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1 Printed in the Republic of Korea "/"-:5*$"- 4$*&/$& 5&$)/0-0(: Vol. 25, No. 6, , Study on stability test of in process sample of recombinant Protein A Yoo Gon Kim, Woo Jong Lee, Chan Hee Won 1 and Chul Soo Shin 2 Korea Institute of Industrial Technology GyeongGi Bio-Center, 864-1, Lui-Dong, Yeongtong-Gu, Suwon-City, Gyeonggi-Do, Korea Department of Environmental Engineering, Chonbuk National University Advanced Protein Technologies(Co.) (Received September 26, 2012; Revised November 30, 2012; Accepted November 30, 2012) w " œ x w ½ š Á Á Á w», 1 w y œw 2 Advanced Protein Technologies(Co.) Abstract: This study is to investigate the issues on how to secure stability during the purification process for the production of recombinant protein A. The final recombinant protein A is produced by passing through the cation exchange column (SP) and the anion-exchange column (Q) during the production process, for which the samples produced by the step-by-step processes can be exposed to trouble in securing stable storage in case the next process cannot be taken within the proper time period. Accordingly, this study aims to evaluate the proper storage conditions and length of time when storing samples produced in the production process. That is, in this study, how to store fair samples, how long the storage period should be set up, and how to evaluate the security of its quality depending on time are dealt with. The items to be experimented with were enodotoxin, SDS-PAGE, HPLC purity and concentration. Experimental results showed that after passing the cation exchange column, when stored at 4 o C or room temperature, SDS-PAGE showed a major, endotoxin is 5.0 Eu/mg or less, and concentration is on average of 8.21 to 8.24 mg/ml and RSD% 0.10~0.62%. In addition, HLPC purity showed somewhat stable results; at the HPLC purity 214 nm, the average is 99.24% to 99.37% and RSD% is 0.22~0.29%, while the average is 89.72% to 89.80% and RSD% 0.62~1.26% at 280 nm. On the contrary, after passing the anion exchange column, when stored at 4 o C or room temperature, SDS-PAGE revealed the major, endotoxin is 0.5 Eu/mg or less, and concentration is on average of 5.59 mg/ml and RSD% 0.03~0.10%. when it comes to HLPC purity, the result showed that at the HPLC purity 214 nm, the average is 99.74% and RSD% is 0.10~0.11%, while the average is 96.16% to 96.85% and RSD% 0.72~1.13%. In conclusion, the stability of fair samples of recombinant protein A during the manufacturing process could be obtained without substance decomposition for 7~8 days at 4 o C or 20~21 days at room temperature. Corresponding author Phone : +82-(0) Fax : +82-(0) kyg00789@hanmail.net 483
