identification of viruses, cancer cells and blood types, and clarification of sugar chain functions is considered as one of the targets of post-genome studies. 
     However, the method of synthesizing an oligonucleic acid and peptide has already been established, and is automated, but sugar chain synthesis method still contains many problems to be solved. Expectations are running high for establishment of sugar chain synthesis method and realization of an effective synthesizer in order to achieve successful clarification of sugar chain functions. At present, the following three sugar chain synthesis methods are practiced:
         (1) Chemical synthesis   (2) Fermentation by genetically recombinant cell and microorganism   (3) Synthesis by glycosyltransferase.       

     According to method (1), the targeted sugar chains are synthesized sequentially while protecting a hydroxyl group other than that for chemical bonding, and reaction steps are numerous and complicated. The method (2) provides a large volume of targeted sugar, but is accompanying by subsequent complicated purification process. 
     The method (3) was developed to solve the problems of methods (1) and (2). It includes the method disclosed in the Japanese Laid-Open Patent Publication No. Hei 11-42096. The method (3) uses the procedure of selective synthesis by glycosyltransferase, and does not require the process of protection of a hydroxyl group, as in method (1). Further, the amount of byproducts is smaller and the purification step subsequent to synthesis is facilitated. 
     In recent years, easy preparation of biologically active protein has been enabled by the development of genetic recombination technology. However, a great portion of biologically active protein is glycoprotein, and a sugar chain to be bonded is different according to each host. Activity may be seriously lost or damaged. 
     It will be very helpful if there is a way of reforming the changed sugar chain into the original one. Physiological function and activity are expected to be improved by modification into the sugar chain different from the originally bonded one. There are two methods of modifying the sugar chain of glycoprotein, and these methods are currently practiced.
         (A) Fermentation by changing the host or using the host modified by injection of glycosyltransferase gene therein   (B) Fermentation of the obtained glycoprotein using endo- or exo-glycosidase and glycosyltransferase.       

     According to method (A), the sugar chain to be bonded is changed but is not always changed into the desired one. To change the sugar chain into a specified one, method (B) is preferred. A method of using the transglycosylation of endoglycosidase includes the method disclosed in the Japanese Laid-Open Patent Publication No. Hei 05-64594. A method of using the transglycosylation of exo-glycosidase and glycosyltransferase includes the method disclosed in Eur. J. Biochem. 191:71-73 (1990). 
     However, these methods modify only the sugar residues of the non-reducing terminus at most, and fail to bring about full-scale modification of sugar chains. There is a further way of using endoglycosidase and glycosyltransferase. For example, there is a method disclosed in J. Am. Chem. Soc. 119:2114-2118 (1997). In this method, glycosyltransferase is used to extend sugar chains onto the non-reducing terminus of the N-acetyl glucosamine residue remaining on the protein subsequent to hydrolysis by endoglycosidase, thereby promoting modification into the glycoprotein bonded with sialyl Lewis×tetraose. The sugar chain bonded is the non-reducing terminus portion of the sugar chain of glycoprotein, and this method is insufficient to achieve modification of the entire sugar chain. 
     A sugar chain synthesizer is disclosed in the Japanese Laid-Open International Patent Publication No. Hei 05-500905. 
     SUMMARY OF THE INVENTION 
     When an actual apparatus is used to synthesize the sugar chain according to the method (3) or (B) described above, separation and purification of the product is carried out for each step, and then the next reaction is carried out. Such a batch system is adopted at present, and human aids have been indispensable to complete the entire processing. 
     In the apparatus disclosed in the aforementioned Japanese Laid-Open International Patent Publication No. Hei 05-500905, glycosyltransferase can be used to extend the sugar chain using the monosaccharide, oligosaccharide and glycoprotein as substrates. However, the reaction column and separation/purification means must be connected in series on a continuous basis according to the order of the sugars to be reacted. To be more specific, even if the sugars of the same type are to be reacted in repetition, the same number of reaction columns and separation/purification means as that of sugars are required. This requires a large-scale apparatus to be manufactured. Further, automatic and continuous performance of sugar chain synthesis is not mentioned in this announcement. Further, the enzyme to be used is restricted to glycosyltransferase alone; glycosidase is not used. 
