Cohn process

Work with Proteins and Development of Fractionation

Cohn was summoned to do work on bovine plasma as a source of protein for treating shock patients. Cohn had previously worked with colleagues using an ammonium sulfate solution to separate normal horse serum into successive fractions. It was the first study where pH and temperature were controlled as variables in the procedure. However, ammonium sulfate did not discriminate well between proteins and was toxic. Therefore, it was not useful within the human body. This led to a change from looking at horse albumin to bovine albumin. In addition, Cohn also switched from ammonium sulfate to ethanol as the protein precipitant. This led to the Cohn Process. Cohn chose ethanol as the protein precipitant for several reasons. First, from previous research, Cohn observed the effects of electrostatic forces on the solubility of peptides being greater when the dielectric constant of the medium was decreased by the addition of ethanol. He tested the effects of sodium chloride on the solubility of egg albumin in ethanol. Egg albumin is a sensitive protein to denaturation, but tolerated the exposure to ethanol at low temperatures. Second, it is non-toxic. Third, it is volatile and thus easily removable from proteins in drying from the frozen state. There was one disadvantage in that the adding alcohol to an aqueous solution of protein; heat is generated. But Cohn made certain that the temperature conditions were closely monitored. Cohn eventually published his paper titled, “Preparation and Properties of Serum and Plasma Proteins. III. Size and Charge of Proteins Separating upon Equilibration across Membranes with Ethanol-Water Mixtures of Controlled pH, Ionic Strength, and Temperature.” The fractionation procedure involved stepwise adjustment of pH, ionic strength, ethanol concentration, and temperature of bovine plasma in four stages, where a protein precipitate, or fraction, is being formed at each stage. There were five in total with the first four removed using centrifugation at each stage. This left a final supernatant solution from the last protein fraction, V, obtained by drying. Fraction V included most of the bovine albumin originally present in the plasma. The directions for the fractionation were said in his paper. It stated that two liters of bovine plasma yields about fifty grams of albumin.

Human Albumin

Cohn realized that the possibility of using human albumin was not out of question. Human blood could be used as a therapeutic agent for military shock, which was important in the time of the Second World War. From then onward, Cohn looked at both the development of bovine albumin and human albumin. Cohn had laboratories in which human albumin was being fractionated from blood using the Cohn Process creating 100 grams of the human albumin a week. These two forms of albumin proved to become important sources to expedite the recovery of soldiers in the Second World War through administration for any wounds and shock.

Albumin was an excellent substitute for human plasma. When administered to wounded soldiers or other patients with blood loss, it helped expand the volume of blood and led to speedier recovery. In addition, Cohn's method was gentle enough so the protein retained its biological activity. Recognizing the importance of this discovery, the government immediately stepped in and asked the major pharmaceutical companies of the time to commercialize Cohn's fractionation process.

The Details of the Process

How does the process work? The goal of the process is to extract and recover albumin from blood plasma. The process is based on the differential solubility of albumin and other plasma proteins based on pH, ethanol concentration, temperature, ionic strength, and protein concentration as mentioned earlier. Albumin has the highest solubility and lowest electric point of all the major plasma proteins. This makes it the final production to be precipitated. The conditions of ethanol concentration change from zero initially to 40%. The pH decreases from neutral at 7 to more acidic at 4.8 over the course of the fractionation. The temperature starts at room temperature and decreases to -5 degrees C. Initially, the blood is frozen. There are five major fractions. Fractions I, II, and III are precipitated out at earlier stages. The conditions of the earlier stages are 8% ethanol, pH 7.2, -3 degrees C, and 5.1% protein for Fraction I; 25% ethanol, pH of 6.9, -5 degrees C, and 3% protein. The albumin remains in the supernatant fraction during the solid/liquid extraction under these conditions. Fraction IV has several unwanted proteins that need to be removed. In order to do this, the conditions are varied in order to precipitate the proteins out. The conditions to precipitate these proteins are raising the ethanol concentration from 18 to 40% and raising the pH from 5.2 to 5.8. Finally, albumin is located in fraction V. The precipitation of albumin is done by reducing the pH to 4.8, which is near the pI of the protein, and maintaining the ethanol concentration to be 40%, with a protein concentration of 1%. Thus, only 1% of the original plasma remains in the fifth fraction. However, at each process stage, albumin is lost, roughly 20% of the albumin is lost through precipitation stages before fraction V. In order to purify the albumin, there is an extraction with water, and adjustment to 10% ethanol, pH of 4.5 at -3 degrees C. Any precipitate formed here is done so by filtration and is an impurity. These precipitates are discarded. Reprecipitation, which improves purity, is done so, by raising ethanol concentration back to 40% from the extraction stage. The pH is 5.2 and it is conducted at -5 degrees C. Several variations of Cohn fraction were created to account for lower cost and higher yield. Generally, if the yield is high, the purity is lowered, to roughly 85-90%.

The Use of Other Fractions

In order to utilize this process, Cohn was able to start the Plasma Fractionation Laboratory after he was given massive funding from the government agencies and the private pharmaceutical companies. This led to the fractionation of human plasma. Human plasma proved to have several useful components other than albumin. Human blood plasma fractionation yielded Serum Albumin, Serum Gamma Globulins, Fibrin Foam and Thrombin, Blood Group Globulins, and a Fibrin Film. Gamma Globulins are found in Fractions II and III and proved to be essential in treating measles for soldiers. Gamma globulin also was useful in treatment for polio, but did not have much effect in treating mumps or scarlet fever. Most importantly, the gamma globulins were useful in modifying and preventing infectious hepatitis during the Second World War. It eventually became a treatment for children exposed to this type of hepatitis. Fibrin Foam and thrombin were found to be useful in controlling blood vessels from oozing especially near tumors. It also minimized bleeding from large veins as well as dealing with blood vessel malformations within the brain. However, it was not useful in controlling arterial bleeding.

