content_image/942633.jpg content_image/942634.jpg content_image/942604.jpg content_image/942631.jpg content_image/942632.jpg
# c12) United States Patent Goklen et al. 

# (54) BUFFER SYSTEM FOR PROTEIN PURIFICATION 

(71) Applicant: GlaxoSmithKline LLC, Wilmington, DE (US) 

(72) Inventors: Kent E. Golden, King of Prussia, PA (US); Eric J. Suda, Phoenixville, PA (US); Antonio Raul Ubiera, King of Prussia, PA (US) 

(73) Assignee: GlaxoSmithKline LLC, Wilmington, DE (US) 

( *) Notice: Subject to any disclaimer, the term ofthis patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days. This patent is subject to a terminal dis› claimer. 

(21) Appl. No.: 15/450,106 

(22) Filed: Mar. 6, 2017 

## (65) Prior Publication Data 

US 2017/0174721 Al Jun. 22, 2017 

(63) Continuation of application No. 14/007,689, filed as application No. PCT/US2012/031076 on Mar. 29, 2012, now Pat. No. 9,624,261. 

(60) Provisional application No. 61/468,814, filed on Mar. 29, 2011. 

(51) Int. Cl. 

<img src='content_image/942633.jpg'>

(52) U.S. Cl. CPC ............ C07K 1122 (2013.01); BOID 1511871 (2013.01); BOID 151362 (2013.01); BOID 151363 (2013.01); BOID 1513809 (2013.01); BOID 1513847 (2013.01); BOID 151426 (2013.01); C07K 1136 (2013.01) 

(58) Field of Classification Search CPC ........ C07K 1/22; C07K 1/36; BOID 15/1871; BOID 15/362; BOID 15/363; BOID 15/3809; BOID 15/3847; BOID 15/426 

See application file for complete search history. 

## (56) References Cited 

<img src='content_image/942634.jpg'>

## Related U.S. Application Data 

## U.S. PATENT DOCUMENTS 

<img src='content_image/942604.jpg'>

# (IO) Patent No.: US 9,868,761 B2 

# (45) Date of Patent: *Jan. 16, 2018 

<img src='content_image/942631.jpg'>

<img src='content_image/942632.jpg'>

Bjorck and Kronvall, J. Innnunol., 133:969 (1984)). 

Bjorck, J. Innnunol., 140:1194 (1988). 

Butler, J. N., Ionic Equilibrium: Solubility and pH Calculations. John Wiley and Sons (1998). 

Forsgren and Sjoquist, J. Innnunol., 97:822 1966. 

Ghose, S.; McNerney, T. M. Hubbard, B. pH transitions in ion› exchange systems: Role in the development of a cation exchange process for a recombinant protein. Biotechnol. Prog. 2002, 18, 530-537. 

Mixed-mode Chromatography Selection Guide. http://www.pall. corn/main/laboratory /Ii terature-library-details. page? id =l 7 50 2 May 30, 2012. 

Ngo, et al., Kosmotropes Enhance the Yield of Antibody Purified by Affinity Chromatography using Innnobilized Bacterial Innnuno› globulin Binding Proteins, Journal oflnnnunoassay&Im› munochemistry, 29: 105-115,2008. 

Pabst, T.M., Carta, G. pH transitions in cation exchange chromato› graphic colunms containing weak acid groups. (2007) Journal of Chromatography A, 1142, pp. 19-31. 

Soto Perez, J. and Frey, D. D. Behavior of the Inadvertent pH Transient Formed by a Salt Gradient in the Ion-Exchange Chroma› tography of Proteins, Biotechnol. Prog. 2005, 21, 902-910. 

Yu, et al., Purification of hCG Monoclonal Antibodies by rProtein A Affinity Chromatography, Chinese Journal of Pharmaceuticals 2010, 41(8). 

Zhou, J. X., et al. pH-conductivity hybrid gradient cation-ex› change chromatography for process-scale monoclonal antibody purification. Journal of Chromatography A, 1175 (2007) 69-80. 

* cited by examiner 

Primary Examiner - Gary Counts 

(74) Attorney, Agent, or Firm - Elizabeth J. Hecht; Edward R. Gimmi 

## (57) ABSTRACT 

A method for purifying a protein using a simplified, sodium chloride-free buffer system that consists of two components (acid and base pairs) for appropriate solution pH control; and a third component for ionic strength control, where the third component is the sodium salt conjugate base. 

US009868761B2 

## FOREIGN PATENT DOCUMENTS 

## OTHER PUBLICATIONS 

## 18 Claims, 6 Drawing Sheets 

