Novel insulin peptides

Insulin analogs characterized by amino acid residue at A21 other than Asn, with a resulting improvement in stability of insulin solutions at acid pH levels. Some insulin analogs may also have amino acid residue changes elsewhere so that these insulin analogs exhibit at least about one charge more than human insulin at a pH value of 7, with preferred substitutions being made for the glutamic acid residues at A4, A17, B13, B21 and/or a basic amino acid residue being substituted at B27. Also contemplated is optional blocking the C-terminal carboxyl group of the B-chain with an amido or ester residue.

BACKGROUND OF THIS INVENTION 
The present invention relates to novel, stabilized insulin analogs and, in 
a preferred embodiment, to novel injectable solutions having prolonged 
insulin action. 
In the treatment of diabetes mellitus, many varieties of insulin 
preparations have been suggested and used. Some of the preparations are 
fast acting and other preparations have more or less prolonged actions. 
Such a prolonged action may be obtained by administering the insulin as a 
suspension of insulin crystals. The crystalline preparations can be 
obtained by crystallization of insulin in the presence of zinc (such as 
LENTE.TM., see Schlichtkrull: Insulin Crystals, Chemical and Biological 
Studies on Insulin Crystals and Insulin Zinc Suspensions, Munksgaard, 
1958) or by crystallization of insulin in the presence of zinc and 
protamine (such as NPH-insulin, see Rep.Steno Mem.Hosp. 1 (1946), 60). 
Acid solution of insulin have been used earlier, both as short-acting 
preparations and as long-acting preparations containing protamine and 
zinc. However, the chemical stability of insulin at pH-values below 4.5 is 
low, as formation of desamidoinsulins (Sundby, F., J.Biol.Chem. 237 
(1962), 3406-3411) and covalent dimers (Steiner et al, Diabetes 17 (1968), 
725-736) takes place. In the pH-range 4.5-6.5, insulin precipitates. 
Hence, in order to make soluble short-acting insulin preparations 
(addition of blood-flow enhancing agent) and long-acting insulin 
preparations (addition of protamine and/or zinc) an insulin stable at low 
pH would be desirable. 
It is known that during the acid ethanol extraction of mammalian insulins 
many dimers are formed (Steiner) and, furthermore, monodesamidoinsulins 
are formed under acid conditions (Sundby). 
One disadvantage in the use of the known suspensions of zinc insulin 
crystals or of zinc protamine insulin is the necessity of shaking the vial 
in order to ensure that the correct amount of insulin is being injected 
and to ensure that the concentration of insulin in the vial remains 
constant throughout its use. In PENEILL .TM. cartridges where air must be 
absent, prolonged acting insulin suspensions require the incorporation of 
a solid body in the cartridge to enable agitation. The shaking of insulin 
suspensions and insulin solutions with air is in itself an undesirable 
process, as insulin has a tendency to denature under formation of fibrills 
at water-air interfaces. Consequently, solutions of insulins with 
prolonged action are desirable. 
Solutions of insulin derivatives having a prolonged action were obtained 
from insulin that had been modified in its amino groups by reaction with 
phenylisocyanate (so-called Isoinsulin, see Hallas-Moeller: Chemical and 
Biological Insulin Studies based upon the Reaction between Insulin and 
Phenylisocyanate, Copenhagen 1945). Similarly, Al,B29-di-Boc substituted 
insulin (Boc designates tertiary butyloxycarbonyl) was reported to show a 
prolonged insulin action after subcutaneous administration (see Geiger & 
Enzmann in: Proinsulin, Insulin, C-peptide; Proceedings of the Symposium 
on Proinsulin, Insulin and C-Peptide, Tokushima 1978; Amsterdam Oxford 
1979, 306-310). The Al,B29-di-Boc substituted insulin was found to exhibit 
a too slightly prolonged action to be clinically useful. 
Solutions of unmodified insulins require large amounts of zinc ions (for 
example, 0.4-1 mg/U insulin) in order to exhibit a prolonged action (see 
J.Pharmacol. 55 (1935), 206). Injection of such large doses of zinc ions 
will probably cause pain and such solutions have, therefore, never been 
used in therapy. 
The isoelectric point of insulin is about 5.5 and attempts have been made 
to decrease the solubility of insulin derivatives at neutral pH by 
shifting the isoelectric point upwards, for example, through additions, in 
the N-terminus of the B-chain, of basic amino acids like lysine or 
arginine (see, for example, German Offenlegungsschrift No. 2,042,299) or 
with the basic dipeptide arginyl-arginine (see Geiger & Enzmann cited 
above). However, near its isoelectric point the solubility of 
Arg.sup.B(-1) -Arg.sup.BO insulin was much higher than that of the parent 
insulin. 
Japanese patent application No. 55-144032 relates to analogues to human 
insulin wherein the B30-amino acid has been replaced by an amino acid 
having at least five carbon atoms, and amides and esters thereof. These 
insulin analogues were to be used in patients who had developed antibodies 
against mammalian insulins. In the Japanese patent application, six 
specific compounds are described, none of which were stated to have 
prolonged action. No specific injectable preparations are described in the 
Japanese patent application. 
European patent application No. 84108442 relates to insulin analogues 
wherein a basic, organic group is attached to the B30-amino acid thereby 
introducing a positive charge at neutral pH. In these analogues, the 
B30-amino acid is neutral and, preferably, threonine as in human insulin. 
German patent application No. 3,327,709 relates to a suspension of 
crystals of the derivatives described in the above-noted European patent 
application as well as an aromatic hydroxy compound. German patent 
application No. 3,326,473 relates to a medicament containing a mixture of 
insulin compounds, of which at least one is described in the above-noted 
European patent application. 
One object of this invention is to prepare insulin derivatives with 
improved properties. 
A second object of this invention is to prepare insulin solutions having an 
improved stability. 
A third object of this invention is to prepare insulin preparations with no 
or low immunogenic action. 
A fourth object of this invention is to prepare insulin analogs which are 
dissolved at pH values below about 5.8. 
The present invention arose within the context of prolonged action insulin 
described above but is not limited thereto. 
Acid insulin solutions, a form that many years ago represented the only 
insulin form employed in diabetes therapy, are relatively unstable (with 
substantial deamidation at A-21 taking place). Substituting a more stable 
amino acid residue for Asn.sup.A21 improves the stability of the insulin 
molecule at pH levels lower than its isoelectric point. Solutions of the 
A-21 substituted insulin analogs of this invention are characterized by 
improved stability at acid pH levels. 
BRIEF STATEMENT OF THIS INVENTION 
The present invention comprises novel analogs of insulin that differ from 
human insulin in that the C-terminal asparagine residue of the A-chain, 
Asn.sup.A21, is substituted by any other naturally occurring amino acid, 
which can be coded for by nucleotide sequences, or by homoserine. Also 
optionally, but preferably: 
(a) an amide or ester residue on the C-terminal carboxyl group of the 
B-chain is present, and 
(b) the insulin analog has at least one charge more than human insulin at a 
pH value at 7, preferably not more than 4 charges more than human insulin 
at a pH value of 7. 
The optional increase in charge is achieved by appropriate substitution of 
a more basic amino acid residue for one or more of the amino acid residues 
in human insulin and, if desired, by the blocking of the carboxylic group 
in the B30 amino acid. 
Thus, the insulin analogs of this invention comprise insulin analogs 
characterized by an A-21 residue which preferably is selected from the 
group consisting of Glu, Asp, Lys, Arg, His, Val, Gln, Ile, Phe, Tyr, Met, 
Gly, Ser, Thr, Ala, Leu, Trp and hSer. 
One preferred set of A21 substituents are the acidic residues of Glu and 
Asp, the latter being the more preferred. 
A second set of preferred A21 substituents are the basic residues of Lys, 
Arg and His, histidine being the more preferred. 
A third set of preferred A21 substituents are the neutral residues of 
valine, glutamine, isoleucine, phenylalanine, tyrosine or methionine and 
more preferably glycine, serine, threonine, alanine or homoserine. 