     The object of the present invention is to provide a sugar chain synthesizer that permits easy synthesis of sugar chains. 
     The present invention is characterized by comprising one or more reaction columns packed with immobilized glycosyltransferase and/or glycosidase, one or more separation/purification means arranged downstream from the reaction column in order to separate reaction products, unreaction products and byproducts contained in the solution eluted from the reaction column, and a circulating flow path for repeated circulation of them on a selective basis. 
     In the present invention, sugar is bonded with a primer made of water-soluble polymer or is disassociated from the primer. This makes it possible to feed the sugar bonded to the primer, to the reaction column required to the intended reaction, and ensures easy synthesis of a desired sugar chain. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a system configuration diagram of Embodiment 1; 
         FIG. 2  is a diagram representing the flow path of a sugar chain synthesizer as Embodiment 1; 
         FIG. 3  is a diagram representing a variation of Embodiment 1; 
         FIG. 4  is a system configuration diagram of Embodiment 2; 
         FIG. 5  is a diagram representing the flow path of a sugar chain synthesizer as Embodiment 2; 
         FIG. 6  is a system configuration diagram of Embodiment 3; 
         FIG. 7  is a diagram representing the flow path of a sugar chain synthesizer as Embodiment 3; 
         FIG. 8  is a system configuration diagram of Embodiment 4; 
         FIG. 9  is a diagram representing the flow path of a sugar chain synthesizer of Embodiment 4; and 
         FIG. 10  is a diagram representing an example of the configuration of an ultrafiltration column. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     [EMBODIMENT 1] 
       FIG. 1  is a system configuration diagram of Embodiment 1. 
     A sugar chain synthesizer comprises:
         pumps  1  and  2  having the function (so-called low pressure gradient function) of selecting a plurality of solvents, and feeding the solvents while mixing these solvents with the lapse of time and changing the composition of the solvent to be fed;   six valves  3  through  8  for selecting a flow path;   reaction columns  18  through  20 ;   separation columns  21  through  23 ;   detectors  9  and  10  for detecting reaction products; and   a controller  11  for controlling these components.       

     For example, a refractive index detector (RI), ultraviolet-visible spectrum detector (UV) or diode array absorbance detector (DAD) is used as the detector  9  used in the present apparatus in order to monitor the product. A diode array absorbance detector (DAD), mass analyzer (MS) or nuclear-magnetic resonance apparatus (NMR) is used as a detector  10  to get information on molecular structure. 
     Glycosyltransferase (e.g. galactosyltransferase, N-acetyl glucosaminyltransferase, N-acetyl galactosaminyltransferase, fucosyltransferase, sialyltransferase, mannosyltransferase) or glycosidase (e.g. mannosidase, galactosidase, fucosidase, sialydase, xylosidase) is immobilized and packed in the reaction column. When a column with immobilized glycosyltransferase therein is used, sugar chain can be extended by adding a new sugar thereto. If a column with immobilized glycosidase therein is used, a specified sugar can be dissociated (separated) from the sugar chain. In the present embodiment, this column is called a reaction column Rn. 
     Further, the reaction product refers to the primer of water soluble polymer (e.g. biological polymer such as protein, glycoprotein, glycopeptide, lipid, glycolipid, oligosaccharide or polysaccharide, or polyacrylamide derivatives disclosed in the Japanese Laid-Open International Patent Publication Nos. Hei 11-42096 and 2001-220399, with its molecular weight preferred to be 10,000 or more; “primer” hereinafter referred to as (P) and the primer is chemically bonded with sugar (Sn)) hereinafter referred to as (P-Sn) in the present embodiment). 
     The separation column used should have a function of separating the reaction product and nucleotide, monosaccharide or oligosaccharide produced from hydrolysis. For example, such a column includes a gel filtration chromatography, cation exchange chromatography, anion exchange chromatography, affinity chromatography, dialysis and ultrafiltration. 
       FIG. 2  is a diagram representing the flow path in the present embodiment. 
