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Protein Instability Following Transport on Dry Ice Implications for Basic and Applied Research Mark Fitchmun Founding Partner and Senior Advisor Somatek Inc. bioprocess development and outsource management for 25 years Slide 2 1)A -80C chest freezer containing critical samples and reagents lost power. 2)Blocks of dry ice were placed in the freezer to prevent the reagents and samples from thawing. 3)In following days, problems arose with a previously routine sandwich ELISA. 4)Subsequent investigation revealed antibodies formulated in DPBS, pH 7.2 are now at pH 5.9. Initial events and observations which lead to this study Somatek Inc. bioprocess development and outsource management for 25 years Slide 3 Carbonic acid formation resulting from exposure to CO 2 damaged one or both antibodies used in the assay. Hypothesis Regarding Assay Failure 1)Both antibodies were sealed in cryogenic storage vials, and presumably were not exposed to CO 2 2)Carbonate and bicarbonate formation require liquid water. 1)Both antibodies were purified using protein-A, and neither appeared to be acid labile. Conflicts with the Hypothesis Somatek Inc. bioprocess development and outsource management for 25 years Slide 4 at -78.5C ? Are common laboratory container closure systems gas tight Both antibodies were sealed in cryogenic storage vials, and presumably were not exposed to CO 2 1) Somatek Inc. bioprocess development and outsource management for 25 years Slide 5 Tris buffered neutral red solution (pH 8.1) was added to container/closure systems. After 48 hours, samples were thawed and pH determined. Testing Container/Closures Against CO 2 at -78.5C - 8.0 - 7.4 - 6.8 pH color scale Somatek Inc. bioprocess development and outsource management for 25 years Slide 6 Testing Container/Closures Against CO 2 at -78.5C Slide 7 Does CO 2 react with frozen samples? Carbonate and bicarbonate formation require liquid water. 2) Somatek Inc. bioprocess development and outsource management for 25 years Slide 8 -80C Dry Ice Thaw Time (minutes) 0 10 20 30 50 pH color indicator - 8.0 - 7.4 - 6.8 Tris/NaCl Tris/Trehalose Interaction Between Sample and Headspace CO 2 Click images for video Slide 9 CO 2 (g) Headspace gasses equilibrate with local environment. Below - 40C CO 2 Enters the Sample in Distinct Phases Loss of seal integrity Below - 40C (reversible) But frozen samples are relatively inert. Somatek Inc. Slide 10 CO 2 (g) Headspace gasses equilibrate with local environment. Below - 40C Above - 20C CO 2 Enters the Sample in Distinct Phases Loss of seal integrity CO 3 = CO 2 (g) H 2 CO 3 CO 2 (l) CO 2 gas dissolves HCO 3 - Below - 40C (reversible)Above - 20C (non-reversible) ionizes hydrates But frozen samples are relatively inert. Slide 11 8 - 0.1 1 10 100 1,000 10,000 Headspace CO 2 Concentration (micrograms per cubic centimeter) Equilibrium pH at 25C 7 - 6 - Calculated pH projections for 150 L samples buffered with 10 mM Tris and sealed in a 1.65 mL microcentrifuge tube 4 - 5 - atmospheric CO 2 at 25C 1% CO 2 at -78.5C 30 x atmospheric CO 2 100% CO 2 at -78.5C 150 L Tris, pH 8.0 150 L Tris, pH 7.3 950 L Tris, pH 7.3 150 L unbuffered - 8 - 7 - 6 - 4 - 5 Slide 12 By what mechanisms can modest changes in pH damage proteins? 1)Disruption of tertiary or quaternary structure 2)Proteolysis (autolytic or from sample impurities) 3)Interfacial damage (aggregation or precipitation) 3) Both antibodies were purified using protein-A, and neither appeared to be acid labile. Somatek Inc. bioprocess development and outsource management for 25 years Slide 13 Native state monomeric protein Native state small soluble aggregates Precipitated protein Denatured small soluble aggregates ( < 70 nm) Denatured large soluble aggregate ( > 70 nm) Aggregation May or May Not Lead to Precipitation Slide 14 In vitro: reduced specific activity altered availability of reactive sites receptor cross linking In vivo: altered biodistribution reduce half-life increase immunogenicity formation of multivalent epitopes toxicity (not seen with monomeric protein) Known Issues with Soluble Aggregates commonly understood digests and conjugations signal transduction drug candidate screening FDA and EMEA guidance documents Aggregates Can Have Their Own Biology In vitro: reduced specific activity altered availability of reactive sites receptor cross linking In vivo: altered biodistribution reduce half-life increase immunogenicity formation of multivalent epitopes toxicity (not seen with monomeric protein) Somatek Inc. bioprocess development and outsource management for 25 years Slide 15 Factors That Drive Aggregation, Precipitation, and Interfacial Damage Factor Effect concentration ionic strength buffer pH temperature freeze/thaw shear air interface plastic interface Near its isoelectric point, a proteins sensitivity to all factors is greatly increased. Therefore small changes in pH can attenuate or amplify the effects of multiple factors. Somatek Inc. bioprocess development and outsource management for 25 years Slide 16 2 hr 5 hr 24 hr rested 0 8 12 16 4 Aggregation Index 