In addition, preferred insulin analogs of interest to practice of this 
invention are characterizable as follows: One or more of the four glutamic 
acid residues at A4, A17, B13 and B21 are replaced by another naturally 
occurring neutral amino acid residue, preferably a glutamine residue; 
and/or the threonine residue at B27 is instead a naturally occurring basic 
amino acid residue, preferably an L-arginine or L-lysine residue; and/or 
the threonine residue at B30 is replaced by one or two basic amino acid 
residues, one being preferred; and/or the C-terminal carboxylic group in 
the B chain may be protected. For example, desirably by --NH.sub.2. 
This invention also comprises solutions of the insulin analogs, optionally 
containing a controlled level of zinc ions therein within a concentration 
of 5 .mu.g to 200 .mu.g per ml. The degree of prolongation of insulin 
action is enhanced and controlled by the addition of zinc ions.

DETAILED PRACTICE OF THIS INVENTION 
It has surprisingly been found that injectable solutions with improved 
stability, most of which also have a surprisingly combined short and 
prolonged insulin action, can be made using, as the active ingredient, a 
single insulin derivative having the general formula I 
##STR1## 
wherein the letters A and B followed by figures in parentheses designate 
the peptide fragments of the A- and B-chains, respectively, indicated by 
the figures in parentheses, E.sup.1, E.sup.2, E.sup.3 and E.sup.4 are the 
same or different each representing a glutamic acid residue or a neutral 
amino acid residue which can be coded for by nucleotide sequences, X 
represents an L-threonine, L-arginine or L-lysine residue, Y and Z are the 
same or different and each represent an amino acid residue wherein any 
side chain amino group may be acylated and wherein any side chain hydroxy 
group may be alkylated, m, n and p are the same or different and each 
represent zero or one, R represents an amido or ester residue which blocks 
the C-terminal carboxyl group of the B-chain, and W represents an amino 
acid residue other than asparagine, with the proviso that if all four 
amino acid residues E.sup.1, E.sup.2, E.sup.3 and E.sup.4 are glutamic 
acid residues, X is Thr, and --Y.sub.m --Z.sub.n --R.sup.p is --Ala, then 
W is different from aspartic acid. 
Furthermore, it is surprising that the compounds of this invention have a 
low formation of dimers. Mammalian insulin contains Asn in the A21 
position. Therefore, it is surprising that the compounds of this invention 
have a satisfactory insulin activity. 
Preferably, substitution is made at one or more of the 7 amino acid 
residues E.sup.1, E.sup.2, E.sup.3, E.sup.4, X, Y and Z and the group R 
causing the compound of formula I to have at least one charge more than 
human insulin at a pH value of 7. 
The novel insulin analogs have the further advantages: 
(1) The formation of the immunogen dimer, i.e. covalently linked insulin 
molecules linked either through the two A-chains, (AA) dimer, or through 
one A-chain and one B-chain, (AB) dimer, (Helbig, H. J., Deutsche 
Wollforschungsinstitut, dissertation, 1976), is substantially decreased. A 
chromatographic fraction of crude porcine insulin, the b-component, 
containing the dimers was shown to be immunogenic in rabbits 
(Schlichtkrull et al., Horm.Metab.Res. Suppl. 5 (1974) 134-143). 
(2) The stability of the novel insulin derivatives is so high that it will 
probably be acceptable to store preparations containing these novel 
insulin derivatives at room temperature for a long period of time. This 
will be a major advantage for the patient. 
(3) It will be possible to prepare dissolved preparations containing the 
novel insulin derivatives at pH values from about 2 to about 5.8. 
(4) It will be possible to prepare preparations containing the novel 
insulin derivatives which, at pH values of about 3, have a substantially 
improved chemical stability. 
(5) It will be possible to prepare soluble, rapidly acting preparations 
containing the novel insulin derivatives by the addition of compounds 
which enhance the absorption. 
(6) It will be possible to prepare soluble, retarded preparations 
containing the novel insulin derivatives by the addition of zinc and/or 
protamine to acid solutions, i.e. solutions having a pH value in the range 
from about 2.5 to about 5.8. 
(7) It will be possible to prepare preparations containing the novel 
insulin derivatives having different absorption profiles. 
A subgroup of compounds of formula I is novel compounds having the general 
formula I wherein R represents an amido residue. 
If, compared with human insulin at a pH value of 7, a change in charge is 
desired in the insulin analogs of this invention, it is obtained by 
substituting the threonine residue in the B27-position with an arginine or 
lysine residue and/or by substituting one or more of the four glutamic 
acid residues in the A4-, A17-, B13-, and B21-position with a neutral 
amino acid residue, preferably with a glutamine residue. In addition, the 
C-terminal carboxyl group of the B-chain may be blocked by an ester group 
or amide group, thereby eliminating the negative charge of this B-30 
carboxyl group. Furthermore, a positive charge may be introduced by 
presence of a basic amino acid residue in the B30- and/or B31-position. 
Since the preferred compounds of formula I can be applied in the clinic as 
solutions having a prolonged action, a decline in immunogenicity as 
compared to the commonly used suspensions of porcine or human insulins may 
occur. 
The degree of prolongation depends on the concentration of zinc ions in the 
preparation. 
Major parameters that control the degree of prolongation of the insulin 
effect are the concentration of zinc and the choice of the compound of 
formula I. The range for the preferred zinc content extends from 0 to 
about 2 mg/ml, preferably from 0 to 200 .mu.g/ml zinc with substitution in 
the B13 and/or B27 position and from about 20 to 200 .mu.g/ml with other 
analogs in a preparation containing 240 nmole of a compound of formula I 
per ml. Using other concentrations of the compound of formula I, the 
content of zinc is to be adjusted correspondingly. 
The prolonged action of solutions of compounds of formula I in the presence 
of zinc ions is ascribed to the low solubility of such compounds at 
neutral pH. 
The pH of the injectable solution of this invention should preferably be 
below the physiological pH, the upper limit being the pH where 
precipitation occurs. At the physiological pH value, compounds of formula 
I of this invention have a low solubility. Stable solutions containing 
about 240 nmole/ml of compounds of formula I have been obtained at pH 
about 5.5. The upper limit depends upon the constituents of the solution, 
i.e. isotonic agent, preservative and zinc concentration, and upon the 
choice of compound of formula I. There is no lower pH limit of the 
solutions and the chemical stability of the compounds of formula I is 
high, even at pH 3. The preferred pH range for the injectable solutions of 
this invention is from about 2.5 to 5.8, more preferred being about 2.8 to 
4.5. 
A further aspect of this invention is that it provides improved flexibility 
for the patients. With two aqueous solutions, one containing a compound of 
formula I and the other containing a zinc salt, the patient can obtain a 
desired degree of prolonged action and a desired profile by mixing the two 
solutions appropriately. Thus, the patient has, using two stock solutions, 
the possibility of choosing one action and profile for the morning 
injection and another action and profile for the evening injection. 
Preferably, the zinc solution contains between about 2 .mu.g and 20 mg 
zinc per ml. Alternatively, both of the stock solutions may contain zinc, 
either in the same or different concentrations, and/or both the stock 
solutions may contain a compound of formula I, either the same or 
different compounds. 
Preferably, the injectable solutions of this invention have a strength of 
between about 60 and 6000 nmole of the compound of formula I per ml. 
It has already been pointed out that W may be a neutral L-amino acid, for 
example valine, glutamine, isoleucine, leucine, phenylalanine, tyrosine, 
methionine or preferably glycine, serine, threonine, alanine or 
homoserine. W may be an acidic amino acid, viz. glutamic acid or 
preferably aspartic acid, or a basic amino acid, viz. lysine, arginine of 
preferably histidine. 
The neutral amino acid (E.sup.1 through E.sup.4) is, for example, glycine, 
valine, isoleucine, leucine, phenylalanine, tyrosine, methionine or 
preferably asparagine, glutamine, alanine, serine or threonine. 