     Pumps  1  and  2  feed solution of bottles  13  through  17 . Here the bottle  13  contains primer (P), the bottle  14  stores buffer solution, and bottles  15  through  17  incorporates sugar nucleotide solution (e.g. uridine-5′-diphosphoglactose, uridine-5′-diphospho-N-acetyl glucosamine, uridine-5′-diphospho-N-acetyl galactosamine, guanosine 5′-diphosphofucose, guanosine 5′-diphosphomannose, cytidine-5′-monophospho-N-acetylneuraminic, etc.; hereinafter referred to as “Xn-Sn” in the present embodiment). 
     Each bottle is assigned with solenoid valves  101  through  106  incorporated in the pump, and the solution for which the valve is opened is fed by the pump. Further, a bottle  12  is a fraction collecting bottle (FC: fraction collector), and drains ( 1 ) and ( 2 ) are drain bottles. 
     The following describes the operation of this apparatus with reference to FIG.  2 : 
     Assume that primer P and sugars S 1 , S 2  and S 3  are bonded in the sequence of P-S 1 -S 2 -S 3 . Also assume that the reaction column used is the one packed with immobilized glycosyltransferase therein. In practice, however, there is no restriction to the sequence of P-S 1 -S 2 -S 3 . The sequence of P-S 1 -S 2 -S 1 -S 3  is also acceptable. 
     However, when the same type of sugar is repeated as in P-S 1 -S 1 , bottle  16  or  17  is replaced by the one containing sugar nucleotide X 1 -S 1 , and the reaction  19  or  20  is replaced by R 1 . Alternatively, another flow path is added; namely, reaction columns and separation means (hereinafter referred to as “separation column”) are extended in four rows. This also applies to the case where S 2  (or S 3 ) is repeated continuously. 
     Further, when sugar is subjected to dissociation, a column packed with immobilized glycosidase therein is added to reaction columns. When the column packed with immobilized glycosidase therein is used for processing, there is no need of using sugar nucleotide solution. 
     When reaction is made in the order of P-S 1 -S 2 -S 3 , the present apparatus basically comprises the following ten steps:
         Step 1: Injection of primer (P) and sugar nucleotide (X 1 -S 1 ) into the reaction column  18  (R 1 ) and their reaction   Step 2: Separation of primer (P-S 1 ), unreacted sugar nucleotide (X 1 -S 1 ) and nucleotide (X 1 ) as a reaction byproduct by the separation column  21  (C 1 )   Step 3: Injection of primer (P-S 1 ) and nucleotide (X 2 -S 2 ) into reaction column  19  (R 2 )   Step 4: Reaction between primer (P-S 1 ) and nucleotide (X 2 -S 2 ) and washing of the separation column  21  (C 1 )   Step 5: Separation of primer (P-S 1 -S 2 ), unreacted sugar nucleotide (X 2 -S 2 ) and nucleotide (X 2 ) as a reaction byproduct by the separation column  21  (C 2 )   Step 6: Injection of primer (P-S 1 -S 2 ) and sugar nucleotide (X 3 -S 3 ) into reaction column  20  (R 3 )   Step 7: Reaction between primer (P-S 1 -S 2 ) and sugar nucleotide (X 3 -S 3 ) and washing of the separation column  22  (C 2 )   Step 8: Separation of primer (P-S 1 -S 2 -S 3 ), unreacted sugar nucleotide (X 3 -S 3 ) and nucleotide (X 2 ) as a reaction byproduct by the separation column  22  (C 3 )   Step 9: Fractionation of primer (P-S 1 -S 2 -S 3 ) (FC)   Step 10: Washing of separation column  23  (C 3 ).       

     The following describes the details of each step with reference to FIG.  2 : Table 1 shows the positions of valves in each step. 