2 484 Yoo Gon Kim, Woo Jong Lee, Chan Hee Won and Chul Soo Shin : w A w» w œ, œ ye (SP) ye (Q) mw w z w A w, œ œ ƒ w w y ƒ š. œ w š w., œ» y t sƒ w. xw m, SDS PAGE, HPLC š. x ye m z þ (4 o C), SDS PAGE major, m 5.0 Eu/mg w, s³ 8.21~8.24 mg/ml, RSD% 0.10~0.62%, š HPLC 214 nm s³ 99.24~99.37%, RSD% 0.22~0.29%, 280 nm s³ 89.72~89.80%, RSD% 0.62~1.26% ùkü. e m z þ (4 o C), SDS PAGE major, m 0.5 Eu/mg w, s³ 5.59 mg/ml, RSD% 0.03~0.10% š HPLC 214 nm s³ 99.74%, RSD% 0.10~0.11%, 280 nm s³ 96.16~96.85%, RSD% 0.72~1.13%.» x w A œ 4 C o 7~8 š 20~21 w p y. Key words: recombinant protein a, stability, endotoxin, SP, Q, HPLC x rprotein A œ x w., ye ye myw œ 4 C o y w ƒ š w, xw m, SDS PAGE, HPLC, x ww. 3FDPNCJOBOU 1SPUFJO " Protein A Staphylococcus aureus x (natural) l w wxk(recombinant)ƒ t y px w w v (Repligen) DNA sww wx 2009 ¾ y x v l w ù px ƒ š j w y wì š. wr w wì ƒ v l t yƒ 4 (USP) š. p w (monoclonal antibody) s, s ƒ w ü û w. j w ƒ w w, j w œ l j w k szw» w w ey (antibody affinity ligand) v l (protein A) w j m v(chromatography)ƒ t k š š protein A w s l w 95% ƒ ƒ wš z. w ey protein A, G, L, v l ƒƒ kv gf (Staphylococcus aureus) p mgf (Streptococcus sp.) w ey ƒ w (immunoglobulin G, IgG) v (constant region, Fc) ey ƒ v l rmpp mg f (Peptostreptococcus magnus) w eq p (kappa light chain) ey š p eq p I, III, IV w w w ƒ w scfvù Fab w r (antibody fragment) w (Fig. 1, Table 1). 1 x t t x w w t x» w w t w» w Analytical Science & Technology
3 Study on stability test of in process sample of recombinant Protein A 485 Table 2. Production process of rprotein A Fig. 1. Structure of antibody domain and affinity ligand. Table 1. Affinity ligand of monoclonal antibody used on purification Ligand Origin microbe Binding domain Protein A Staphylococcus aureus Fc region of IgG Protein G Streptococcus sp. Fc region of IgG Protein L Peptostreptococcus magnus kappa light chain of IgG š. ù t y, y, Ÿ ( ) y w, w y wš w» w w w v. w t z t w e š w ( z)» w w. t» w» w, y t sƒw x w. t x ƒ ƒ xw. š wš þ t 5±3 o C, þ t -20±5 C ³ w o š. 2-5 w " œ (E.Coli) w z w cell cell lysis w z y e (SP Column) w z y e (Q Column) z (UF) w. œ w Table 2 ù kü. 6,7 y j m v y j m v ƒ ƒƒ w w w» ƒ» w,, w. y j m v v w e ƒ w,»» ƒ. y ph j w. š yj m v y ( ) y y 2 ƒ, ƒƒ y (Na +, Cl )» w. (+) w» ( ) w ƒ ( y ) w j š, ( ) w (+) w ƒ ( y ) w k. w w j» w. ww ( ) w w l w w. 8 m ƒ w ³ m. ³ m ³ s s Vol. 25, No. 6, 2012
4 486 Yoo Gon Kim, Woo Jong Lee, Chan Hee Won and Chul Soo Shin (lipopolysaccharide) w. ³ s td w. m x ³ w m w w x, x ³ w m x y yw y j» w w. w x w» w ù. 9 x» x HPLC system Waters HPLC w, Column Phenomenex mm (BioSep- Sec-S2000) wš UV GE GeneQuant w. Endotoxin Gel Clot LONZA, Limulus Amebocyte Lysate PYROGENTPlus Single Test Kit š Electrophoresis system BIO-RAD PowerPac, Invitrogen Novexminicell & XCell SureLock w, SDS Pre made gel KOMA 4-12% Tris, KG5012 š Autopipete (20~1000 µl), Incubator, Volumetricflask, ph meter, Clean bench x» w. )1-$»» Sol. 1, 0.3 M Dibasic anhydrous sodium phosphate (21.3 g/500 ml water), Sol. 2, 0.3M Monobasic anhydrous sodium phosphate (18.0 g/500 ml water) ƒƒ Sol ml 1 L f š phƒ 7.0 ¾ Sol. 2 z 0.45 µm membrane filter w. q 214 nm, 280 nm (Mode: Dual), e L35 (BioSep-Sec-S2000), 1 ml/min, 20 µl š 20. Anhydrous sodium