Examples of R are ester moieties, for example, lower alkoxy, preferably 
methoxy, ethoxy and most preferred tertiary butoxy. 
Furthermore, R can be a group of the general formula --NR.sup.1 R.sup.2 
wherein R.sup.1 and R.sup.2 are the same or different and each represents 
hydrogen or lower alkyl. Hereinafter the term "lower" designates that the 
group in question contains less than 7 carbon atoms, preferably less than 
5 carbon atoms. In a preferred embodiment of this invention, R is 
--NH.sub.2. Furthermore, R may be a lactam residue which preferably 
contains less than 8 atoms in the lactam ring, for example a lactam of a 
diaminocarboxylic acid. 
In a preferred embodiment of this invention, R is uncharged. 
According to one preferred embodiment of this invention, the amino acid 
residues designated Y and Z are residues from L-amino acids which are 
coded for by nucleotide sequences. 
Any side chain amino group in the amino acid residues designated Y and Z 
may be acylated by an acid containing from 2 to 18 carbon atoms, 
preferably a fatty acid containing from 6 to 18 carbon atoms, for example, 
lauric acid. Thus, --Y.sub.m --Z.sub.n --R.sub.p may be 
--Lys(Lau)-NH.sub.2. 
Examples of preferred alkylated hydroxy groups are methoxy, ethoxy and 
tertiary butoxy. 
In one group of preferred compounds of formula I Y and/or Z is a basic 
amino acid residue wherein the side chain amino group optionally is 
acylated (m=1). 
In another group of preferred compounds of formula I n is zero and Y is a 
basic amino acid residue (m=1). 
In a further group of preferred compounds of formula I Y and Z are both 
basic amino acid residues (m=1, n=1). 
Another preferred embodiment of this invention is preparations containing a 
compound of formula I wherein El, E2, E.sup.3 and/or E.sup.4 is a 
glutamine residue, and/or X is Lys or Arg, and W is Gly, Ser, Thr, Ala, 
His, Asp or hSer, and within this subclass of compounds of formula I, a 
further preferred embodiment is preparations containing a compound of 
formula I wherein the group --Y.sub.m --Z.sub.n --R.sub.p --Thr--NH or 
--Lys--NH.sub.2. 
Specific preferred compounds of formula I are each of the following: 
Gly.sup.A21,Lys.sup.B27,Thr.sup.B30 --NH.sub.2 human insulin, 
Ser.sup.A21,Lys.sup.B27,Thr.sup.B30 --NH.sub.2 human insulin, 
Thr.sup.A21,Lys.sup.B27,Thr.sup.B30 --NH.sub.2 human insulin, 
Ala.sup.A21,Lys.sup.B27,Thr.sup.B30 --NH.sub.2 human insulin, 
His.sup.A21,Lys.sup.B27,Thr.sup.B30 --NH.sub.2 human insulin, 
Asp.sup.A21,Lys.sup.B27,Thr.sup.B30 --NH.sub.2 human insulin, 
Gly.sup.A21,Arg.sup.B27,Thr.sup.B30 --NH.sub.2 human insulin, 
Ser.sup.A21,Arg.sup.B27,Thr.sup.B30 --NH.sub.2 human insulin, 
Thr.sup.A21,Arg.sup.B27,Thr.sup.B30 --NH.sub.2 human insulin, 
Ala.sup.A21,Arg.sup.B27,Thr.sup.B30 --NH.sub.2 human insulin, 
His.sup.A21,Arg.sup.B27,Thr.sup.B30 --NH.sub.2 human insulin, 
Asp.sup.A21,Arg.sup.B27,Thr.sup.B30 --NH.sub.2 human insulin, 
Gln.sup.B13,Gly.sup.A21,Arg.sup.B27,Thr.sup.B30 --NH.sub.2 human insulin, 
Gln.sup.B13,Ser.sup.A21,Arg.sup.B27,Thr.sup.B30 --NH.sub.2 human insulin, 
Gln.sup.B13,Thr.sup.A21,Arg.sup.B27,Thr.sup.B30 --NH.sub.2 human insulin, 
Gln.sup.B13,Ala.sup.A21,Arg.sup.B27,Thr.sup.B30 --NH.sub.2 human insulin, 
Gln.sup.B13,His.sup.A21,Arg.sup.B27,Thr.sup.B30 --NH.sub.2 human insulin, 
Gln.sup.B13,Asp.sup.A21,Arg.sup.B27,Thr.sup.B30 --NH.sub.2 human insulin, 
Gln.sup.B13,Gly.sup.A21,Lys.sup.B27,Thr.sup.B30 --NH.sub.2 human insulin, 
Gln.sup.B13,Ser.sup.A21,Lys.sup.B27,Thr.sup.B30 --NH.sub.2 human insulin, 
Gln.sup.B13,Thr.sup.A21,Lys.sup.B27,Thr.sup.B30 --NH.sub.2 human insulin, 
Gln.sup.B13,Ala.sup.A21,Lys.sup.B27,Thr.sup.B30 --NH.sub.2 human insulin, 
Gln.sup.B13,His.sup.A21,Lys.sup.B27,Thr.sup.B30 --NH.sub.2 human insulin 
and 
Gln.sup.B13,Asp.sup.A21,Lys.sup.B27,Thr.sup.B30 --NH.sub.2 human insulin 
Another preferred embodiment of this invention is preparations containing a 
compound of formula I in which m is one, n is zero, Y is Thr, Ala or Ser, 
R is hydroxy, and W is Gly, Ser, Thr, Ala, His or Asp, and examples of 
such compounds are as follows: 
Ser.sup.A21,Lys.sup.B27 human insulin, 
Thr.sup.A21,Lys.sup.B27 human insulin, 
Ala.sup.A21,Lys.sup.B27 human insulin, 
His.sup.A21,Lys.sup.B27 human insulin, 
Asp.sup.A21,Lys.sup.B27 human insulin, 
Gly.sup.A21,Lys.sup.B27 human insulin, 
Ser.sup.A21,Arg.sup.B27 human insulin, 
Thr.sup.A21,Arg.sup.B27 human insulin, 
Ala.sup.A21,Arg.sup.B27 human insulin, 
His.sup.A21,Arg.sup.B27 human insulin, 
Asp.sup.A21,Arg.sup.B27 human insulin, 
Gly.sup.A21,Arg.sup.B27 human insulin, 
Gln.sup.A21,Ser.sup.A21,Arg.sup.B27 human insulin, 
Gln.sup.A21,Thr.sup.A21,Arg.sup.B27 human insulin, 
Gln.sup.A21,Ala.sup.A21,Arg.sup.B27 human insulin, 
Gln.sup.A21,His.sup.A21,Arg.sup.B27 human insulin, 
Gln.sup.A21,Asp.sup.A21,Arg.sup.B27 human insulin, 
Gln.sup.A21,Gly.sup.A21,Arg.sup.B27 human insulin, 
Gln.sup.A21,Ser.sup.A21,Gln.sup.B13 human insulin, 
Gln.sup.A21,Thr.sup.A21,Gln.sup.B13 human insulin, 
Gln.sup.A21,Ala.sup.A21,Gln.sup.B13 human insulin, 
Gln.sup.A21,His.sup.A21,Gln.sup.B13 human insulin, 
Gln.sup.A21,Asp.sup.A21,Gln.sup.B13 human insulin, 
Gln.sup.A21,Gly.sup.A21,Gln.sup.B13 human insulin, 
Arg.sup.B27,Ser.sup.A21,Gln.sup.B13 human insulin, 
Arg.sup.B27,Thr.sup.A21,Gln.sup.B13 human insulin, 
Arg.sup.B27,Ala.sup.A21,Gln.sup.B13 human insulin, 
Arg.sup.B27,His.sup.A21,Gln.sup.B13 human insulin, 
Arg.sup.B27,Asp.sup.A21,Gln.sup.B13 human insulin, 
Arg.sup.B27,Gly.sup.A21,Gln.sup.B13 human insulin, 
Gln.sup.A21,Ser.sup.A21,Lys.sup.B27 human insulin, 
Gln.sup.A21,Thr.sup.A21,Lys.sup.B27 human insulin, 
Gln.sup.A21,Ala.sup.A21,Lys.sup.B27 human insulin, 
Gln.sup.A21,His.sup.A21,Lys.sup.B27 human insulin, 
Gln.sup.A21,Asp.sup.A21,Lys.sup.B27 human insulin, 
Gln.sup.A21,Gly.sup.A21,Lys.sup.B27 human insulin, 
Gln.sup.B13,Ser.sup.A21,Lys.sup.B27 human insulin, 
Gln.sup.B13,Thr.sup.A21,Lys.sup.B27 human insulin, 
Gln.sup.B13,Ala.sup.A21,Lys.sup.B27 human insulin, 
Gln.sup.B13,His.sup.A21,Lys.sup.B27 human insulin, 
Gln.sup.B13,Asp.sup.A21,Lys.sup.B27 human insulin 
Gln.sup.B13,Gly.sup.A21,Lys.sup.B27 human insulin, and 
Ser.sup.A21,Arg.sup.B27,Thr.sup.B30 --NH.sub.2 human insulin. 