                                                                                             TABLE 1                   (EMBODIMENT 1)                Step                1 (1)   2 (2)   2   3 (1)   3 (2)   4   5   6 (1)   6 (2)   7   8   9 (1)                   Injec-   Re-   Sepa-   Injec-   Re-   Wash-   Sepa-   Injec-   Re-   Wash-   Sepa-   Fraction-       10       Valve   tion   action   ration   tion   action   ing   ration   tion   action   ing   ration   ation   9 (2)   Washing               Pump 1                                                               101   Open       (Primer)       102       Open   Open   Open   Open   Open   Open   Open   Open   Open   Open   Open   Open   Open       (Buffer)       V3   P2   P2   P2   P2   P2   P2   P3   P3   P3   P3   P4   P4   P4   P4       Pump 2       103       Open   Open       Open   Open   Open       Open   Open   Open   Open   Open   Open       (Buffer)       104   Open       (X1-S1)       105               Open       (X2-S2)       106                               Open       (X3-S3)       V3   P2   P2   P2   P3   P3   P3   P3   P4   P4   P4   P4   P4   P4   P4       for       fraction-       ation       V4   P1   P1   P1   P3   P1   P1   P1   P4   P1   P1   P1   P5   P1   P1       Before       detector       W6   Detect   Detect   Detect   Detect   Detect   Detect   Detect   Detect   Detect   Detect   Detect   Detect   Detect   Detect       (Column       21)       W7   Detect   Detect   Detect   D   Detect   Detect   Detect   Detect   Detect   Detect   Detect   Detect   Detect   Detect       (Column       22)       W8   Detect   Detect   Detect   Detect   Detect   Detect   Detect   D   Detect   Detect   Detect   Detect   Detect   Detect       (Column       23)                    
In Table 1, valves  101  through  106  are in the “Close” position except for “Open”. Valves  3  through  5  (V) indicate the connection positions in terms of “P 1 ” through “P 4 ”. Valves  6  through  8  (W) indicate the drain side as a detector side in terms of “D”.
 
     &lt;Explanation of Step 1&gt; 
     (1) Open the valve  101  of the pump  1  and valve  104  of the pump  2 , and connect each of the valves  3  and  5  to the position (2), so that primer (P) and nucleotide (X 1 -S 1 ) is injected into the reaction column  18  (R 1 ). The amount of instruction is determined by the flow rate and feed time as given in equations (1) and (2):
 
Amount of  P  injected ( ml )=flow rate ( ml/min .)×time ( min .)  [1]
 
Amount of  X   1 - S   1  injected ( ml )=flow rate ( ml/min .)×time ( min .)  [2]
 
     When the solution feed time is the same, the ratio of primer (P) to nucleotide (X 1-S   1 ) is determined by the flow ratio. In the case of 50%/50%, for example, the flow rate of pump  1  is equal to the flow rate of pump  2 . 
     (2) Open the valve  102  of the pump  1  and valve  103  of the pump  2 , and let the buffer  14  flow at the same flow rate so that the primer (P) and sugar nucleotide (X 1 -S 1 ) is fed into the reaction column  18  (R 1 ). Then reduce the flow rate and allow reaction to continue, for example, at the flow rate of 0 ml/min. for a certain period of time. 
     &lt;Explanation of Step 2&gt; 
     Upon termination of reaction, raise the pump flow rate. The primer (P-S 1 ) as a reaction product in the reaction column  18  (R 1 ), unreacted nucleotide (X 1 -S 1 ) and nucleotide as reaction byproduct ((X 1 ), which is removed sugar nucleotide (S 1 ) from sugar sugar nucleotide (X 1 -S 1 )) are led to the separation column  21  (C 1 ) and separated. 
     The following separation modes can be considered:
         (a) Gel filtration: If the molecular weight of the primer (P-S 1 ) exceed the exclusion limit of the GPC column, the primer is eluted earlier than sugar nucleotide (X 1 -S 1 ) or nucleotide (X 1 ). (Retention capacity is smaller).   (b) Anion exchange: The neutral primer (P-S 1 ) is not absorbed but the sugar nucleotide (X 1 -S 1 ) or nucleotide (X 1 ) as anion is absorbed by the column and leaching occurs later.   (c) Cation exchange: The neutral primer (P-S 1 ) is not absorbed but the sugar nucleotide (X 1 -S 1 ) and nucleotide (X 1 ) as anion is eluted earlier because they are ion-excluded.   (d) Ultrafiltration: The unreacted sugar nucleotide and nucleotide as a reaction byproduct, they have smaller molecular size, are filtrated and separated from the primer with larger molecular size.       

     The following steps are followed when the separation mode (b) is used: 
     &lt;Explanation of Step 3&gt; 
     (1) When the primer (P-S 1 ) has been detected by a detector  9 , set the valve  4  to the position (3) of the reaction column  19  (R 2 ), and the valve  7  to the drain side. At the same time, open the valve  105  of the pump  2  and set the valve  5  to the position (3) of the reaction column  19  (R 2 ) to feed the sugar nucleotide (X 2 -S 2 ). If the ratio of the sugar nucleotide (X 2 -S 2 ) is 50%, set the flow rates of the pumps  1  and  2  to the same level. (Same as in step 1). 