phosphate, Monobasic anhydrous, sodium phosphate, š Lal water w. x w A w» w e, ye (SP) m k œ 4 o C 20 ¾, 7 ¾ w Éü x ww. š ye (Q) m k œ 4 o C 21 ¾, 8 ¾ w Éü x ww. xw HPLC,, SDS Page, m 4 x w w x ww. )1-$ x x x 214 nm, 280 nm HPLC Chromatogram y w x., Chromatogram peak peakƒ % w w y wš w. š x», ye m k 4 o C xw» š w, 1, 2, 3, 6, 7, 10, 15 š 20 ƒƒ þ Éü x wwš», 1, 2, 3, 6, 7 x ww. š ye m k 4 o C», 1, 2, 3, 7, 8, 11, 15 š 21» x ww», 1, 2, 3, 7, 8 x ww. x š w A œ 1 mg/ml ( )z Blank ( )w HPLC ( : 50 µl)w 241 nm, 280 nm dual mode w. x x UV GE, GeneQuant d q 280 nm.» 5.0 mg/ml, ye»» 4 o C 20~21, 7~8 y r. w w x (USP) UV w. x 1 mg/ml ( ) z UV q 280 nm Ÿ w, 3 d w š ƒ %CV, 5% ü y w. 4%4 1"(& x x x major ƒ Á Analytical Science & Technology
5 Study on stability test of in process sample of recombinant Protein A 487 r x ye z 4 C o», 6, 10 š 20 x w w,», 3, 7 x ww. š ye z 4 C o», 3, 11, š 21,», 3 š 8 ƒƒ x ww. x» e 130V q wš Maker 15 µl gel loading z 15 µl gel loading w. Loading 50 z y w. m x x m» ye z 5.0 EU/mg w, yf z 0.5 EU/mg w. ye z 4 C o», 6, 10 š 20 x ww,», 3, 7 x ww. š ye z 4 C o», 3, 11, š 21,», 3 š 8 ƒƒ x ww. x Gel clot LONZA, Limulus Amebocyte Lysate PYROGENT Plus Single Test Kit w. x UV (mg/ml) z w Serial dilution wš 250 µl m kit w z Incubation (37 o C, 60 min) k y w. m z þ (4 o C) w y wš w. xw SDS PAGE, m, š HPLC, HPLC x 214 nm, 280 nm x ww, x»» 20 x w w. x SDS PAGE» major 20 major ùkû, m» 5.0 Eu/mg w Eu/mg w. š» 8.28 mg/ml mg/ml s³ 8.21 mg/ml, RSD% 0.1% ùkü yw ùkü. w HPLC» x 214 nm 99.12%, % 98.86~99.65% RSD% 0.29%, 280 nm» 90.58% % s³ 89.72%, RSD% 0.62% ùkü x yù w p x ye œ 41 P $ x rprotein A w» w ye Fig. 2. Result of SP sample at 4 o C storage. Table 3. Raw data of SP sample at 4 o C storage <D : Day, : Not Test> Items Time SDS PAGE <Criteria: > Endotoxin <Criteria: 5.0 EU/mg> Concentration <Criteria: 5.0 mg/ml> HPLC Purity 214 nm <Criteria: 90%> 280 nm <Criteria: 70%> Initial 1D 2D 3D 6D 7D 10D 15D 20D Vol. 25, No. 6, 2012
6 488 Yoo Gon Kim, Woo Jong Lee, Chan Hee Won and Chul Soo Shin.» x ye m z 4 o C 20 yù yw p w. Fig. 2 SP 4 o C w x š. š Table 3 x Raw data š. ye œ x ye m z w y wš w. x w SDS PAGE, m, š HPLC, HPLC x 214 nm, 280 nm x ww, x»» 7 x ww. x SDS PAGE» major 7 major ùkû, m» 5.0 Eu/mg w Eu/mg w. š» 8.28 mg/ml mg/ml s³ 8.24 mg/ml, RSD% 0.11% ùkü yw ùkü. w HPLC» x 214 nm 99.12%, % 99.12~99.71% s³ 99.37%, RSD% 0.22%, 280 nm» 90.58% % s³ 89.80%, RSD% 1.26% ùkü x yù w p.» x ye m z Fig. 3. Result of SP sample at room temperature storage. 7 yù yw p w. Fig. 3 SP 4 o C w x š. š Table 4 x Raw data š š. e œ 2 P $ x ye m z þ (4 o C) w y wš w. xw SDS PAGE, m, š HPLC, HPLC x 214 nm, 280 nm x ww, x»» 21 x ww. x SDS PAGE» major 21 major ùkû, m» 0.5 Eu/mg w Eu/mg w. š» 5.62 mg/ml mg/ml s³ 5.59 Table 4. Raw data of SP sample at room temperature storage <D : Day, : Not Test> Time Items Initial 1D 2D 3D 6D 7D SDS PAGE <Criteria: > Endotoxin <Criteria: 5.0 EU/mg> Concentration <Criteria: 5.0 mg/ml> HPLC Purity 214 nm <Criteria: 90%> 280 nm <Criteria: 70%> Analytical Science & Technology