Further examples of specific preferred compounds according to this 
invention are the following: GlyA21 human insulin, Ala.sup.A21 human 
insulin, Ser.sup.A21 human insulin, Thr.sup.A21 human insulin, 
hSer.sup.A21 human insulin, Gly.sup.A21 porcine insulin, Ala.sup.A21 
porcine insulin, SerA21 porcine insulin and ThrA21 porcine insulin. 
In one group of preferred compounds of formula I, E.sup.2 and E.sup.3 is a 
glutamine residue. 
In another group of preferred compounds of formula I, X is an arginine or 
lysine residue. 
In a further group of preferred compounds of formula I, W is Gly, Ser, Thr, 
Ala, His, Asp or hSer. 
As is well known in the art, not all of the amino acid residues in human 
insulin are essential for the insulin action. 
Indeed, porcine insulin and bovine insulin which differs from human insulin 
in amino acid residues have been employed to treat diabetics. Considerable 
species to species variations exist in the insulin molecule. Thus, many 
amino acid residues in the human insulin molecule may be changed without 
undue diminution in insulin activity, including some residues influencing 
the isoelectric point of the molecule. 
It is obvious that the groups designated E.sup.1, E.sup.2, E.sup.3, 
E.sup.4, R, W, X, Y and Z are to be selected so that the resulting 
compound of formula I is pharmaceutically acceptable. 
In the known biphasic insulin preparations, it is common to combine fast 
acting, soluble insulin with prolonged acting, crystalline insulin in the 
same injection. Using compounds of formula I of this invention, a similar 
combined short and prolonged action can be obtained with a solution of a 
single compound of formula I. The ratio between short and long acting 
effect decreases as the concentration of zinc ions in the solution is 
increased. 
Compounds of formula I may be prepared by a transpeptidation reaction in 
which a biosynthetic precursor compound having the correct insulin 
disulfide bridges and having the general formula II: 
##STR2## 
wherein Q is a peptide chain with q amino acids, q is an integer from 0 to 
33, T is Lys or Arg, r is zero or one, and A, B, E.sup.1, E.sup.2, 
E.sup.3, E.sup.4, W and X each are as defined above, is reacted with an 
amino compound of the general formula III: 
EQU H--Y.sub.m --Z.sub.n --R.sub.p (III) 
wherein Y, Z, R, m, n and p each are as defined above, and wherein side 
chain amino groups and hydroxy groups in Y and Z optionally are blocked 
with amino and hydroxy protecting groups, using trypsin or a trypsin like 
enzyme as a catalyst in a mixture of water and organic solvents 
analogously as described in U.S. Pat. No. 4,343,898. When W is hSer, 
nucleotides coding for Met are introduced at the A21 site in the gene. In 
the protein expressed conversion of Met into hSer is accomplished by 
cyanogen bromide. Preferred compounds of formula III for use in this 
process are Thr--NH.sub.2 , Lys(Boc)--NH.sub.2 , Thr(Bu.sup.t)--OBu.sup.t, 
Thr--OBu.sup.t, Ala--NH.sub.2 and Arg(Boc)--NH.sub.2 . Amino groups may be 
derivatized by acylation with a fatty acid. Hydroxy groups may be 
protected by alkylation. If Y and Z contain groups which are reversibly 
blocked by amino protecting groups, these groups may be removed at a later 
stage, after the amino protected intermediate has been separated from the 
trypsin or trypsin like enzyme. Of the trypsin like enzymes, lysyl 
endopeptidase from Achromobacter lyticus is useful. 
The compound of formula II may be expressed in a host organism such as 
yeast similar to the description in European patent application 
publication No. 163,529 of which the U.S. counterpart is S.N. 739,123, 
filed May 29, 1985 now U.S. Pat. No. 4,916,212, issued Apr. 10, 1990 using 
a gene having the correct codons for the amino acids in question. The gene 
encoding the novel insulin derivative is then inserted into a suitable 
expression vector which when transferred to yeast is capable of expressing 
the desired compound. The product expressed is then isolated from the 
cells or the culture broth depending on whether it is secreted from the 
cells or not. 
An example of a reversible amino protecting group is tertiary 
butoxycarbonyl and a reversible hydroxy protecting group is tertiary 
butyl. Such groups are removed under conditions which do not cause 
undesired alteration in the compound of formula I, for example, by 
trifluoroacetic acid. 
Changes in the A4, A17, A21, B13, B21 or B27 position may conveniently be 
introduced by genetic engineering, leaving for trypsin catalyzed 
semisynthesis to introduce the desired C-terminal residue of the B-chain. 
The advantage in introducing the additional positive charges within the 
frame of the 51 amino acids of the insulin molecule to form the novel 
compounds of formula I rather than by prolongation of the B-chain beyond 
the 30 residues of the mammalian insulins relates to ease in preparation. 
In the semisynthetic transpeptidation, a large molar excess of the amino 
acid amide or amino acid ester is employed. If a dipeptide amide or ester 
were to be used in the transpeptidation reaction, either price or 
solubility or both are prohibitive for use in large.excess, and 
consequently the yield of the product becomes lower. Even when the same 
equimolar excess of, for example, Lys(Boc)--NH.sub.2 or 
Lys(Boc)--Lys(Boc)--NH.sub.2 is used in the transpeptidation reaction 
under similar conditions, the yield with the amino acid amide becomes 
substantially higher than with the dipeptide amide. 
Insulin preparations of this invention are prepared by dissolving a 
compound of formula I in an aqueous medium at slightly acidic conditions, 
for example, in a concentration of 240 or 600 nmole/ml. The aqueous medium 
is made isotonic, for example, with sodium chloride, sodium acetate or 
glycerol. Furthermore, the aqueous medium may contain zinc ions in a 
concentrations of up to about 30 .mu.g of Zn++ per nmol of compound of 
formula I, buffers such as acetate, citrate and histidine and 
preservatives such as m-cresol, nipagin or phenol. The pH value of the 
final insulin preparation depends upon the number of charges that have 
been changed in the compound of formula I, the concentration of zinc ions, 
the concentration of the compound of formula I and the compound of formula 
I selected. The pH value is adjusted to a value convenient for 
administration such as about 2.5-4.5, preventing precipitation. The 
insulin preparation is made sterile by sterile filtration. 
The insulin preparations of this invention are used similarly to the use of 
the known insulin preparations. 
Any novel feature or combination of features described herein is considered 
essential to this invention. 
Herein the abbreviations used for the amino acids are those stated in 
J.Biol.Chem. 243 (1968), 3558. The amino acids stated herein are in L 
configuration. In formula I and elsewhere herein, A(1-3) is Gly-Ile-Val, 
A(5-6) is Gln-Cys etc., cf. the amino acid sequence of human insulin. 
