Procedure for the chromatographic purification of insulins

Details Procedure for the chromatographic purification of insulins Procedure for the chromatographic purification of insulins

1999-08-24

2004-03-23

Carlson, Karen Cochrane (Department: 1653)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

Insulin; related peptides

C530S303000

Reexamination Certificate

active

06710167

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to an improved procedure for the chromatographic purification of insulins.
BACKGROUND
In addition to enzymatic and/or genetic engineering procedures, the procedures for preparing insulins essentially comprise chromatographic procedures in order to fulfill the extremely high purity demands.
The term “insulins” is understood here as meaning insulins originating from natural sources or recombinant insulins (i.e., expressed by genetically modified microorganisms) of animal or human origin (e.g., porcine insulin, bovine insulin or human insulin), proinsulins (e.g., insulin precursors, preinsulins), or insulin derivatives.
Insulin derivatives are designated below as derivatives of naturally occurring insulins, namely human insulin or animal insulins, which differ by substitution of at least one naturally occurring amino acid and/or addition of at least one amino acid and/or organic residue from the corresponding, otherwise identical naturally occurring insulin.
Human insulin is a polypeptide which is constructed of 51 amino acids. The so-called A (acidic) chain consists of 21 amino acids, and the B (basic) chain consists of 30 amino acids. In both amino acid chains, 6 cysteine residues occur, each two cysteine residues being bonded to one another via a disulfide bridge (the two chains are linked to one another by two cysteine bridges). In biologically active human insulin, the A and B chains are bonded to one another via two cysteine bridges, and a further bridge occurs in the A chain. The following cysteine residues are linked to one another in the (biologically active) human insulin:
A6-A11
A7-B7
A20-B19.
The letters A and B represent the particular insulin amino acid chain and the number represents the position of the amino acid which is counted from the amino to the carboxyl end of the respective amino acid chain.
The preparation of recombinant insulin is customarily carried out in the steps of fermentation and cell disruption, followed by protein chemistry and process technology processes, customarily chromatographic processes, for the purification of the product.
Genetic engineering procedures allow human proinsulin or proinsulin (proinsulin of insulin derivatives) which has an amino acid sequence and/or amino acid chain length differing from human insulin, to be prepared in microorganisms. The proinsulins prepared from genetically modified
Escherichia coli
cells do not have correctly bonded cysteine bridges. A procedure for obtaining human insulin having correctly bonded cysteine bridges using
E. coli
is disclosed, for example, in EP 0 055 945. Improved procedures for the preparation of human insulin and insulin derivatives having correctly bonded cysteine bridges are described in EP 0 600 372 A1 (U.S. Pat. No. 5,473,049) and in EP 0 668 292 A2 (U.S. Pat. No. 5,663,291).
Proinsulin, a precursor of insulin, prepared from genetically modified microorganisms is first isolated from the cells, correctly folded, and then converted enzymatically to human insulin. In addition to undesired by-products, the cleavage mixture obtained in the enzymatic peptidation processes contains both the valuable substance and the undesired insulin-like impurities, which do not markedly differ either in molecular weight or in other physical properties from the valuable product, thereby making separation and purification very difficult, particularly on a large industrial scale.
The process technology processes for purification are a series of various chromatography procedures (e.g., adsorption chromatography, ion-exchange chromatography, reversed phase or reverse-phase high-pressure chromatography or combinations thereof) in some cases in a number of stages using different support materials, in some cases with subsequent crystallization, the actual purification being achieved by chromatography. The removal of the insulin-like impurities in this case takes place on ion exchangers or on reversed phase silica supports.
The end-polishing (removal of very minor impurities, as the last purification stage) is customarily carried out in the high-pressure range using chromatography on reversed phase silica gel (RP-HPLC=reversed phase high-pressure liquid chromatography).
“Reversed phase silica gel” (or reverse-phase, i.e., lipophilically modified, that is hydrophobic) is understood as meaning a silica material to which a hydrophobic matrix has been applied. Examples of a hydrophobic matrix are alkanes having a chain length of 3 to 20 carbon atoms, in particular 4 to 18 carbon atoms. The particle sizes are in the range from 10 to 50 &mgr;m, the pore widths 50 to 300 A.
Examples of chromatography procedures that, according to the prior art, utilize RP-silica gels (lipophilically modified silica gels) are EP 0 547 544 A2 (U.S. Pat. No. 5,621,073) or EP 0 474 213 A1 (U.S. Pat. No. 5,245,008). According to the prior art, the high demands on the purity of the insulins to be prepared can only be fulfilled by the use of reversed phase silica gels. The use of reversed phase silica gel, however, has crucial disadvantages:
Reversed phase silica gels are only stable in the range from pH 2 to pH 10. In the chromatography of fermentation products, high molecular weight by-products are always contained which are persistently adsorbed and cannot be desorbed using the customary elution. These by-products concentrate on the RP silica gel with time (referred to as aging of the adsorbent).
Regeneration or cleaning in place (CIP) is usually carried out only by rinsing with dilute sodium hydroxide solution. Thus, in each CIP process, a part of the RP silica gel is destroyed requiring continuous replacement which is very cost-intensive. The danger of denaturation furthermore exists for insulins on silica gels.
Many attempts have been made to replace RP gels based on silica without complete success. Attempts using RP material based on alumina or titanium dioxide (both materials are not completely pH-stable, but at least more stable than silica gel) have shown that the separation is inadequate and that the required purity cannot be achieved.
A further necessary property of chromatography materials is their pressure stability.“Pressure-stable polymeric chromatography materials” are understood as meaning particles of organic polymers, which can occur in all possible forms, e.g., rod form, fragments, or preferably, spherical form, and preferably have diameters between from about 10 &mgr;m to about 35 &mgr;m, and whose deformation under the action of pressure (up to 70 bar) is only slight. The material located in the chromatography column must be so well packed that no cavities are present (the quality of the packing determines the separation result). For the packing of columns, in principle, two different techniques are known, which can also be used in combination. The first technique is the method of compressing the packing by means of a ram that is usually hydraulically operated (DAC=direct axial compression). The second technique is a method of packing the column hydrodynamically by means of a high-pressure pump, i.e. pressing a suspension of liquid and particles into the column. In both cases, it is necessary for pressures to reach about 70 bar on the cross section of the column in order to avoid cavity formation and to pack the particles as tightly as possible.
Many organic polymer particles are not pressure-stable and deform under high pressure resulting in flat disk spheres that overlap and suppress the flow through the packing. In contrast, reversed phase silica gels are considerably more pressure-stable by nature, and barely deform under the pressures mentioned.
SUMMARY OF THE INVENTION
The object of the present invention is to provide a process for the chromatographic purification of insulins on suitable chromatography materials that are pressure-stable and can be employed in the entire pH range from about 1 to about 14. Due to the high separation efficiency of this purification process only one stage is needed.
The object is achieved by a procedure for the

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