specify the invention by examples and comparison examples ; however , the invention is not limited by the following examples . strain : the original strain of recombinant hansenula polymorpha hepatitis b vaccine expressing hbsag that is constructed with dna recombinant technique ( researched and developed by dalian hanxin biological pharmaceutical co ., ltd .) enjoys a strain no . of hbsagu35 - 16 - 9 . ( chinese pharmacopoeia , 2010 version , three volumes , recombinant hepatitis b vaccine ( hansenula polymorpha yeast ), page 132 ). it is preserved by dalian hanxin biological pharmaceutical co ., ltd . the fermentation growth medium comprises the following ingredients : glycerin , magnesium sulfate , potassium chloride , sodium chloride and ammonium hydrogen phosphate . the mass ratio in 1 l is 5 : 2 : 1 : 0 . 1 : 4 . it is prepared by injection water and sterilized under 121 ° c . for 30 minutes . sodium chloride solution : concentration is 3 mol / l ; it is prepared by injection water ; it is sterilized by filtration with 0 . 22 μm membrane filter ; pge6000 solution : concentration is 50 %; it is prepared by injection water ; it is sterilized under 121 ° c . for 30 minutes ; silica gel solution : concentration is 7 . 5 %; it is prepared by injection water ; it is sterilized under 121 ° c . for 30 minutes ; tris - hcl + 2 mol / l nacl solution : tris ( batch no . : wf0131la01 , usa ); concentration is 0 . 1 mol / l ; ph value is 8 . 5 ; it is prepared by injection water ; it is sterilized by filtration with 0 . 22 μm membrane filter ; potassium bromide solution : density is 1 . 04 g / ml , 1 . 28 g / ml , 1 . 34 g / ml respectively ; it is prepared by injection water ; it is sterilized by filtration with 0 . 22 μm membrane filter ; sodium chloride solution : concentration is 0 . 9 %; it is prepared by injection water ; it is sterilized by filtration with 0 . 22 μm membrane filter ; phosphate buffer solution : sodium hydrogen phosphate and monosodium phosphate have a concentration of 3 mmol / l ; it is prepared by injection water ; it is sterilized by filtration with 0 . 22 μm membrane filter ; alcl 3 solution : concentration is 10 %; it is prepared by injection water ; sodium hydroxide solution : concentration is 0 . 5 mol / l ; it is prepared by injection water ; aluminum potassium sulfate solution : concentration is 10 %; it is prepared by injection water ; it is sterilized by filtration with 0 . 22 μm membrane filter ; preparation of concentrate of recombinant ( hansenula polymorpha yeast ) hepatitis b surface antigen fermentation : one batch strain of working seed of recombinant hansenula polymorpha yeast was prepared ( it is obtained by amplification of the original strain of hepatitis b vaccine of recombinant hansenula polymorpha yeast ( strain no . : hbsagu35 - 16 - 9 )) and the batch strain of working seed was seeded in 300 ml growth medium and cultured for 24 h at 35 ° c . then the medium was transferred to 3 l growth medium further cultured for 24 h at 35 ° c . and the resulting medium was transferred to 30 l growth medium cultured for 15 h at 35 ° c ., finally , the culture medium was transferred to 200 l growth medium and cultured at 35 ° c . for 65 h a in a condition of a dissolved oxygen for above 20 %, a ph value at 6 . 8 and a air flow between 30 ˜ 200 ml / h , obtained fermentation broth of hansenula polymorpha cell expressing hepatitis b surface antigen for about 240 l ; preliminary purification : grinding and crushing the fermentation broth hansenula polymorpha cell by physical crushing method to realize a crushing rate of 92 %, removing cell debris by centrifugation at 4000 rpm and the supernatant was collected . the supernatant was performed two aqueous phase extraction by treating with nacl / peg6000 for 9 h and centrifuged with a speed of 6000 rpm , collected about 230 l supernatant , and then the supernatant was absorbed by silica gel solution for 15 h and desorpted under 60 ° c . for 1 h , then centrifuged at a rotational speed of 4000 rpm and collected about 110 l supernatant ; fulfill the preliminary purification . fine purification : purificating the preliminarily purified sample by deae sepharose ff anionic column chromatography ; the sample loading quantity is 10 times of the column volume , after loading the sample to the column , the column was eluted with 100 mmol / l tris buffer solution , velocity flow of 60 l / h . the elutes was monitored at absorbance of 280 nm , and collected the protein peak with od value of above 1 ; concentrating the collected protein peak and implemented a centrifugal process to the belt in the equivalent density area ; monitored at absorbance of 280 nm and collected the protein peak with od value of above 1 . the collected protein peak was further purified by molecule sieve column chromatography , the column was eluted with 0 . 