Unless otherwise indicated, the species of insulins stated herein is 
human. 
Synthesis of the insulin compounds 
The source of insulin was an insulin precursor expressed in yeast as 
described in European patent application publication No. 163.529 of which 
the U.S. counterpart is S.N. 739,123, filed May 29, 1985 now U.S. Pat. No. 
4,916,212. issued Apr. 10, 1990. 
The insulin precursors were recovered from the fermentation broths by 
adsorption to LICHROPREP .TM. RP-18 as described in Example 7 of the same 
European patent application. The precursors were eluted from the column 
with 0.2 M KCl, 0.001 M HCl in 33% (v/v) ethanol. The insulin precursors 
were crystallized from the pool by successive additions of water (1 volume 
per volume of pool), solid trisodium citrate to obtain a molarity of 0.05 
M and finally zinc acetate to obtain a molarity of 0.006 M. The pH value 
was adjusted to 6.8 and the mixture was left overnight at 4.degree. C. The 
crystals were isolated by centrifugaton, washed with water and dried in 
vacuo. 
Protected amino acids and protected peptides for enzymatic semisynthesis 
were either prepared by standard methods or purchased (custom synthesis) 
from either Nova Biochem or Bachem, both Switzerland. 
The letters .TM. after a name indicates that it is a trade mark. 
In the starting material in Examples 1 through 14, (Q.sub.q --T).sub.r of 
formula II was chosen to Ala-Ala-Lys and constructed as described for 
yeast plasmid pMT610 in Example 10 in European patent application 
publication No. 163.529. Nucleotides coding for Gln.sup.B13, Gln.sup.A17, 
Arg.sup.B27, Lys.sup.B27, Asp.sup.A21, Gly.sup.A21, His.sup.A21, 
Ser.sup.A21 and Thr.sup.A21 were substituted in pMT610 by site specific 
mutagenesis using the procedure in Nucl.Acids.Res. 11 (1983), 5103-5112. 
EXAMPLE 1 
Synthesis of His.sup.A21, Arg.sup.B27 human insulin 
The title compound was synthesized from the corresponding single chain 
insulin precursor, viz. His.sup.A21,Arg.sup.B27, 
B(1-29)-Ala-Ala-Lys-A(1-21), using methods analog to those described in 
Example A. Yields, charges relative to human insulin, rates of migration 
relative to insulin in DISC PAGE electrophoresis at pH 8.9 and deviations 
in amino acid compositions from human insulin appear from Table I, below. 
EXAMPLES 2-8 
Synthesis of Asp.sup.A21,Arg.sup.B27, Thr.sup.B30 --NH.sub.2 human insulin, 
Gly.sup.A21,Arg.sup.B27,Thr.sup.B30 --NH.sub.2 human insulin, 
His.sup.A21,Arg.sup.B27,Thr.sup.B30 --NH.sub.2 human insulin, 
Gly.sup.A21,Arg.sup.B27,Gln.sup.B13 Thr --NH.sub.2 2 human insulin, 
Ser.sup.A21,Arg.sup.B27,Gln.sup.B13 Thr.sup.B30 --NH.sub.2 human insulin, 
Thr.sup.A21,Arg.sup.B27,Gln.sup.B13 Thr.sup.B30 --NH.sub.2 human insulin 
and 
Ser.sup.A21,Arg.sup.B27,Thr.sup.B30 --NH.sub.2 human insulin 
Asp.sup.A21,Arg.sup.B27,B(1-29) --Ala--Ala--Lys--A(1-21), 
Gly.sup.A21,Arg.sup.B27,B(1-29) --Ala--Ala--Lys--A(1-21), 
His.sup.A21,Arg.sup.B27,B(1-29) --Ala--Ala--Lys--A(1-21), 
Gly.sup.A21,Arg.sup.B27,B(1-29) --Ala--Ala--Lys--A(1-21), 
Ser.sup.A21,Arg.sup.B27,B(1-29) --Ala--Ala--Lys--A(1-21), 
Thr.sup.A21,Arg.sup.B27,B(1-29) --Ala--Ala--Lys--A(1-21) and 
Ser.sup.A21,Arg.sup.B27,B(1-29) --Ala--Ala--Lys--A(1-21) transpeptidation 
in organic aqueous solution in the presence of Thr--HN.sub.2 as described 
in European patent application publication No. 194.864, Examples 4 and 6. 
Yields, charges relative to human insulin, rates of migration relative to 
insulin in DISC PAGE electrophoresis at pH 8.9 and deviations in amino 
acid compositions from human insulin appear from Table I. 
EXAMPLE 9 
Synthesis of Asp.sup.A21,Arg.sup.B27,Lys.sup.B30 --NH.sub.2 human insulin 
The title compound was synthesized from the corresponding single chain 
insulin precursor, viz. Asp.sup.A21,Arg.sup.B27, 
B(1-29)--Ala--ala--Lys--A(1-21) by tryptic transpeptidation in organic 
aqueous solution int he presence of Lys(Boc)--NH.sub.2, purification of 
the intermediate, Lys(Boc).sup.B30 --NH.sub.2 human insulin, followed by 
removal of the Boc protecting grou by TFA as described in European patent 
application publication No. 194.864, Examples 5 and 7. Yield and 
analytical data are shown in Table I. 
TABLE I 
__________________________________________________________________________ 
Charge 
Rate of 
Deviations in amino 
relative 
migration 
acid compositions 
to human 
at pH 8.9, % 
from human insulin 
Substitution in Yield, 
insulin 
relative to 
after acid hydrolysis, 
human insulin % at pH 7 
human insulin 
residues/molecule 
__________________________________________________________________________ 
His.sup.A21, Arg.sup.B27 
16 +1.1 
75 +1 His, +1 Arg, -1 Asp, -1 Thr 
Asp.sup.A21, Arg.sup.B27, Thr.sup.B30 --NH.sub.2 
23 +1 75 +1 Arg, -1 Thr 
Gly.sup.A21, Arg.sup.B27, Thr.sup.B30 --NH.sub.2 
32 +2 55 +1 Gly, +1 Arg, -1 Asp, -1 Thr 
His.sup.A21, Arg.sup.B27, Thr.sup.B30 --NH.sub.2 
20 +2.1 
55 +1 His, +1 Arg, -1 Asp, -1 Thr 
Gly.sup.A21, Arg.sup.B27, Gln.sup.B13, Thr.sup.B30 --NH.sub.2 
23 +3 35 +1 Gly, +1 Arg, -1 Asp, -1 Thr 
Ser.sup.A21, Arg.sup.B27, Gln.sup.B13, Thr.sup.B30 --NH.sub. 
21 +3 35 +1 Ser, +1 Arg, -1 Asp, -1 Thr 
Thr.sup.A21, Arg.sup.B27, Gln.sup.B13, Thr.sup.B30 --NH.sub.2 
29 +3 35 +1 Arg, -1 Asp 
Ser.sup.A21, Arg.sup.B27, Thr.sup.B30 --NH.sub.2 
+2 55 +1 Ser, +1 Arg, -1 Asp, -1 Thr 
Asp.sup.A21, Arg.sup.B27, Lys.sup.B30 --NH.sub.2 
13 +2 55 +1 Arg, +1 Lys, -2 Thr 
__________________________________________________________________________ 
EXAMPLE 10 
Preparation of injectable solutions of compounds of formula I 
Sterile injectable solutions of the compounds of formula I for testing of 
the degree of prolonged action were made using 1.6% (w/v) glycerol as the 
isotonicum, using 0.3% (w/v) m-cresol as the preservative, and being 
buffered with 0.01 M sodium acetate. The concentration of zinc ions was 8 
or 80 .mu.g/ml. the pH values of the solutions were adjusted sufficiently 
off the isoelectric point of the compounds of formula I to keep the 
solutions clear upon storage at 4.degree.C. The solutions contained 240 
nmole/ml of the compounds of formula I. The concentration of 240 nmole/ml 
was established by measurement of the absorbance at 276 nm of a more 
concentrated stock solution devoid of m-cresol, using the molar extinction 
coefficient for porcine insulin of 6100 for these derivatives (see 
Handbuch der Inneren Mdiizin, vol. 7/Part 2A, Editor: oberdisse, 1975, 
113). For monocomponent procine insulin, the established potency is 28.5 
U/mg dry substance (see Diabetes Care, Vol. 6/Supplement 1 (1983), 4), 
viz. 1 U corresponds to 5.95 nmole. 