Variations to the Process

The Cohn Fractionation method was also introduced to several other groups interested in separations of proteins within blood. Several groups made their own modifications to the Cohn Process. The modifications were done in order to lower cost and improve yield as well as purity. The Gerlough method, developed in 1955 used variations to improve process economics by reduced consumption of ethanol. Instead of 40% in certain steps, Gerlough used 20% ethanol for precipitation. This is especially used for Fractions II and III. In addition, Gerlough combined the two fractions of IV into one step to reduce the number of fractionations required. While this method proved to be more economically friendly, it was not adopted by industry because of this combination of fractions II, III, and IV, for fear of mixing and high impurities. Another method was the Hink method developed in 1957. This method enabled higher yields to be obtained through recovery of some of the plasma proteins discarded in the Fractions of IV. The improved yields, however, compensated for lower purities that were within the 85% range. Mulford came up with a similar method where fractions II and III supernatant was used as the last step before finishing and heat treatment. The method essentially combined fractions IV and V, but in this case, the albumin would not be as pure even though the yields may be higher. Another variation was developed by Kistler and Nitschmann. The goal of this method was to develop a purer form of albumin. However, the higher purity is offset by lower yields. Similar to Gerlough, the Precipitate A, which is equivalent to Cohn’s Fraction II and III, was done at a lower ethanol concentration of 19%, but the pH, in this case, was also lower to 5.85. Also similar to Gerlough and Mulford, the fraction IV was combined and precipitated at conditions of 40% ethanol, pH of 5.85, and temperature of -8 degrees C. The albumin, which is recovered in fraction V, is recovered in Precipitate C at a pH adjustment to 4.8. Similar to the Cohn Process, the albumin is purified by extraction into water followed by precipitation of the impurities at 10% ethanol, pH 4.6, and -3 degrees C. Akin to the Cohn Process, the precipitate formed here is filtered out and discarded. Then Precipitate C (fraction V) is reprecipitated at pH 5.2 and stored ad a paste at -40 degrees C. This process has been more widely accepted because it separates the fractions and makes each stage independent of each other. Another variation involved a heat ethanol fractionation. It was originally developed to inactivate the hepatitis virus. In this process, recovery of high yield, high purity albumin is the most important goal, while the other plasma proteins are neglected. In order to make sure the albumin does not denature in the heat, there are stabilizers such as sodium octanoate, which allow the albumin to tolerate higher temperatures for long periods. In heat ethanol, the plasma is heat treated at 68 degrees C with sodium octanoate with 9% ethanol at pH of 6.5. This results in improved albumin recovery with yields of 90%, and purities of 100%. It is not nearly as expensive as cold ethanol procedures such as the Cohn Process. One drawback is the presences of new antigens due to possible heat denaturation of the albumin. In addition, the other plasma proteins have practical uses and to neglect them would not be worth it. Finally, the expensive heat treatment vessels offset the lower cost compared to the cold ethanol format that do not need it. For these reasons, several companies haven not adopted this method even though it has the most impressive results. However, one prominent organization that uses it is the German Red Cross. The latest variation was developed by Hao in 1979. This method is significantly simplified compared to the Cohn Process. Its goal is to create high albumin yields as long as albumin is the sole product. Through a two stage process, impurities are precipitated directly from fractions II and III supernatant at 42% ethanol, pH 5.8, temperature -5 degrees C, 1.2% protein, and 0.09 ionic strength. Fraction V is precipitated at pH 4.8. Fractions I, II, III, and IV are coprecipitated at 40% ethanol, with pH of 5.4 to 7.0, and temperature -3 to -7 degrees C. Fraction V is then precipitated at pH 4.8 and -10 degrees C. The high yields are due to a combination of a simplified process, with lower losses due to coprecipitation, and use of filtration. Higher purities were also achieved at 98% because of the higher ethanol levels, but the yields were lowered with the high purity.

Consequences and Influences of Cohn Process

The Cohn Process was a major development in the field of blood fractionation. As mentioned before, it has several practical uses in treating diseases such as hepatitis and polio. It was most useful during the Second World War where soldiers recovered at a faster rate because of the transfusions with albumin. The Cohn Process has been modified over the years as seen above. In addition, it has influenced other processes with the blood fractionation industry. This has led to new forms of fractionation such as chromatographic plasma fractionation in ion exchange and albumin finishing processes. In general, the Cohn Process and its variations have given a huge boost to and serve as a foundation for the fractionation industry to this day. However, the process has not been studied well because it is archaic. Most importantly, it has never been modernized by manufacturing companies. In addition, the conventional process can be environmentally unfriendly because ethanol is a highly explosive substance. It is unsanitary because of the open vessels and tanks, thus the possibility of contamination is high. Finally, the cold ethanol format may be too gentle to kill off certain viruses that require heat inactivation. Since is process remains unchanged for so long several built in inefficiencies and inconsistencies affect the economics of the process for pharmaceutical and manufacturing companies. Nevertheless, this process still serves as a major foundation for the blood industry in general and its influence can be seen at it is referred to in the development of newer methods. Although it has its drawbacks depending on the variation, the Cohn Process’ main pro is its practical uses and its endless possibilities within pharmacological and medical industries.