USOO8609416B2 (12) United States Patent (10) Patent No.: US 8,609,416 B2 Barnett (45) Date of Patent: Dec. 17, 2013 (54) METHODS AND COMPOSITIONS OTHER PUBLICATIONS COMPRISING HEAT SHOCKPROTEINS Novoselova et al., “Treatment with extracellular HSP70/HSC70 pro (75) Inventor: Michael E. Barnett, Manhattan, KS tein can reduce polyglutamine toxicity and aggregation.” J (US) Neurochem 94:597-606, 2005.* Johnson et al., (1993) Exogenous HSP70 becomes cell associated but (73) Assignee: Ventria Bioscience, Fort Collins, CO not internalized, by stressed arterial Smooth muscle cell. In vitro (US) Cellular and Developmental Biology—Animal, vol. 29A. No. 10, pp. 807-821. (*) Notice: Subject to any disclaimer, the term of this Bethke et al., (2002) Different efficiency of heat shock proteins patent is extended or adjusted under 35 (HSP) to activate human monocytes and dendritic cells; Superiority of U.S.C. 154(b) by 244 days. HSP60, The Journal of Immunology, vol. 169, pp. 6141-6148. Khan et al., (2008) Toll-like receptor 4-mediated growth of (21) Appl. No.: 12/972,112 endometriosis by human heat-shock protein 70, Human Reproduc tion, vol. 23, No. 10, pp. 2210-2219. (22) Filed: Dec. 17, 2010 Lasunskaia E.B., et al., (2003) Transfection of NSO myeloma fusion partner cells with HSP70 gene results in higher hybridoma yield by (65) Prior Publication Data improving cellular resistance to apoptosis, Biotechnology and Bioengineering 81 (4):496-504. US 2011 FO189751A1 Aug. 4, 2011 * cited by examiner Related U.S. Application Data (60) Provisional application No. 61/288,234, filed on Dec. Primary Examiner — Rosanne Kosson 18, 2009. (74) Attorney, Agent, or Firm — Dennis A. Bennett (51) Int. Cl. CI2N 5/02 (2006.01) (57) ABSTRACT (52) U.S. Cl. USPC ........................................... 435/405:435/.407 Compositions, and uses thereof, which are beneficial for (58) Field of Classification Search eukaryotic cells in culture, and methods for their use in pro USPC .................................................. 435/405, 407 moting cell growth, viability and recombinant protein expres See application file for complete search history. Sion. The methods disclosed in the present application are useful, for example, for improving cell viability and in accel (56) References Cited erating the rate of cell growth of cells grown in culture. In one aspect, the Supplements of the invention are useful for U.S. PATENT DOCUMENTS improving or enhancing the yield of the recombinant proteins 2004/OO72347 A1 4/2004 Schuler et al. from the cell cultures. 2010, OO15713 A1 1/2010 Deeter 2012,0315697 A1 12/2012 Pettit et al. 2013. O157356 A1 6, 2013 Barnett 25 Claims, 15 Drawing Sheets Seracare + Hsp70 and Cellastim neg control Sera + Hsp70 Cellastim 1.0 ug/ml 5.0 ug/ml 10.0 ug/ml conc. of Hsp70 U.S. Patent Dec. 17, 2013 Sheet 1 of 15 US 8,609,416 B2 Figure 1 Sigma Albumin 1600 14OO 2OO OOO 8OO 6OO 2 41 61 81 01 121 141 16 81 201 22 Mw (kDa) Celastin PO107 101 126 151 Mw (kDa) Celastin PO71 as -"T"- 13 25 37 49 61 73 85 97 09 12 133 45 157 169 181 1932O5 2.17 229 Mw (kDa) U.S. Patent Dec. 17, 2013 Sheet 2 of 15 US 8,609,416 B2 Figure 1 continued Cellastim P0171 (solid line) and Sigma Albumin (dotted line) 13 25 37 49 61 73 85 97 109 121 133 145 157 169 18, 1932O5 217 229 Mw (kDa) P0171 (solid line) and PO107 (dotted line) 1500 OOO 500 3 25 37 49 61 73 85 97 O9 12 133 45 157 69 18, 1932O5 2.17 229 Mw (kDa) U.S. Patent Dec. 17, 2013 Sheet 3 of 15 US 8,609,416 B2 Igure 1 continue fee.8 28:8-8-2ss 3-4-3 is : sess st . rts ---------- 5. i.3 s 33 ir U.S. Patent Dec. 17, 2013 Sheet 4 of 15 US 8,609,416 B2 Figure 2 2 3 4 S 6 7 8 9 0. 1 2 3 4 5 6 7 S 9 () U.S. Patent Dec. 17, 2013 Sheet 5 Of 15 US 8,609,416 B2 Figure 3 Cell Prime, Seracare, and Cellastim Albumin in cell Culture neg Cont Cell Prime SeraCare Cellastim 0.5 mg/ml 1.0 mg/ml 5 mg/ml 10 mg/ml album in Concentration Endotoxin levels in old (B000) vs. new (B0000C) process U.S. Patent Dec. 17, 2013 Sheet 6 of 15 US 8,609,416 B2 Figure 3 continued Cell Viability from old (B000) and new (B0000C) process 5 10 - S.S. S.So S s°ss Soss So deSS NS.S. S Se. Sg. SSC S CSS & 5 g g g g g g (5 & &SSSSSSSSSS) (S&S (S S S S S S &S U.S. Patent Dec. 17, 2013 Sheet 7 Of 15 US 8,609,416 B2 Figure 4 A LANE: 2 3 4. 