Injectable solutions containing 240 nmole/ml of the compounds of formula I 
stated in Table II and having the pH values and content of zinc stated 
therein were made. 
Test for prolongation of insulin effect 
The prolongation of the hypoglycemic effect produced by the injectable 
solutions of insuline was tested according to British Pharmacopoeia 1980, 
a 142, in fasted rabbits. Each test solution was administered 
subcutaneously in a dosis of 14.3 nmole per rabbit in 12 animals weighing 
3-4 kg, and the course of the hypoglycemia was followed for 6 hours. For 
comparison the fast acting preparation ACTRAPID.TM. porcine insulin and 
the intermediate acting MONOTARD.TM. human insulin, were included in the 
tests. The results of the tests are shown in Table II. 
TABLE II 
______________________________________ 
Glucose in 
Compound of Zn.sup.++, percent of initial 
formula I .mu.g/ml 
pH 1 h 2 h 4 h 6 h 
______________________________________ 
Asp.sup.A21, Arg.sup.B27, Thr.sup.B30 -NH.sub.2 
80 4 60 54 58 60 
human insulin 
Asp.sup.A21, Arg.sup.B27, Lys.sup.B30 -NH.sub.2 
80 4 72 67 61 59 
human insulin 
Gly.sup.A21, Arg.sup.B27, Thr.sup.B30 -NH.sub.2 
8 4 59 62 71 74 
human insulin 
Gly.sup.A21, Arg.sup.B27 , Thr.sup.B30 -NH.sub.2 
80 4 72 73 74 74 
human insulin 
His.sup.A21, Arg.sup.B27 human insulin 
80 4 65 53 66 88 
His.sup.A21, Arg.sup.B27, Thr.sup.B30 -NH.sub.2 
80 4 61 52 52 72 
human insulin 
Gly.sup.A21, Arg.sup.B27, Gln.sup.B13, 
80 4 82 86 85 90 
Thr.sup.B30 --NH.sub.2 human insulin 
Ser.sup.A21, Arg.sup.B27, Gln.sup.B13, 
80 4 90 91 88 92 
Thr.sup.B30 --NH.sub.2 human insulin 
Thr.sup.A21, Arg.sup.B27, Gln.sup.B13, 
80 4 90 90 88 93 
Thr.sup.B30 --NH.sub.2 human insulin 
Ser.sup.A21, Arg.sup.B27, Thr.sup.B30 --NH.sub.2 
80 4 60 62 64 68 
human insulin 
ACTRAPID .TM. porcine insulin 
15 7 46 44 74 91 
MONOTARD .TM.human insulin 
80 7 54 43 50 74 
______________________________________ 
The potencies of insulin compounds were assessed in the mouse blood sugar 
depletion test (British Pharmacopoeia 1980, A 141-A 142). In order to 
minimize the problem of estimating potency of insulins having a timing 
different from the standard, insulin solutions for potency determinations 
were made up without additions of zinc. Solutions were made up to contain 
240 nmole/ml based on the absorbance at 276 nm. The zinc content of 
solutions were 8-10 .mu.g/ml, arizing from the crystalline derivatives. 
The estimated potencies of some insulin compounds are shown in Table III, 
below. 
TABLE III 
______________________________________ 
Potency Confidence 
relative to 
limits 
insulin, % 
(P = 0.05), % 
______________________________________ 
Asp.sup.A21, Arg.sup.B27, Thr.sup.B30 --NH.sub.2 
83 92-74 
human insulin 
Asp.sup.A21, Arg.sup.B27, Lys.sup.B30 --NH.sub.2 
69 77-62 
human insulin 
Gly.sup.A21, Arg.sup.B27, Thr.sup.B30 --NH.sub.2 
75 83-68 
human insulin 
His.sup.A21, Arg.sup.B27 human insulin 
71 79-63 
His.sup.A21, Arg.sup.B27, Thr.sup.B30 --NH.sub.2 
72 81-64 
human insulin 
Gly.sup.A21, Arg.sup.B27, Gln.sup.B13, 
49 54-44 
Thr.sup.B30 --NH.sub.2 human insulin 
Ser.sup.A21, Arg.sup.B27, Gln.sup.B13, 
47 54-40 
Thr.sup.B30 --NH.sub.2 human insulin 
Thr.sup.A21, Arg.sup.B27, Gln.sup.B13, 
28 32-24 
Thr.sup.B30 --NH.sub.2 human insulin 
Ser.sup.A21, Arg.sup.B27, Thr.sup.B30 --NH.sub.2 
76 83-68 
human insulin 
______________________________________ 
EXAMPLE 11 
Di- and polymerization products formed per month after storage at 25 C. 
The compositions tested were as follows: 0.24 mM insulin analog, 0.3% /w/v) 
m-cresol, 1.6% (w/v) glycerol, 0.01 M sodium acetate and 3 Zn.sup.++ per 
insulin hexamer. The determination was made using HPSEC (high performance 
size exclusion chromatography). 
The results obtained appears from Table IV. A reference insulin having 
Asn.sup.A21 is included for comparison. 
TABLE IV 
______________________________________ 
pH of formulation 
Insulin analog 3.0 4.0 5.0 
______________________________________ 
Arg.sup.B27 Thr.sup.B30 --NH.sub.2 
0.15% 0.62% 1.4% 
human insulin 
Gly.sup.A21, Arg.sup.B27, Thr.sup.B30 --NH.sub.2 
0.01-0.03% 
0.02-0.05% 
0.20% 
human insulin 
His.sup.A21, Arg.sup.B27 
0.01-0.04% 
human insulin 
Asp.sup.A21, Arg.sup.B27, Thr.sup.B30 --NH.sub.2 
0.03% 0.15% 0.14% 
human insulin 
______________________________________ 
EXAMPLE 12 
Deamidation products formed per month after storage 25 at 25.degree. C. and 
a pH value of 3. 
The compositions tested were as follows: 0.24 mM insulin analog, 0.3% (w/v) 
m-cresol, 1.6% (w/v) glycerol, 3 Zn.sup.++ per insulin hexamer and 0.01 M 
sodium acetate to obtain a pH value of 3. The determinations were made 
using DISC PAGE analysis. 
The results obtained appears from Table V. A reference insulin having 
Asn.sup.A21 is included for comparison. 
