Separation ofpyrogens from proteins
Electrophoresis 1990,11,981-982
J. Lucas M. Faupel C. Goecking Ciba-Geigy Biotechnology and Exploratory Research Services, Base1
98 1
Preparative separation of pyrogens from proteins by isoelectric focusing using a multicompartment electrolyser with Immobiline membranes Due to the nature of lipopolysaccharide endotoxin structures of bacterial pyrogens, their removal from solutions containing therapeutic proteins is often a problem in the pharmaceutical industry. In this report we describe the application of electromotive force to dislodge lipopolysaccharide endotoxins from proteins. This was performed by employing a multicompartment electrolyzer fitted with Immobiline membranes of specified PIS. A thousand-fold reduction of endotoxin could be achieved in the model test system described. This contribution describes the use of a new recycling isoelectric focusing approach without the use of carrier ampholytes.
The term pyrogen refers to agents which induce fever. The most common pyrogenic contaminants of parenteral drugs and medical devices are cell wall components shed from gramnegative bacteria. Potential sources of endotoxins may be solvents such as water, chemicals and starting materials, as well as glassware and instruments used in the manufacturing process 111. In the case of proteins made by rDNA technology, the major source can be from the bacteria expressing the protein products. In addition to their properties of inducing pyrogenic and shock reactions, endotoxins can also cause other diverse and profound effects on the vascular, nervous and immune systems [ 2 ] .Another detrimental property of endotoxins may be their known adjuvant effect which could potentially intensify immune responses against therapeutic drugs. The endotoxin limit set by F D A guidelines for most pharmaceutical products is for a single dose 0.5 ng endotoxin per kilogram body weight or 25 ng endotoxinldose for a 50 kg adult [ l l . Due to their size and charge heterogeneity, separation of endotoxins from proteins in solution can often be troublesome. Endotoxin inactivation by chemical methods are unsuitable because they are stable under extremes oftemperature and pH which would destroy proteins [31. Furthermore, due to their amphipathic nature, endotoxins tend to adhere to proteins in a fashion similar to detergents. In such cases, endotoxin activity often coelutes with the protein when chromatographic procedures such as ion exchange chromatography or gel filtration are employed. Additional procedures for endotoxin reduction include ultrafiltration for separating endotoxin from low molecular weight drugs and peptides smaller that 10 kDa. However, larger proteins cannot be treated by this procedure due to micellar endotoxin forms, ranging in size from 20 kDa up to 1000 kDa [31. For proteins, polymixin B immobilized onto Sepharose beads is frequently used to reduce endotoxin [41. However, in the majority of cases, these supports are unsatisfactory in reducing endotoxin beneath FDA limits. Another approach is detergent treatment to displace endotoxins from proteins, either in the presence or absence of polymixin B Sepharose, but here the problems arise in the subsequent removal of the detergents from the protein products. Two reports describe the removal of pyrogens from protein solutions by means of recycling isoelectric focusing in presence of carrier ampholytes [S,61.
Recently, a device has been described for the preparative fractionation of proteins by isoelectric focusing using a multicompartment electrolyzer fitted with Immobiline membranes of predetermined pls [7, 81. Basically, a single unit chamber of the device contains two Immobiline gels of differing pls, cast onto glass-fiber filters, a flow chamber, and adjacent chambers for anode and cathode electrolytes. The cathodic membrane of higher PIand the anodic membrane of lower p l are clamped onto each end of the flow chamber. Upon application of current, only molecules having a PImore acidic than the acidic membrane or more basic than the basic Immobiline membrane pls can traverse the membranes, due to their retained charge, and thus leave the sample chamber. Samples with PIS lying between thepls ofthe membranes remain within the flow chamber. Here we have evaluated the electrolyzer for its ability to remove the negatively charged endotoxins from proteins. Before use, the apparatus was depyrogenated by circulating 0.1 N NaOH through the sample and electrolyte chambers overnight, without the membranes installed. Thereafter the device was flushed with pyrogen-free water, the membranes installed, and run at constant 500 V, under circulation of pyrogen-free water with a peristaltic pump for 4 h. After this treatment the water in the sample chamber had an endotoxin level of 60 pg/mL as determined by the limulus amoebocyte lysate (LAL) [9, 101. For the test, an 8 mg/mL solution of
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Correspondence: Dr. J. Lucas, Genetics Institute, One Burtt Rd., Andover, MA 01810, USA 0VCH Verlagsgesellschaft mbH, D-6940Weinheim, 1990
Figure 1. Endotoxin concentration of the myoglobin solution in the sample flow chamber during the course ofthe run. Current was applied at time =0, as indicated by the arrow. 0173-0835/90/1111-0981 %3.50+.25/0
982
K. Otsuka and Sh. Terabe
Electrophoresis 1990,11, 982-984
myoglobin (Serva, Heidelberg, FRG), p17.3, was dissolved in pyrogen-free water. This solution was found to have an endotoxin level of 6000 pg/mL (determined by LAL). The solution was then continuously circulated between the pH 6.98 and pH 8.04 membranes. Anolyte and catholyte solutions on theoppositesideofthemembranewere 1 mMHEPES,pH 5.1, and 1 mM NaOH, pH 10, respectively. As can be seen from Fig. 1 endotoxin levels remained unchanged at 6000 pg/mL for the first 4 h before current application (t = -4 to 0). However, upon application of current at a constant voltage of 500 V (time =0), the level of endotoxin decreased within 3 h to 6 pg/mL and remained at this level afterwards, even after being run overnight. When the electrolytes were tested for endotoxin activity, the anolyte contained 600 pg/mL, whereas the catholyte
Electrophoresis 1990,11,981-982
J. Lucas M. Faupel C. Goecking Ciba-Geigy Biotechnology and Exploratory Research Services, Base1
98 1
Preparative separation of pyrogens from proteins by isoelectric focusing using a multicompartment electrolyser with Immobiline membranes Due to the nature of lipopolysaccharide endotoxin structures of bacterial pyrogens, their removal from solutions containing therapeutic proteins is often a problem in the pharmaceutical industry. In this report we describe the application of electromotive force to dislodge lipopolysaccharide endotoxins from proteins. This was performed by employing a multicompartment electrolyzer fitted with Immobiline membranes of specified PIS. A thousand-fold reduction of endotoxin could be achieved in the model test system described. This contribution describes the use of a new recycling isoelectric focusing approach without the use of carrier ampholytes.