     Solution is injected until the detection of primer (P-S 1 ) terminates. If the volume of the primer (P-S 1 ) is such that “the flow rate of the pumps  1  and  2 ×solution injection time” has exceeded “the volume of the reaction column  19  (R 2 )” due to the expansion of the band of primer, then suspend solution injection halfway (pump flow rate=0) and allow the reaction to terminate. Then re-inject the remaining solution. 
     (2) When the primer (P-S 1 ) has moved to the reaction column  19  (R 2 ), set the valve  4  to the drain position (1), and set the valve  7  back to the original position. Open the valve  103 , switch the solution to be fed by the pump  2  and select the buffer  14 . Inject all the primer (P-S 1 ) and sugar nucleotide (X 2 -S 2 ) to the reaction column  19  (R 2 ). After that, reduce the flow rate and continue a flow rate of 0 ml/min. for example, for a certain period of time. In this case, only the pump  2  is used to feed the buffer to the reaction column. 
     &lt;Explanation of Step 4&gt; 
     While the primer (P-S 1 ) and sugar nucleotide (X 2 -S 2 ) are reacting with each other in the reaction column  19  (R 2 ), the pump  1  continues to feed buffer  14  to the separation column  21  (C 1 ). The unreacted nucleotide (X 1 -S 1 ) and nucleotide of reaction byproduct (X 1 ) absorbed in the column are washed out of the column. 
     &lt;Explanation of Step 5&gt; 
     After reaction between the primer (P-S 1 ) and sugar nucleotide (X 2 -S 2 ), set the valve  3  to the position (3). Increase the flow rate of the buffer  14  using the pumps  1  and  2  and separate the primer (P-S 1 -S 2 ) from the unreacted sugar nucleotide (X 2 -S 2 ), nucleotide (X 2 ) as a reaction byproduct in the separation column  22  (C 2 ). 
     &lt;Explanation of Step 6&gt; 
     (1) When the primer (P-S 1 -S 2 ) has been detected by the detector  9 , set the valve  4  to the position (4) and the valve  8  to the drain position (2). At the same time, set the valve  5  to the position (4) to feed the sugar nucleotide (X 3 -S 3 ). If the sugar nucleotide (X 3 -S 3 ) has a ratio of 50%, set the flow rates of the pumps  1  and  2  to the same level. (Same as in step 1). Solution is injected until the detection of the primer (P-S 1 -S 2 ) terminates. 
     (2) When the primer (P-S 1 -S 2 ) has been moved to the reaction column  20  (R 3 ), set the valve  4  to the drain position (1) and set the valve  8  to the original position. Set the pump  2  to the buffer  14 , and inject all the primer (P-S 1 -S 2 ) and sugar nucleotide (X 3 -S 3 ) to the reaction column  20  (R 3 ). After that, use only the pump  2  to feed the buffer and reduce the flow rate. Continue a flow rate of 0 ml/min. for example, for a certain period of time. 
     &lt;Explanation of Step 7&gt; 
     While the primer (P-S 1 -S 2 ) and sugar nucleotide (X 3 -S 3 ) are reacting with each other in the reaction column  20  (R 3 ), the pump  1  continues to feed buffer  14  to the separation column  22  (C 3 ). The unreacted nucleotide (X 2 -S 2 ) and nucleotide of reaction byproduct (X 2 ) absorbed in the column are washed out of the Column. 
     &lt;Explanation of Step 8&gt; 
     After reaction between the primer (P-S 1 -S 2 ) and sugar nucleotide (X 3 -S 3 ) set the valve  3  to the position (4). Increase the flow rate of the buffer  14  using the pumps  1  and  2  and separate the water-soluble polymer (P-S 1 -S 2 -S 3 ) from the unreacted sugar nucleotide (X 3 -S 3 ) and nucleotide (X 3 ) as a reaction byproduct in the separation column  23  (C 3 ). 