7 Study on stability test of in process sample of recombinant Protein A 489 Fig. 4. Result of Q sample at 4 o C storage. mg/ml, RSD% 0.10% ùk ü yw ùkü. w HPLC» x 214 nm 99.55%, % 99.55~99.80% RSD% 0.10%, 280 nm» 95.84% % s³ 96.16%, RSD% 1.13% ùkü x yù w p.» x ye m z 21 yù yw p w. Fig. 4 Q 4 o C w x š. š Table 5 x Raw data š š. e œ 2 x ye m z Fig. 5. Result of Q sample at room temperature storage. w y wš w. x w SDS PAGE, m, š HPLC, HPLC x 214 nm, 280 nm x ww, x»» 8 x ww. x SDS PAGE» major 8 major ùkû, m» 0.5 Eu/mg w Eu/mg w. š» 5.62 mg/ ml mg/ml s³ 5.59 mg/ ml, RSD% 0.03% ùkü yw ùkü. w HPLC» x 214 nm 99.55%, % 95.55~99.84% RSD% 0.11%, 280 nm» 95.84% % s³ 95.91%, RSD% 0.72% ùkü x yù w p. Table 5. Raw data of Q sample at 4 o C storage <D : Day, : Not Test> Items Time SDS PAGE <Criteria: > Endotoxin <Criteria: 0.5 EU/mg> Concentration <Criteria: 5.0 mg/ml> HPLC Purity 214 nm <Criteria: 90%> 280 nm <Criteria: 70%> Initial 1D 2D 3D 7D 8D 11D 15D 21D Vol. 25, No. 6, 2012
8 490 Yoo Gon Kim, Woo Jong Lee, Chan Hee Won and Chul Soo Shin Table 6. Raw data of Q sample at room temperature storage Time HPLC Purity SDS PAGE <Criteria: > Endotoxin <Criteria: 0.5 EU/mg> Concentration <Criteria: 5.0 mg/ml> 214 nm <Criteria: 90%> 280 nm <Criteria: 70%> Items <D : Day, : Not Test> Initial 1D 2D 3D 7D 8D » x ye m z 8 yù yw p w. Fig. 5 Q 4 o C w x š. š Table 6 x Raw data š. rprotein A w» w œ q w» w x ye ye z þ (4 o C) š» þ 20~21, 7~8 w, xw SDS PAGE, m, HPLC,. ƒ œ x mw» y t s ƒ w x ye m z þ (4 o C) SDS PAGE major, m 5.0 Eu/mg w, s³ 8.21 mg/ml, RSD% 0.10% ùk ü. š HPLC» x 214 nm 99.12%, % 98.86~99.65% RSD% 0.29%, 280 nm» 90.58% % s³ 89.72%, RSD% 0.62% ùkü. ye m z x x SDS PAGE major, m 5.0 Eu/mg w, s³ 8.24 mg/ml, RSD% 0.11% ùkü š HPLC 214 nm 99.12~99.71% s³ 99.37%, RSD% 0.22%, 280 nm s³ 89.80%, RSD% 1.26% ùkü. e m z þ (4 o C) z SDS PAGE major, m 0.5 Eu/mg w,» 5.62 mg/ml mg/ml s³ 5.59 mg/ml, RSD% 0.10% š HPLC» x 214 nm 99.55%, % 99.55~99.80% RSD% 0.10%, 280 nm» 95.84% % s³ 96.16%, RSD% 1.13%. e m z x SDS PAGE major, m 0.5 Eu/mg w,» 5.62 mg/ml mg/ml s³ 5.59 mg/ml, RSD% 0.03% š HPLC» x 214 nm 95.55~ 99.84% RSD% 0.11%, 280 nm» 95.84% % s³ 95.91%, RSD% 0.72% ùkü.» x rprotein A œ (SP) þ (4 o C) 20, 7 w, w œ (Q) þ (4 o C) 21, 8 w.» w p š, y. š x 1. S. Aldington and J. Bonnerjea, Scale-up of monoclonal antibody purification processes. J. Chrom B, 848, (2007). Analytical Science & Technology
9 Study on stability test of in process sample of recombinant Protein A Guideline on Stability Testing of Biological Product - KFDA(2008). 3. ICH guideline Q5C - Quality of Biotechnological Products: Stability Testing of Biotechnological/Biological Products. 4. ICH guideline Q1A(R2) - Stability Testing of New Drug Substance and Products. 5. ICH guideline Q1E - Evaluation for Stability Data. 6. G. Strohlein, L. Aumann, T. Muller-Spath and M. Morbidelli, The multicolumn counter current solvent gradient purification process., BioPharm Int., (2007). 