TABLE V 
______________________________________ 
Arg.sup.B27 Thr.sup.B30 --NH.sub.2 
approx. 10% 
human insulin 
Gly.sup.A21, Arg.sup.B27, Thr.sup.B30 --NH.sub.2 
below 0.5% 
human insulin 
His.sup.A21, Arg.sup.B27 
below 0.5% 
human insulin 
Asp.sup.A21, Arg.sup.B27, Thr.sup.B30 --NH.sub.2 
below 0.5% 
human insulin 
______________________________________ 
EXAMPLE A 
Synthesis of Gln.sup.13, Arg.sup.B27 human insulin 
To a suspension of 5 g of Gln.sup.B13 Arg.sup.B27, 
B(1-29)-Ala-Ala-Lys-A(1-21) insulin precursor in 50 ml of 2 M 
Thr-OBu.sup.t,CH.sub.3 COOH (L-threonine tert.butyl ester, hydroacetate 
salt) in DMF, 25 ml of 25.5% (v/v) water in DMF (25.5 ml water, DMF to 
make 100 ml) was added. The suspension was cooled to 12.degree. C. under 
stirring. A solution of 0.5 g of porcine trypsin in 12.5 ml of a 0.05 M 
aqueous solution of calcium acetate was added. Stirring was continued 
until dissolution. After 48 hours at 12.degree. C., the proteins were 
precipitated by pouring the mixture into 600 ml of acetone. The 
precipitate was isolated by centrifugaton, washed once with 200 ml of 
acetone, isolated by centrifugation and dried in a stream of nitrogen. The 
precipitate was dissolved in 100 ml of 0.04 N hydrochloric acid, the pH 
value was adjusted to 2.5 and the solution was applied to a 5.times.30 cm 
preparative high pressure liquid chromatography (hereinafter designated 
HPLC) column packed with silica particles substituted with 
octadecyldimethylsilyl (mean particle size 15 micron, pore size 100 
Angstrom). The column was equilibrated with ethanol/0.3 M aqueous solution 
of potassium chloride, 0.001 N hydrochloric acid, in a ratio of 35.5/64.5 
(parts per volume). The proteins were eluted from the column with the same 
buffer at a rate of 2 liter/h. Gln.sup.B13 Arg.sup.B27, Thr.sup.B30 
--OBu.sup.t human insulin was found in a peak emerging from the column 
between 55 and 100 min. The Gln.sup.B13 Arg.sup.B27, Thr.sup.B30 
-OBu.sup.t human insulin was isolated from the pool by successive 
additions of water to make ethanol concentration 15% (v/v), solid 
trisodium citrate to obtain a molarity of 0.05 M with respect to citrate 
and solid zinc chloride to obtain a molarity of 0.006 M with respect to 
zinc. The pH value was adjusted to 6.8 and after 1 hour at room 
temperature, the crystallisation was continued at 4.degree. C. for 24 
hours with stirring. The crystals were spun down, washed twice with 20 ml 
of ice-cold water, spun down and dried in vacuo. Yield: 2.51 g of 
Gln.sup.B13 Arg.sup.B27, Thr.sup.B30 -OBu.sup.t human insulin. 
Gln.sup.B13 Arg.sup.B27, Thr.sup.B30 --OBu.sup.t human insulin was 
dissolved in 100 ml of trifluoroacetic acid and left for 2 hours at room 
temperature. The trifluoroacetic acid was removed by lyophilization. The 
lyophilisate was dissolved in 100 ml of water, the pH value adjusted to 
2.5 and 20 g of sodium chloride was added. The salt cake consisting of 
Gln.sup.B13 Arg.sup.B27 human insulin was isolated by centrifugation. The 
salt cake was dissolved in 850 ml of water and Gln.sup.B13 Arg.sup.B27 
human insulin was crystallized by successive additions of 150 ml of 
ethanol, 14.7 g of trisodium citrate, dihydrate and 0.82 g of zinc 
chloride followed by adjustment of the pH value to 6.8. After 1 hour at 
room temperature, the crystallisation was continued at 4.degree. C. for 24 
hours with gentle stirring. The crystals were spun down, washed twice with 
20 ml of ice-cold water, spun down and dried in vacuo. yield: 1.71 g of 
Gln.sup.B13 Arg.sup.B27 human insulin, corresponding to 36%. 
The amino acid composition was in agreement with the theory, arginine and 
threonine both being 2 residues/molecule. The product was pure in DISC 
PAGE electrophoresis, the rate of migration being 55% of that of human 
insulin corresponding to a difference in charges of about 2. For details 
of the DISC PAGE electrophoresis see Horm.Metab.Res. Supplement Series No. 
5 (1974), 134. The content of zinc in the crystals was 0.42% 
(weight/weight). 
For completeness it is noted that novel insulin analogs of non-human 
sequence fall within practice of this invention. Both a proper but not 
necessarily the human peptide sequence and an insulin activity are 
contemplated herein as within the term insulin. As has already been 
indicated all amino acid residues in the peptide sequence that is human 
insulin are not required for insulin activity. A notable instance in the 
nominal difference between porcine insulin and human insulin, B30 being 
Ala in porcine insulin. Ser is at B30 in rabbit insulin. Both these animal 
insulins contain Asn at A21. A different residue than Asn at A21 may be 
substituted advantageously in these non-human insulins. Although human 
insulin analogs are preferred modes of this invention, many years of use 
and experience with porcine insulin demonstrate their utility and 
accordingly that a substitution for Asn at A21, e.g., by Gly, Ser, Thr, 
Ala or hSer, is advantageous in such insulins. 
1. Preparation of a gene coding for human proinsulin B-C-A 
Total RNA purified (Chirgwin, J. M. Przybyla, A. E., McDonald, R. J. & 
Rutter, W. J., Biochemistry 18, (1979) 5294-5299) from human pancreas was 
reverse transcribed (Boel, E., Vuust, J., Norris, F., Norris, K., Wind, 
A., Rehfeld, J. F. & Marcker, K. A., Proc.Nacl.Acad.Sci. U.S.A. 80, 
(1983), 2866-2869) with AMV reverse transcriptase and d(GCTTTATTCCATCTCTC) 
as 1. strand primer. After preparative urea-polyacrylamide gel 
purification of the human proinsulin cDNA, the second strand was 
synthesized on this template with DNA polymerase large fragment and 
d(CAGATCACTGTCC) and 2nd strand primer. After Sl nuclease digestion the 
human proinsulin ds. cDNA was purified by polyacrylamide gel 
electrophoresis, tailed with terminal transferase and cloned in the PstI 
site on pBR327 (Sorberon et al., Gene 9, (1980), 297-305) in E. coli. A 
correct clone harbouring a plasmid containing a gene encoding human 
proinsulin B-C-A was identified from the recombinants by restriction 
endonuclease analysis and confirmed by nucleotide sequencing (Maxam, A., 
and Gilbert, W., Methods in Enzymology, 65 (1980), 499-560. Sanger, F., 
Nicklen, S. & Coulson, A. R., Proc.Natl.Acad.Sci. U.S.A. 74, (1977), 
5463-5467). 
2. Preparation of genes coding for precursors of human insulin 
The gene encoding B(1-29)-A(1-21) of human insulin was made by side 
specific mutagenesis of the human proinsulin sequence with a 75 bp in 
frame deletion in the C-peptide coding region inserted into a circular 
single stranded M-13 bacteriophage vector. A modified procedure (K. Norris 
et al., Nucl.Acids.Res. 11 (1983) 5103-5112) was used in which a 
chemically synthesized 19-mer deletion primer was annealed to the M13 
template. After a short enzymatic extension reaction a "universal" 15-mer 
M13 dideoxy sequencing primer was added followed by enzymatic extension 
and ligation. A double stranded restriction fragment (BamHl-Hind III) was 
cut out of the partly double stranded circular DNA and ligated into pBR322 
cut with BamHI and Hind III. 
The obtained ligation mixture was used to transform E. coli and 
transformants harbouring a plasmid pMT319 containing the gene encoding 
B(1-29)-A(1-21) of human insulin were identified. 
Genes encoding B(1-29)-Ala-Ala-Lys-A(1-21) and B(1-29)-Ser-Lys-A(1-21) were 
made accordingly by insertion of a fragment encoding MF.alpha.l-B-C-A in 
the M-13 bacteriophage and site specific mutagenesis of the human 
proinsulin sequence with chemically synthesized 30-mer and 27-mer deletion 
primers, respectively, and the above mentioned "universal" 15-mer M13 
dideoxy sequencing primer. A double stranded restriction fragment 
(Xbal-EcoRl) was cut out of the partly double stranded circular DNA and 
ligated into pUC13 and pT5, respectively. By transformation and 
retransformation of E. coli, transformants harbouring a plasmia pMT598 
containing the gene encoding B(1-29)-Ala-Ala-Lys-A(1-21) and pMT630 
containing the gene encoding B(1-29)-Ser-Lys-A(1-21) were identified. 