7 8 9 () () 75 MW S) (kDa)kD 25 2) S B Hsp- Hisp-2 1) SDBE 2) SDBE 3) Hsp70/HSA 4) HSA 5) HSA 6) Hsp70 7) C.-amylase 8) 01-amylase U.S. Patent Dec. 17, 2013 Sheet 8 of 15 US 8,609,416 B2 Figure 4 continued Enzymatic activity of ATP-agarose purified Hsp70 from Cellastim has 4. & ox o o o ovu r c D N C O has C ce e d E dialysate 1 dialysate 2 sample 1 sample 2 U.S. Patent Dec. 17, 2013 Sheet 9 Of 15 US 8,609,416 B2 Figure 5 Hsp70 and Seracare Albumin in Cell Culture neg cont Hsp70 Seracare Hsp70 + Seracare S& 0.5ug/ml 1 ug/ml 5ug/ml 10 ug/ml X1000 for Sera care U.S. Patent Dec. 17, 2013 Sheet 10 of 15 US 8,609,416 B2 Figure 6: Seracare vs. Hsp70 1600 -- 1400 1200 - 1000 -a-Seracare 8OO --Hsp70 600 -- HSp 400 - 200 O Atom-A-T-A-I-A 0.5 1 5 10 mg/ml product added U.S. Patent Dec. 17, 2013 Sheet 11 of 15 US 8,609,416 B2 Figure 7 Seracare + Hsp70 and Cellastim neg control a Sera + Hsp70 Cellastim 1.0 ug/ml 5.0 Ug/ml 10.0 ug/ml conc. of Hsp70 U.S. Patent Dec. 17, 2013 Sheet 12 of 15 US 8,609,416 B2 Figure 8 A Removal of ATP binding Proteins from Cellastim gd neg control Starting Part A G 0.5 mg/ml 1.0 mg/ml 5.0 mg/ml 10.0 mg/ml Album in Concentration Rescue and recovery of lost performance E dr s CD o ce d s > Control (n=4) A (n=4) A+ Hsp70 (n=5) Sample Description U.S. Patent Dec. 17, 2013 Sheet 13 of 15 US 8,609,416 B2 Figure 9 Lipid Comparison Cellastim vs. Plasma vs. Yeast 1.8 1.6 1.4 1.2 s O.8 O.2 Cellastim HSA(n=3) Il pHSA (n=1) Yeast HSA(n=2) Lipid Comparison Cellastim vs. Plasma vs. Yeast O.9 0.8 O.7 O6 O.5 s O.4 O.3 O.2 O.1 O Cellastim pHSA (n=1) Yeast HSA(n=2) U.S. Patent Dec. 17, 2013 Sheet 14 of 15 US 8,609,416 B2 Figure 9 continued Effect of adding exogenous Lipids 25 Celastim (n=3) O Defatted Celastim (n=3) Defatted Celastim + Linoleic acid Novazyme + Linoleic acid U.S. Patent Dec. 17, 2013 Sheet 15 Of 15 US 8,609,416 B2 Figure 10 Differential Scanning Calorimetry d w 7 O 9. Cellastim e s p-HSA Oy-HSA s E l R 73 oO Transition Temperature US 8,609,416 B2 1. 2 METHODS AND COMPOSITIONS fatty acids. Bovine serum albumin (BSA) has long been used COMPRISING HEAT SHOCKPROTEINS as a Supplement in cell culture media as it is a component of fetal bovine serum (FBS) which is commonly added to a basal CROSS-REFERENCE TO RELATED media at 1-20% total volume. BSA is a major component in a APPLICATIONS number of defined serum free media formulations since it is readily available in bulk, is relatively cheap, and can be puri This application claims the benefit of U.S. provisional fied to homogeneity relatively easily. Representative sources patent application No. 61/288.234 filed on Dec. 18, 2009, the of albumin include for example, plasma derived from bovine, entire contents of which are incorporated herein by reference. horse, pig and other mammalian species. 10 With the advent of the large scale production of recombi STATEMENT REGARDING FEDERALLY nant proteins, vaccines and other products destined for human SPONSORED RESEARCH ORDEVELOPMENT clinical use, stricter requirements on the formulations used in the production of those products have been instigated. Not Applicable. Because of the threat of animal-derived materials harboring 15 pathogens that may affect the safety of the products, many TECHNICAL FIELD existing recombinant production processes have been modi fied such that all materials or culture components used in the The invention relates to compositions, and uses thereof, entire process are devoid of animal-derived products. That is, which are beneficial for eukaryotic cells in culture, and meth the cell culture components cannot have been isolated or ods for their use in promoting cell growth, viability and purified from whole animal sources. Therefore, the recombi recombinant protein expression. nant production of media Supplements, as an alternative to the purification of these supplements directly from the whole BACKGROUND animal is preferred. Accordingly, human and cell culture components from other species can be manufactured using Investigation of biological processes often requires the 25 recombinant means, using defined tissue culture media, and examination of those processes in cells, tissues and organs using highly characterized tissue culture cells, which are cer that comprise less than the entire organism.