9 % nacl solution for protein separation and desalination ; the elutes was monitored at absorbance of 280 nm and collecting hbsag protein peak with od value of above 0 . 8 for 28 . 2 l ; adopt hplc method to inspect the purity of above 99 . 0 %; ultimately dilute hbsag protein content until 220 μg / ml by phosphate buffer solution ; adopt 0 . 22 μm sterilization filter membrane for sterilization and filtration for 60 l . therefore , it will obtain the stock solution of recombinant hansenula polymorpha yeast hepatitis b surface antigen . preparation of semi - finished hepatitis b vaccine ( aluminum adjuvant + hbsag process — prior art ); batch no . of semi - finished product is s201001 preparation of aluminum adjuvant : mix up , adding 54 ml of 10 % alcl 3 solution into the flask , then adding 0 . 5 mol / l naoh solution at a speed of 50 ml / min and the mixture ( reaction solution ) was subjected to measure ph value at any time ; stop adding solution once ph value is 7 . 00 , and 110 ml of 0 . 5 mol / l naoh solution was further added , supplement 436 ml of 0 . 9 % nacl solution until the ultimate volume of 600 ml , the solution was sterilized by moist heat sterilization under 121 ° c . for 30 minutes , obtaining the aluminum adjuvant . dilution of stock solution : adopting 54 . 5 ml stock solution with a protein content of 220 μg / ml ( prepared in example 1 ) and put into 500 ml conical flask ; adding 300 ml of 0 . 9 % nacl solution so that protein content is 40 μg / ml . absorption of semi - finished product : mix up , 300 ml aluminum adjuvant was added to the other flask , then the 300 ml diluted stock solution was added and mixed for 30 minutes , obtaining the semi - finished product of hepatitis b vaccine ; the batch no . of semi - finished product is s201001 . preparation of semi - finished hepatitis b vaccine ( aluminum adjuvant + hbsag process — prior art ); batch no . of semi - finished product is s201002 preparation of aluminum adjuvant : mix up , adding 54 ml of 10 % alcl 3 solution into the flask , then adding 0 . 5 mol / l naoh solution at a speed of 50 ml / min and the mixture ( reaction solution ) was subjected to measure ph value at any time ; stop adding solution once ph value is 6 . 95 , and 108 ml of 0 . 5 mol / l naoh solution was further added , supplement 438 ml of 0 . 9 % nacl solution until the ultimate volume of 600 ml , the solution was sterilized by moist heat sterilization under 121 ° c . for 30 minutes , obtaining the aluminum adjuvant . dilution of stock solution : adopting 54 . 5 ml stock solution with a protein content of 220 μg / ml ( prepared in example 1 ) and put into 500 ml conical flask ; adding 300 ml of 0 . 9 % nacl solution so that protein content is 40 μg / ml . absorption of semi - finished product : mix up , 300 ml aluminum adjuvant was added to the other flask , then the 300 ml diluted stock solution was added and mixed for 30 minutes , obtaining the semi - finished product of hepatitis b vaccine ; the batch no . of semi - finished product is s201002 . preparation of semi - finished hepatitis b vaccine ( aluminum adjuvant + hbsag process — prior art ); batch no . of semi - finished product is s201003 preparation of aluminum adjuvant : mix up , adding 54 ml of 10 % alcl 3 solution into the flask , then adding 0 . 5 mol / l naoh solution at a speed of 50 ml / min and the mixture ( reaction solution ) was subjected to measure ph value at any time ; stop adding solution once ph value is 7 . 00 , and 115 ml of 0 . 5 mol / l naoh solution was further added , supplement 431 ml of 0 . 9 % nacl solution until the ultimate volume of 600 ml , the solution was sterilized by moist heat sterilization under 121 ° c . for 30 minutes , obtaining the aluminum adjuvant . dilution of stock solution : adopting 54 . 5 ml stock solution with a protein content of 220 μg / ml ( prepared in example 1 ) and put into 500 ml conical flask , adding 300 ml of 0 . 