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Human albumin : Production processes and safety

This chapter gives an overview of current production methods of human albumin , and the safety of modern albumin preparations
Connie Broumis Global Pathogen Safety CSL Behring , Australia
Wilfried Freudenberg Director Manufacturing , CSL Behring
Philipp Schumann Process Engineering , CSL Behring AG , Bern
Human albumin has been used successfully in volume replacement and many other indications for over 60 years . It is used in larger quantities than any other biopharmaceutical solution and the annual consumption of albumin is still increasing . Many studies of human albumin were conducted from the 1970s to the 1990s when impurities in earlier preparations of albumin and plasma protein fraction were associated with side effects . However , manufacturing processes have been upgraded considerably since then and are continually evolving . Modern albumin products of greater purity have demonstrated excellent long-term safety profiles .
Manufacturing goals The main goal for manufacturers is to produce therapeutic human albumin preparations with high purity that contain albumin that is as close to the native plasma protein as possible . Therapeutic albumin should be monomeric and free from aggregates , which may influence tolerability . Tolerability is also affected by the presence of additional plasma proteins , which might be modified upon pasteurisation ( heat treatment at 60 ° C for 10 hours ) and potentially cause adverse reactions .
Experience with older generation albumin products suggests that high levels of prekallikrein activator ( PKA ), which can cause hypotension , and high levels of endotoxins , which are implicated in febrile reactions should be avoided . Levels of metal ions , in particular aluminium , should be kept very low to avoid accumulation in patients with impaired renal function , or in neonates .
Current manufacturing processes Albumin is prepared from pooled human plasma , which contains around 60g / l of total protein . Albumin is present at concentrations around 40g / l and is the most abundant protein present in plasma . Compared with other major plasma proteins , albumin has a very high solubility and low isoelectric point ( that is , the pH at which a molecule carries no net charge ). In addition , 17 disulphide bonds along the albumin polypeptide chain confer structural stability , so that under conditions where other proteins may be denatured , albumin remains in the native form . These unique properties allow albumin to be purified using relatively simple , but effective , fractionation methods .
Plasma proteins are separated or fractionated by changing the pH , temperature and / or ionic strength of the plasma intermediates , so that normally soluble proteins precipitate and can be removed by centrifugation or filtration .
An effective way to precipitate proteins is to add ethanol to the plasma pool , while simultaneously cooling ( cold ethanol fractionation ), and this process was defined by Edwin Cohn in 1946 , specifically for the purification of albumin . 1 Modifications of this basic cold ethanol fractionation process include the stepwise increase in ethanol concentration from 0 % to approximately 40 %, and lowering the pH from neutral to around pH 4.8 , which is approximately equal to the isoelectric point of albumin . At each stage , precipitated proteins such as immunoglobulins and α1-proteinase inhibitor are recovered by centrifugation or filtration .
The majority of manufacturers continue to base their processes for albumin production on the original Cohn method , or a modified version of the Cohn method – the Cohn-Oncley process – which involves a gradient of up to 43 % ethanol .
An alternative but equally effective method of ethanol fractionation is based on the process defined by Kistler and Nitschmann in 1962 . 2 This process uses fewer precipitation steps , and precipitated proteins are removed by filtration or centrifugation .
Manufacturing process and safety of current albumin products For some processes , the first step is removal of the cryoprecipitate fraction containing coagulation factors . The resulting cryo-poor plasma may be treated with chromatography resins to adsorb Factor IX , Factor X , Prothrombin Complex Concentrate and C1-Inhibitor . For the Cohn or modified Cohn methods , this is followed by a precipitation step with 8 % ethanol , whereby the precipitate ( Fraction I ) contains Factor XIII , which can be adsorbed from the supernatant by dedicated resins .
A further increase of ethanol precipitates immunoglobulins ( Fraction II and III or precipitate A ); the Cohn process uses around 25 % ethanol at pH 6.9 and the Kistler and Nitschmann process uses 19 % ethanol at around pH 5.9 . Albumin remains in solution and addition of ethanol to approximately 40 % results in the precipitation of further proteins including α1-proteinase inhibitor and transferrin ( Fraction IV ). Finally , albumin is precipitated at a pH near to its isoelectric point , often by a further slight increase in the ethanol concentration . All ethanol precipitation steps are performed at defined
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