7. D. Agalloco, Control, et al., Process simulation testing for steril bulk pharmaceutical chemicals, PDA Technical Report No. 28, PDA J. Pharm. Sci. Technol., 52, Supplement S3 (1998). 8. S. R. Gallant, S. Vunnum and S. M. Cramer, Optimization of preparative ion-exchagen chromatography of proteins: linear gradient separations, J. Chromato A, 725, (1996). 9. S. Liu, R. Tobias and G. Jackowski, Removal of Endotoxin from Recombinant Protein Preparations, Clinical Biochem., 30(6), (1997). Vol. 25, No. 6, 2012
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Journal of the Korean Ceramic Society Vol. 45, No. 10, pp. 625~630, 2008. Effect of Storage Conditions on the Setting Properties of Brushite Bone Cement Containing Granular β-tricalcium Phosphate Sun-Ae
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w y wz 8«( 3y) 137~142, 2005 J. of the Korean Society for Environmental Analysis sw n w Polychlorinated Biphenyls»y Áwx Á yá w w yw Analysis of Polychlorinated Biphenyls from Pohang and Gangseo, Busan
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Journal of the Korea Concrete Institute Vol. 23, No. 3, pp. 393~400, June, 2011 GGGGG DOI 10.4334/JKCI.2011.23.3.393 x RC { sƒ y y 1) *Á½ 1) Á 2) 1) y w œw 2) w œw Bond Strength Evaluation of RC Beams
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w y wz 8«( 2y) 00~00, 2005 J. of the Korean Society for Environmental Analysis Calix[6]arene w k š w *Á x Á Á«ƒm w yw, *w w yw Cesium Ion Selective Solid Contact Electrodes Based on Calix[6]arene Won-sik
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w y wz 8«( 4y) 00~00, 2005 J. of the Korean Society for Environmental Analysis HPLC f y e t p»wá½»x w y w»y Detection Characteristics of a HPLC Method on Carbonyl-DNPH Derivatives in Relation with Column
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J. Korean. Soc. Living. Environ. Sys. Vol. 14, No. 4, pp 343~350(2007) w y y w z 14 «4 y 2007 yw» l s p»xá ³ *w w w œw, **w w w The Property of Pressure Distribution of Hybrid Underfloor Air Distribution
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w wz 8«3y 2008 6 pp. 51 ~ 58 m qp yp š w k sƒ Evaluation of Dynamic Modulus based on Aged Asphalt Binder y*á **Á***Á**** Lee, Kwan-HoÁCho, Kyung-RaeÁLee, Byung-SikÁSong, Yong-Seon Abstract Development
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ª Œª Œ 31ƒ 3B Á 2011 5œ pp. 265 ~ 276 ª w w s³ The Correlation Between the Moving Average of Precipitation and Groundwater Level in Korea Á½û» Yang, Jeong-SeokÁKim, Nam-Ki Abstract Precipitation data and
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w m y Vol. 16(4), p. 53~61, 2011 œ 광물찌꺼기에함유된오염물질의안정화를위한표면차폐재의성능평가 ½ ³ 1 Á 1 *Á½ 2 Á½ 2 Á 3 Á 3 1 w œ w œw, 2 ( ) f lp, 3 w Ÿw œ Ÿw» Evaluation of Surface Water-preventing Materials on Stabilization of
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1) KIGAS Vol. 16, No. 5, pp 21~27, 2012 (Journal of the Korean Institute of Gas) http://dx.doi.org/10.7842/kigas.2012.16.5.21 실험실의 사례 분석에 관한 연구 이근원 이정석 한국산업안전보건공단 산업안전보건연구원 (2012년 9월 5일 투고, 2012년 10월 19일