9 % nacl solution so that protein content is 40 μg / ml . absorption of semi - finished product : mix up , 300 ml aluminum adjuvant was added to the other flask , then the 300 ml diluted stock solution was added and mixed for 30 minutes , obtaining the semi - finished product of hepatitis b vaccine ; the batch no . of semi - finished product is s201003 . production process of semi - finished hepatitis b vaccine ( in - situ absorption — the invention ); batch no . of semi - finished product is s201004 mix up ; adding 200 ml pbs solution into the flask ; adding 54 . 5 ml of 220 μg / ml stock solution ( prepared in example 1 ); then adding 150 ml pbs solution and adding 54 . 7 ml of 10 % kal ( so 4 ) 2 solution ; then adding 0 . 9 % nacl solution until total weight 520 ml , start adding 0 . 5 mol / l naoh solution into the flask at a speed of 50 ml / min ; in the addition of 45 ml of 0 . 5 mol / l naoh solution , adopt sample and measure ph value ; once ph value is 5 . 50 , control the addition speed of 0 . 5 mol / l naoh solution at 20 ml / min ; adopt sample and measure ph value at any time ; stop adding 0 . 5 mol / l naoh solution once ph value is 7 . 00 ; add 56 ml solution in total ; ultimately supplement 0 . 9 % nacl solution until 600 ml ; mix up for 10 minutes and then stop mixing ; seal up and sediment . first washing and sedimentation : upon sedimentation for 19 h , removing the supernatant , then adding 0 . 9 % nacl into the flask until 600 ml and it was mixed for 10 minutes , close the mixing function and seal up and sediment . second washing and sedimentation : upon sedimentation for 12 h , removing the supernatant , then adding 0 . 9 % nacl into the flask until 600 ml and it was mixed for 10 minutes , close the mixing function and seal up and sediment . third washing and sedimentation : upon sedimentation for 12 h , removed the supernatant , then supplement 0 . 9 % nacl into the flask until 600 ml and it was mixed for 10 minutes , close the mixing function and seal up and sediment . preparation of semi - finished products : upon sedimentation for 20 h , removing the supernatant , then supplement 0 . 9 % nacl into the flask until 600 ml ; mix up evenly for 30 minutes , obtaining the semi - finished hepatitis b vaccine . the batch no . of semi - finished product is s201004 . production process of semi - finished hepatitis b vaccine ( in - situ absorption — the invention ); batch no . of semi - finished product is s201005 mix up ; adding 200 ml pbs solution into the flask ; adding 54 . 5 ml of 220 μg / ml stock solution ( prepared in example 1 ); then adding 150 ml pbs solution and adding 54 . 7 ml of 10 % kal ( so 4 ) 2 solution ; then adding 0 . 9 % nacl solution until total weight 520 ml , start adding 0 . 5 mol / l naoh solution into the flask at a speed of 50 ml / min ; in the addition of 45 ml of 0 . 5 mol / l naoh solution , adopt sample and measure ph value ; once ph value is 5 . 5 , control the addition speed of 0 . 5 mol / l naoh solution at 20 ml / min ; adopt sample and measure ph value at any time ; stop adding 0 . 5 mol / l naoh solution once ph value is 6 . 88 ; add 52 ml solution in total ; ultimately supplement 0 . 9 % nacl solution until 600 ml ; mix up for 10 minutes and then stop mixing ; seal up and sediment . first washing and sedimentation : upon sedimentation for 19 h , removing the supernatant , then adding 0 . 9 % nacl into the flask until 600 ml and it was mixed for 10 minutes , close the mixing function and seal up and sediment . second washing and sedimentation : upon sedimentation for 12 h , removing the supernatant , then adding 0 . 9 % nacl into the flask until 600 ml and it was mixed for 10 minutes , close the mixing function and seal up and sediment . third washing and sedimentation : upon sedimentation for 12 h , removing the supernatant , then supplement 0 . 9 % nacl into the flask until 600 ml and it was mixed for 10 minutes , close the mixing function and seal up and sediment . preparation of semi - finished products : upon sedimentation for 20 h , removing the supernatant , then supplemented 0 . 9 % nacl into the flask until 600 ml ; mix up evenly for 30 minutes , obtaining the semi - finished hepatitis b vaccine . the batch no . of semi - finished product is s201005 . production process of semi - finished hepatitis b vaccine ( in - situ absorption — the invention ); batch no . of semi - finished product is s201006 mix up ; adding 200 ml pbs solution into the flask ; adding 54 . 