7.3 Control 6.85.86.34.85.3 pH of test solution -lactoglobulin (pI = 5.2) 40 mg/mL stock diluted to 2 mg/mL at indicated pH Proteins Tend to Aggregate or Precipitate Near Their Isoelectric Points Slide 17 2 hr 5 hr 24 hr rested 0 8 12 16 4 Aggregation Index 7.3 Control 6.85.86.34.85.3 pH of test solution -lactoglobulin (pI = 5.2) 40 mg/mL stock diluted to 2 mg/mL at indicated pH vortexed Proteins are Often Sensitive to Stress Near Their Isoelectric Points Slide 18 0.1 pH units 0.1% of genome 7.4 - 8 - 7 - 6 - 4 - 5 pH 5.0 - 7.0 Slide 19 histones pepsin -lactoglobulin lysozyme carbonic anhydrase antibodies 0.1 pH units 0.1% of genome Slide 20 Dry ice causes a drop in pH of 1.8 0.1 units. But the original pH can be recovered if the sample is not thawed. Protein Aggregation/Precipitation as a Function of Storage and Shipping Conditions Somatek Inc. bioprocess development and outsource management for 25 years Slide 21 Aggregation corresponds to a drop in pH. And can be prevented if sample is not thawed. Protein Aggregation/Precipitation as a Function of Storage and Shipping Conditions Somatek Inc. bioprocess development and outsource management for 25 years Slide 22 Similar behavior for other acidic proteins and buffer systems. Protein Aggregation/Precipitation as a Function of Storage and Shipping Conditions Somatek Inc. bioprocess development and outsource management for 25 years Slide 23 Dry ice is less likely to cause aggregation of basic proteins. Protein Aggregation/Precipitation as a Function of Storage and Shipping Conditions Somatek Inc. bioprocess development and outsource management for 25 years Slide 24 Dry ice is less likely to cause basic proteins to aggregate. Dry ice is more likely to cause aggregation of acidic proteins. Approximately 55% of eukaryotic and 60% prokaryotic proteins have acidic isoelectric points. Protein Aggregation/Precipitation as a Function of Storage and Shipping Conditions Somatek Inc. bioprocess development and outsource management for 25 years Slide 25 Summary and Conclusions: 1)Common laboratory container/closure systems do not provide a reliable barrier to CO 2 at -78.5C. 2)Subsequent carbonic acid formation can cause a fall in pH by as much as 2.5 pH units. 3)Proteins with acidic isoelectric points are most susceptible to aggregation and interfacial stress. Damage to proteins placed into dry ice shippers may be overlooked, thereby compromising interpretations of experimental results. Some Out-Of-Box-Failures of protein based products may be a direct result of transport on dry ice. Somatek Inc. bioprocess development and outsource management for 25 years Slide 26 Preliminary Recommendations to Researchers Do 1)Place samples and reagents into a -80C freezer for several days after being exposed to dry ice. Do Not 1)Thaw samples or reagents shortly after exposure to dry ice. 2) Move samples from dry ice to a -20C freezer. 3) Place dry ice into any freezer. Somatek Inc. bioprocess development and outsource management for 25 years Slide 27 Parameters That Obscure Cause and Effect Relationships Samples moved to a -80C freezer for a few days are inadvertently rescued. Container volume influences acidification as well as interfacial stress. pH is almost never measured in samples less than 500 L (those most effected). Aggregation is almost never measured. PERIOD. Strong buffers can stabilize pH but tend to increase salt induced aggregation. The effects of aggregation can be indistinguishable from other factors such as impurities, freeze/thaw damage, improper folding, etc. Its not possible to detect aberrant activity, when a proteins real activity is the subject of investigation. ~ 45% of proteins have a pI > 7.4, and are unlikely to be effected. Lacking an a priori suspicion of the phenomenon described here, its effects may appear random, be unattributable to their cause, or go unnoticed, compromising interpretations of experimental results. Somatek Inc. bioprocess development and outsource management for 25 years Slide 28 Preliminary Observations and Ideas for Next Steps Secondary barriers that did not reliably protect samples from CO 2 1)Parafilm 1)Polyethylene zip-seal bags 1)Laminated aluminized Mylar zip-seal bags Secondary barriers that did reliably protect samples from CO 2 1)Laminated aluminized Mylar heat-seal bags. Small vessels containing moist litmus paper or equivalent could accompany actual samples to confirm bag integrity during shipping. Somatek Inc. bioprocess development and outsource management for 25 years Slide 29 Brian M. MurphyLegacy BioDesign, Johnstown, CO Mark C. ManningUniversity of Colorado, Denver Barbara M. MuellerTorrey Pines Institute for Molecular Studies Spencer SwartsSan Diego State University Peter van der GeerSan Diego State University Contributors: Somatek Inc. bioprocess development and outsource management for 25 years Slide 30 Mark Fitchmun Founding Partner & Senior Advisor Somatek Inc. 3937 Southview Drive San Diego CA 92117 phone: 858-449-1310 email: fitchmun@somatek.comfitchmun@somatek.com Somatek Inc. bioprocess development and outsource management for 25 years