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J. of the Korean Sensors Society Vol. 18, No. 2 (2009) pp. 168 172 p k ù p p l xá xá ³ Á *Á w * Fabrication of the CNT-FET biosensors with a double-gate structure Byunghyun Cho, Byounghyun Lim, Jang-Kyoo
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Investigation of the Korean Traditional Hobun Manufacturing Technique NATIONAL RESEARCH INSTITUTE OF CULTURAL HERITAGE 2008 Investigation of the Korean Traditional Hobun Manufacturing Technique - Centering
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y, 38«, 6y, 699-705, 2005 Econ. Environ. Geol., 38(6), 699-705, 2005 t sÿ y xk 1 Á s 2Á z 1 Á½ 1 Á e 3 1 w y w,» w, 2w y w, 3š w y w Chemical Speciation of Heavy Metals in Geologic Environments on the
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Printed in the Republic of Korea "/"-:5*$"- 4$*&/$& 5&$)/0-0(: Vol. 26, No. 1, 80-85, 2013 http://dx.doi.org/10.5806/ast.2013.26.1.080 Study on the analytical method using GC-MS for the accident preparedness
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Journal of the Korean Ceramic Society Vol. 48, No. 1, pp. 63~68, 2011. DOI:10.4191/KCERS.2011.48.1.063 Effects of Substituting B 2 O 3 for P 2 O 5 on the Structure and Properties of SnO-P 2 O 5 Glass Systems
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The e-business Studies Volume 17, Number 4, August, 30, 2016:319~332 Received: 2016/07/28, Accepted: 2016/08/28 Revised: 2016/08/27, Published: 2016/08/30 [ABSTRACT] This paper examined what determina
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Journal of the Korean Ceramic Society Vol. 8, No. 1, pp. 0~5, 011. DOI:10.191/KCERS.011.8.1.00 Solidification of Heavy Metal Ions Using Magnesia-phosphate Cement HunG Choi, Hyun Ju Kang, Myung Shin Song,
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Journal of the Korean Ceramic Society Vol. 48, No. 4, pp. 323~327, 2011. DOI:10.4191/KCERS.2011.48.4.323 Electrical Properties and Temperature Stability of Dysprosium and Erbium Co-doped Barium Titanate
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J. Fd Hyg. Safety 21(4), 204 212 (2006) ŸŸ w» ƒ, ƒ, w ½ Á Á½ ¼Á½ Á Á½ Ášy Á» y Quantitative Analysis of Acid Value, Iodine Value and Fatty Acids Content in Sesame Oils by NIRS Jae-Kwan Kim, Myung-Jin Lee,
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Journal of the Korea Concrete Institute Vol. 23, No. 2, pp. 177~184, April, 2011 GGGGG DOI 10.4334/JKCI.2011.23.2.177 x w w MRS w p s y 1) Á z 2) Á x 3) * 1) wû w œw 2) w œw 3) w Nonlinear Analysis for
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w wz 16«2y Kor. J. Clin. Pharm., Vol. 16, No. 2. 2006 ù txƒ š x B Á xk B Á C Á z B B w w w C w Regulatory System of Quasi-drugs in Korea Hyun Soon Sohn a, Hyun Taek Shin a, In Sook Song b, and Hyo Jung
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