A gene encoding B(1-29)-Thr-Arg-Glu-Ala-Glu-Asp-Leu-Gln-Lys-A(1-21) was 
made in a similar way as described above by insertion of a fragment 
encoding MF.alpha.l-B(1-29)-A(1-21) in a M13 mp11 bacteriophage and site 
specific mutagenesis of the B(1-29)-A(1-21) sequence with a chemically 
synthesized 46-mer deletion primer 
(5'-CACACCCAAGACTAAAGAAGCTGAAGACTTGCAAAGAGGCATTGTG-3') and the "universal" 
primer. Also, by a similar procedure a gene encoding 
B(1-29)-Thr-Arg-Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gl 
y-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-A(1-2 
1) was constructed. 
3. Plasmid constructions 
The gene encoding B(1-29)-A(1-21) of human insulin (B'A) was isolated as a 
restriction fragment from pMT319 and combined with fragments coding for 
the TPI promoter (TPI.sub.p) (T. Alber and G. Kawasaki. Nucleotide 
Sequence of the Triose Phosphate Isomerase Gene of Saccharomyces 
cerevisiae. J.Mol. Applied Genet. 1 (1982) 419-434), the MF.alpha.l leader 
sequence (J. Kurjan and I. Herskowitz., Structure of a Yeast Pheromone 
Gene (MF.alpha.): A Putative .alpha.-Factor Precursor Contains four Tandem 
Copies of Mature .alpha.-Factor. Cell 30 (1982) 933-943) and the 
transcription termination sequence from TPI of S.cerevisiae (TPI.sub.T). 
These fragments provide sequences to ensure a high rate of transcription 
for the B'A encoding gene and also provide a presequence which can effect 
the localization of B'A into the secretory pathway and its eventual 
excretion into the growth medium. This expression unit for B'A (TPI.sub.P 
-MF.alpha.l leader - B'A - TPI.sub.T was then placed on a plasmid vector 
containing the yeast 2.mu. origin of replication and a selectable marker, 
LEU 2, to give pMT344, a yeast expression vector for B'A. 
During in vivo maturation of .alpha.-factor in yeast, the last (C-terminal) 
six amino acids of the MF.alpha.l leader peptide (Lys-Arg-Glu-Ala-Glu-Ala) 
are removed from the .alpha.-factor precursor by the sequential and an 
aminodipeptidase which removes the Glu-Ala residues (Julius, D. et al. 
Cell 32 (1983) 839-852). To eliminate the need for the yeast 
aminodipeptidase, the sequence coding for the C-terminal Glu-Ala-Glu-Ala 
of the MF.alpha.l leader was removed via in vitro mutagenesis. The 
resulting yeast expression plasmid, pMT475, contains the insert coding for 
TPI.sub.P -MF.alpha.l leader (minus Glu-Ala-Glu-Ala) - B'A - TPI.sub.T. 
In a preferred construction the modified expression unit was transferred to 
a stable, high copy number yeast plasmid CPOT, (ATCC No. 39685), which can 
be selected merely by the presence of glucose in the growth medium. The 
resulting yeast expression vector for B'A was numbered pMT479. 
The fragment encoding MF.alpha.l leader (minus 
Glu-Ala-Glu-Ala)-B(1-29)-Ala-Ala-Lys-A(1-21) was isolated as a restriction 
fragment from pMT598 and combined with fragments coding for the TPI 
promoter and the TPI terminator and transferred to the above mentioned 
high copy number yeast plasmid CPOT. The resulting yeast expression vector 
for B(1-29)-Ala-Ala-Lys-A(1-21) was numbered pMT610. 
The fragment containing the insert TPI.sub.P - MF.alpha.l leader (minus 
Glu-Ala-Glu-Ala)-B(1-29)-Ser-Lys-A(1-21)-TPI.sub.T was isolated as a 
restriction fragment from pMT630 and transferred into CPOT. The resulting 
yeast expression vector for B(1-29)-Ser-Lys-A(1-21) was numbered pMT639. 
The fragment containing the insert TPI.sub.P - MF.alpha.l leader-(minus 
Glu-Ala-Glu-Ala)B(1-29)-Thr-Arg-Glu-Ala-Glu-Asp-Leu-Gln-Lys-A(1-21)-TPI.su 
b.T was inserted into a high copy number yeast plasmid DPOT, being a CPOT 
derivative containing a Sphl-BamHI-fragment of pBR322 inserted into a 
SpHl-BamHI fragment of CPOT. The resulting yeast expression vector for 
B(1-29)-Thr-Arg-Glu-Ala-Glu-Asp-Leu-Gln-Lys-A(1-21) was numbered p1126. 
4. Transformation 
Plasmids pMT344 and pMT475 were transformed into S. cerevisiae leu 2 
mutants by selection for leucin prototrophy as described by Hinnen et al. 
(A. Hinne, J. B. Hicks and G. R. Fink. Transformation of Yeast. 
Proc.Nat.Aca.Sci 75 (1978) 1929). 
Plasmids pMT479, pMT610, pMT639 and p1126 were transformed into S. 
cerevisiae strains carrying deletions in the TPI gene by selecting for 
growth on glucose. Such strains are normally unable to grow on glucose as 
the sole cardon source and grow very slowly on galactose lactate medium. 
This defect is due to a mutation in the triose phosphate isomerase gene, 
obtained by deletion and replacement of a major part of this gene with the 
S. cerevisiae LEU 2 gene. Because of the growth deficiencies there is a 
strong selection for a plasmid which contains a gene coding for TPI. 
pMT479 contains the Schizo. pombe TPI gene. 
5. Expression of human insulin precursors in yeast 
Expression products of human insulin type were measured by radioimmunoassay 
for insulin as described by Heding, L. (Diabetologia 8, 260-66, 1972) with 
the only exception that the insulin precursor standard in question was 
used instead of an insulin standard. The purity of the standards were 
about 98% as determined by HPLC and the actual concentration of peptide in 
the standard was determined by amino acid analysis. The expression levels 
of immunoreactive human insulin precursors in the transformed yeast 
strains are summarized in Table 1. 
TABLE 1 
__________________________________________________________________________ 
Expression levels of immunoreactive human insulin precursors in yeast. 
Immunoreactive 
insulin precursor 
Yeast strain 
Plasmid 
Construct (nmol/l supernatant) 
__________________________________________________________________________ 
MT 350 (DSM 2957) 
pMT 344 
B(1-29)-A(1-21) 100 
MT 371 (DSM 2958) 
pMT 475 
B(1-29)-A(1-21) 192 
MT 519 (DSM 2959) 
pMT 479 
B(1-29)-A(1-21) 2900 
MT 620 (DSM 3196) 
pMT 610 
B(1-29)-Ala--Ala--Lys--A(1-21) 
1200-1600 
MT 649 (DSM 3197) 
pMT 639 
B(1-29)-Ser--Lys--A(1-21) 
1600 
ZA 426 p1126 
B(1-29)-Thr--Arg--Glu--Ala--Glu-- 
Asp--Leu--Gln--Lys--A(1-21) 
200 
__________________________________________________________________________ 
6. Conversion of human insulin precursor into B30 esters of human insulin 
The conversion of the human insulin precursors into human insulin esters 
can be followed quantitatively by HPLC (high pressure liquid 
chromatography) on reverse phase. A 4.times.300 mm ".mu.Bondapak C18 
column" (Waters Ass.) was used and the elution was performed with a buffer 
comprising 0.2 M ammonium sulphate (adjusted to a pH value of 3.5 with 
sulphuric acid) and containing 26-50% acetonitrile. The optimal 
acetonitrile concentration depends on which ester on desires to separate 
from the insulin precursor. In case of human insulin methyl ester 
separation is achieved in about 26% (v/v) of acetonitrile. 
Before the application on the HPLC column the proteins in the reaction 
mixture was precipitated by addition of 10 volumes of acetone. The 
precipitate was isolated by centrifugation, dried in vacuo, and dissolved 
in 1 M acetic acid. The depository DSM is Deutsche Sammlung von 
Mikroorganismen, Grisebochstrasse 8, D-3400 Gottingon, West Germany.