     &lt;Explanation of Step 9&gt; 
     (1) When the primer (P-S 1 -S 2 -S 3 ) has been detected by the detector  9 , set the valve  4  to the fractionation position (5) and fractionate the sugar chain compound into the bottle  12  (FC). 
     (2) When detection by the detector  9  has terminated, set the valve  4  back to the drain position (1). 
     &lt;Explanation of Step 10&gt; 
     The pumps  1  and  2  continue to feed buffer  14  to the separation column  23  (C 3 ). The unreacted nucleotide (X 3 -S 3 ) and nucleotide of reaction byproduct (X 3 ) absorbed in the column are washed out of the column. 
     The aforementioned explanation refers to the procedure for creating the synthesized sugar chain (P-S 1 -S 2 -S 3 ) in the present embodiment. 
       FIG. 3  is a diagram representing a variation of the present embodiment. It shows the flow path when a detector  10  is added. The detector  10  is connected upstream from the detector  9  via the splitter (Sp), and captures the molecular structure of the component eluted from each separation column. The procedure for synthesizing the sugar chain In  FIG. 3  is the same as that in FIG.  2 . 
     According to the configuration given in  FIG. 3 , there is a detector  10  provided for detecting the information on the molecular structure of the reaction product. This makes it possible to check for each detector to see if synthesis reaction is carried out as planned or not. If reaction yield fails to reach the expected level, the next reaction for synthesis can be suspended to prevent to waste reaction reagent and time. 
     In the example shown with reference to the present embodiment, the time of switching the flow path by valves is based on the result of detection by the detector  9 . When planned sugar modification is to be processed and the time of passage of the solution eluted from the reaction column and separation column is known, valve switching time may be controlled according to the lapse of time, not according to the result of the detector  9 . 
     [EMBODIMENT 2] 
       FIG. 4  is a system structure diagram of Embodiment 2. 
     The sugar chain synthesizer comprises six pumps  1 ,  2 ,  24  through  27  capable of feeding the solvents of bottles  13  through  17  at a certain flow rate for a certain time, six valves  3  through  8  for switching the flow paths, two detectors  9  and  10  for detecting the reaction product and a controller  11  for controlling these components. The detectors  9  and  10  used in the present embodiment are the same as those used in embodiment 1. 
       FIG. 5  is a diagram representing the flow path of a sugar chain synthesizer as Embodiment 2. This flow path shown in  FIG. 5  in the one when one detector is used. The six pumps  1 ,  2 ,  24  through  27  feed the solution of bottles  13  through  17  at a certain flow rate for a certain period of time according to the control by the controller  11 . The difference from the embodiment 1 is that sugar nucleotides (X 1 -S 1 , X 2 -S 2  and X 3 -S 3 ) are fed directly to the reaction columns  18  through  20  (R 1 , R 2  and R 3 ) by the pumps  25 ,  26  and  27 , respectively. 
     In the present embodiment, the pumps  1  and  2  perform the function of sending the same buffer  14  alone; so-called low-pressure gradient function is not necessary. Further, the pumps  25 ,  26  and  27  can feed the sugar nucleotide solutions (X 1 -S 1 ), (X 2 -S 2 ) and (X 3 -S 3 ) to the reaction columns  18  through  20  without passing through respective solenoid valves. Accordingly, unlike the low-pressure gradient function where open/close operation of the solenoid valves is synchronized with the pump suction process, it is possible to provide more accurate control of the time of feeding the primer (P) and sugar nucleotide solutions (X 1 -S 1 ), (X 2 -S 2 ) and (X 3 -S 3 ). 
     [EMBODIMENT 3] 
       FIG. 6  is a system structure diagram of Embodiment 3; 
     The sugar chain synthesizer comprises:
         two pumps  1  and  2  capable of feeding the buffers (B 1  and B 2 )  14  and  30  at a certain flow rate for a certain time;   a sample injector  28  for injecting the primer (P)  13  into a flow path;   a sample injector  29  for injecting the sugar nucleotides (X 1 -S 1 , X 2 -S 2  and X 3 -S 3 )  15 ,  16  and  17  into the flow path;   six valves  3  through  8  for switching the flow path;   two detectors  9  and  10  for detecting reaction products; and   a controller  11  for controlling these components. The detectors  9  and  10  used in the present embodiment are the same as those used in embodiment 1.       