5 ml of 220 μg / ml stock solution ( prepared in example 1 ); then adding 150 ml pbs solution and adding 54 . 7 ml of 10 % kal ( so 4 ) 2 solution ; then adding 0 . 9 % nacl solution until total weight 520 ml , start adding 0 . 5 mol / l naoh solution into the flask at a speed of 50 ml / min ; in the addition of 45 ml of 0 . 5 mol / l naoh solution , adopt sample and measure ph value ; once ph value is 5 . 5 , control the addition speed of 0 . 5 mol / l naoh solution at 20 ml / min ; adopt sample and measure ph value at any time ; stop adding 0 . 5 mol / l naoh solution once ph value is 6 . 92 ; add 58 ml solution in total ; ultimately supplement 0 . 9 % nacl solution until 600 ml ; mix up for 10 minutes and then stop mixing ; seal up and sediment . first washing and sedimentation : upon sedimentation for 19 h , removing the supernatant , then adding 0 . 9 % nacl into the flask until 600 ml and it was mixed for 10 minutes , close the mixing function and seal up and sediment . second washing and sedimentation : upon sedimentation for 12 h , removing the supernatant , then added 0 . 9 % nacl into the flask until 600 ml and it was mixed for 10 minutes , close the mixing function and seal up and sediment . third washing and sedimentation : upon sedimentation for 12 h , removing the supernatant , then supplement 0 . 9 % nacl into the flask until 600 ml and it was mixed for 10 minutes , close the mixing function and seal up and sediment . preparation of semi - finished products : upon sedimentation for 20 h , removing the supernatant , then supplemented 0 . 9 % nacl into the flask until 600 ml ; mix up evenly for 30 minutes , obtaining the semi - finished hepatitis b vaccine . the batch no . of semi - finished product is s201006 . determination the value of semi - finished hepatitis b vaccine for absorption completeness , mice ed 50 , sedimentation speed of aluminum particle and appearance reference product : the frozen reference product of recombinant ( yeast ) hepatitis b vaccine comes from national institute for the control of pharmaceutical and biological products . diagnostic reagent kit of hepatitis b surface antigen is purchased from shanghai kehua biotechnology co ., ltd . the test sample was centrifuged for 5 minutes with the speed of 6500 rpm , collecting the supernatant . measure hbsag content in the reference product , test sample and its supernatant by the testing method of in vitro relative potency of recombinant hepatitis b vaccine ( yeast ). adopt the logarithm of hbsag content in the reference product as horizontal coordinate and adopt logarithm of corresponding absorbance as vertical coordinate for linear regression ; correlation coefficient is not less than 0 . 99 . the hbsag content was calculated by subjecting the absorbance value of test sample and supernatant to the equation of linear regression and the absorption rate was calculated with following formula . wherein : p refers to the absorption rate of test sample , %; c s refers to hbsag content of supernatant in the test sample in the unit of μg / ml ; c t refers to hbsag content of test sample in the unit of μg / ml . detection methods for the median effective dose ( ed 50 ) in mice : diagnostic reagent kit of antigen on hepatitis b surface comes from shanghai kehua biotechnology co ., ltd . the vaccine was diluted continuously . 14 ˜ 16 g balb / c mice were intraperitoneally injected with 1 . 0 ml dilution of vaccine . 10 mice were used to injection in each dilution degree of vaccine . after raising for 4 - 6 weeks , the blood was collected from eyeballs of mouse about 1 ml and immune serum was prepared from the blood . measuring the anti - hbs in the immune serum using the diagnostic reagent kit of hepatitis b surface antigen and calculating the seroconversion transfer rate according to the seroconversion quantity in each dilution degree ; further calculating ed 50 . ed 50 value = 10 terminal logarithm of 50 % seroconversion transfer rate wherein : terminal logarithm of 50 % seroconversion transfer rate = logarithm of dilution degree ( content ) of above 50 % seroconversion transfer rate + distance ratio × dilution series logarithm the detection results of absorption completeness and ed 50 of semi - finished hepatitis b vaccine are shown in table 1 . the detection results of aluminum particle sedimentation speed and appearance of semi - finished hepatitis b vaccine are shown in table 2 .