The term pyrogen refers to agents which induce fever. The most common pyrogenic contaminants of parenteral drugs and medical devices are cell wall components shed from gramnegative bacteria. Potential sources of endotoxins may be solvents such as water, chemicals and starting materials, as well as glassware and instruments used in the manufacturing process 111. In the case of proteins made by rDNA technology, the major source can be from the bacteria expressing the protein products. In addition to their properties of inducing pyrogenic and shock reactions, endotoxins can also cause other diverse and profound effects on the vascular, nervous and immune systems [ 2 ] .Another detrimental property of endotoxins may be their known adjuvant effect which could potentially intensify immune responses against therapeutic drugs. The endotoxin limit set by F D A guidelines for most pharmaceutical products is for a single dose 0.5 ng endotoxin per kilogram body weight or 25 ng endotoxinldose for a 50 kg adult [ l l . Due to their size and charge heterogeneity, separation of endotoxins from proteins in solution can often be troublesome. Endotoxin inactivation by chemical methods are unsuitable because they are stable under extremes oftemperature and pH which would destroy proteins [31. Furthermore, due to their amphipathic nature, endotoxins tend to adhere to proteins in a fashion similar to detergents. In such cases, endotoxin activity often coelutes with the protein when chromatographic procedures such as ion exchange chromatography or gel filtration are employed. Additional procedures for endotoxin reduction include ultrafiltration for separating endotoxin from low molecular weight drugs and peptides smaller that 10 kDa. However, larger proteins cannot be treated by this procedure due to micellar endotoxin forms, ranging in size from 20 kDa up to 1000 kDa [31. For proteins, polymixin B immobilized onto Sepharose beads is frequently used to reduce endotoxin [41. However, in the majority of cases, these supports are unsatisfactory in reducing endotoxin beneath FDA limits. Another approach is detergent treatment to displace endotoxins from proteins, either in the presence or absence of polymixin B Sepharose, but here the problems arise in the subsequent removal of the detergents from the protein products. Two reports describe the removal of pyrogens from protein solutions by means of recycling isoelectric focusing in presence of carrier ampholytes [S,61.
Recently, a device has been described for the preparative fractionation of proteins by isoelectric focusing using a multicompartment electrolyzer fitted with Immobiline membranes of predetermined pls [7, 81. Basically, a single unit chamber of the device contains two Immobiline gels of differing pls, cast onto glass-fiber filters, a flow chamber, and adjacent chambers for anode and cathode electrolytes. The cathodic membrane of higher PIand the anodic membrane of lower p l are clamped onto each end of the flow chamber. Upon application of current, only molecules having a PImore acidic than the acidic membrane or more basic than the basic Immobiline membrane pls can traverse the membranes, due to their retained charge, and thus leave the sample chamber. Samples with PIS lying between thepls ofthe membranes remain within the flow chamber. Here we have evaluated the electrolyzer for its ability to remove the negatively charged endotoxins from proteins. Before use, the apparatus was depyrogenated by circulating 0.1 N NaOH through the sample and electrolyte chambers overnight, without the membranes installed. Thereafter the device was flushed with pyrogen-free water, the membranes installed, and run at constant 500 V, under circulation of pyrogen-free water with a peristaltic pump for 4 h. After this treatment the water in the sample chamber had an endotoxin level of 60 pg/mL as determined by the limulus amoebocyte lysate (LAL) [9, 101. For the test, an 8 mg/mL solution of
1
-4
0
1
2
3
14
TIME (hours) -
Correspondence: Dr. J. Lucas, Genetics Institute, One Burtt Rd., Andover, MA 01810, USA 0VCH Verlagsgesellschaft mbH, D-6940Weinheim, 1990
Figure 1. Endotoxin concentration of the myoglobin solution in the sample flow chamber during the course ofthe run. Current was applied at time =0, as indicated by the arrow. 0173-0835/90/1111-0981 %3.50+.25/0
982
K. Otsuka and Sh. Terabe
Electrophoresis 1990,11, 982-984
myoglobin (Serva, Heidelberg, FRG), p17.3, was dissolved in pyrogen-free water. This solution was found to have an endotoxin level of 6000 pg/mL (determined by LAL). The solution was then continuously circulated between the pH 6.98 and pH 8.04 membranes. Anolyte and catholyte solutions on theoppositesideofthemembranewere 1 mMHEPES,pH 5.1, and 1 mM NaOH, pH 10, respectively. As can be seen from Fig. 1 endotoxin levels remained unchanged at 6000 pg/mL for the first 4 h before current application (t = -4 to 0). However, upon application of current at a constant voltage of 500 V (time =0), the level of endotoxin decreased within 3 h to 6 pg/mL and remained at this level afterwards, even after being run overnight. When the electrolytes were tested for endotoxin activity, the anolyte contained 600 pg/mL, whereas the catholyte