       FIG. 7  is a diagram representing the flow path of a sugar chain synthesizer as Embodiment 3: This diagram shows the case where one detector is used. Two pumps  1  and  2  feed the solution of the buffers  14  and  30  (need not always be arranged for each pump; one common buffer may be sufficient as shown in  FIG. 7 ) at a certain flow rate for a certain period of time according to the control by the controller  1 . The difference from the Embodiment 1 is that the primer (P) and sugar nucleotides (X 1 -S 1 , X 2 -S 2  and X 3 -S 3 ) are injected into the flow path through the sample injectors  28  and  29  and are fed into the reaction columns  18 ,  19  and  20  (R 1 , R 2  and R 3 ) by the buffer  14  (or  30 ). 
     In the present embodiment, pumps  1  and  2  are feed only the buffer  14  or  30 , without the need of using a so-called low-pressure gradient function. Further, a required amount of the primer (P) and sugar nucleotides (X 1 -S 1 , X 2 -S 2  and X 3 -S 3 ) are injected into the flow path through the sample injection. Accordingly, unlike the case of a low-pressure gradient function where the open/close operation of the solenoid valve is synchronized with the pump suction, the present embodiment provides the advantage of eliminating the waste of the primer (P) and sugar nucleotides (X 1 -S 1 , X 2 -S 2  and X 3 -S 3 ). 
     [EMBODIMENT 4] 
       FIG. 8  is a system configuration diagram of Embodiment 4.  FIG. 9  is a diagram representing the flow path. In the present embodiment, either one or two detectors may be used. When the analysis time is known in advance, control may be carried out without using a detector. 
     The difference from the Embodiment 3 is that the separation columns and valves connected in series to respective reaction columns in Embodiment 3 are connected centrally to one separation column, and an ultrafiltration column  40  is used for the separation column in the present embodiment. 
     Shared use of the separation column allows a valve  41  to be arranged instead of valves  6  through  8  in the present invention. 
     The configuration of the ultrafiltration column  40  is given in FIG.  10 . The ultrafiltration column  40  has a cylindrical ultrafiltration membrane  48  inside and two outputs; an outlet  47  for discharging the solution, injected through an inlet  45 , of such a great molecular weight that they cannot pass through the membrane, and an outlet  46  for discharging the solution that has passed through the membrane. Such a membrane that allows the sugar nucleotide or nucleotide to pass by but not the primer is selected as an ultrafiltration membrane  48 . The molecular weight of the primer is about 10,000 or more and that of the sugar nucleotide or nucleotide is about 400 or more. Such a considerable difference in size ensures easy separation between them. 
     A three-way valve  42  is arranged downstream from the outlet  47 . This valve is designed to permit selective connection between the flow path on the detector side and one of the outlets  46  and  47 . For the outlet on the non-connection side, the flow path is closed by the valve. 
     The following describes the method of separation by the ultrafiltration column  40 : When the solution eluted from the reaction column has been injected, the three-way valve  42  is set to the outlet  46 . Only the solution having passed through the ultrafiltration membrane  48  is discharged. The primer and sugar bonded with the primer are left in the ultrafiltration membrane  48 . When the three-way valve  42  is switched over to the outlet  47  side after the lapse of the specified time, the primer and sugar having bonded with the primer are discharged to the detector side. 
     The operation in the present embodiment is basically the same as that shown in reference to Embodiment 1. Use of the separation column is shared. The moment when the valves  6  through  8  in the Embodiment 1 are switched over to the drain side, the valve  41  is switched over the flow path on the side of the dotted line. This arrangement permits the same operation as that of the Embodiment 1, despite shared use of the separation column. 
     In the Embodiment 1, shared use of the separation column allows the configuration of the apparatus to be simplified. Further, use of the ultrafiltration column as a separation column causes the primer (P) to be concentrated in the ultrafiltration column. This eliminates the problem of expansion of the band of the primer (P) as described in the step 3 of the Embodiment 1, and facilities the injection into the next reaction column. 
     As described above, the present invention ensures easy sugar chain synthesis even in the case of complicated sugar chain synthesis. 
     INDUSTRIAL FIELD OF APPLICATION 
     Application of the present invention to a sugar chain synthesizer facilitates synthesis or separation of sugar chains.