Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2002-01-18
2003-07-22
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S123120, C536S124000
Reexamination Certificate
active
06596861
ABSTRACT:
The present invention relates to (i) a method for the reductive amination of polysaccharides, particularly useful in a process for conjugating polysaccharides to polypeptides, and (ii) produced—by—process polysaccharide-polypeptide conjugates and use thereof in pharmaceutical compositions e.g., vaccines.
In recent years, there has been considerable interest in conjugates of polysaccharide and polypeptide molecules and methods of preparation of such conjugates. In the area of vaccine development, for example, purified bacterial capsular polysaccharides have been covalently attached to protein molecules to produce protein-polysaccharide conjugate vaccines. Purified capsular polysaccharide from
Haemophilus influenzae
type b has been covalently attached to a number of protein molecules, e.g. diphtheria toxoid and tetanus toxoid protein, and these conjugates are known to elicit a Tell dependent immune response in the infant population. This feature has allowed the development and licensure of effective vaccines against disease caused by the bacterium
Haemophilus influenzae
type. This approach of preparing conjugate vaccines has also been extended to other capsular polysaccharides, such as those purified from
Neisseriae meningitidis
and
Streptococcus pneumoniae.
Polysaccharides usually are T-independent antigens unable to elicit an anamnestic (memory) response. Additionally, they are not at all or poorly immunogenic in infants. It is well-known that such disadvantages may be overcome by conjugating the polysaccharides to a carrier polypeptide.
Polysaccharides may be coupled to polypeptides directly or via spacer/linker molecules, according to a variety of methods. Some of them take advantage of the reducing end present in all polysaccharides. Indeed, this extremity may be easily reacted with functional groups such as amino groups. In particular, it may be subjected to reductive amination under mild conditions that is, at low temperature (usually at about 50° C.), in the presence of an appropriate reducing agent. Reductive amination is a classical reaction of carbonyl and amino groups, and is the result of 2 successive reactions:
Formation of imine: R—CHO+H
2
N—R′→R—CH═NH—R′
Reduction of the double bond in the presence of a reducing agent: R—CH═NH—R′→R—CH
2
—NH—R′;
wherein R is the polysaccharide and R′ an amino donor. Imine formation usually evolves toward an equilibrium state, the final product being therefore partially aminated (some molecules remaining non-aminated).
The simplest method involves reacting the terminal reducing sugars of polysaccharides with amino groups of a polypeptide (e.g., side chain amino groups of lysine residues), in the presence of a reducing agent. As a result, the polysaccharide is coupled to the polypeptide via a direct covalent bound.
More sophisticated procedures involve reacting the terminal reducing sugars of the polysaccharide with a linker or a spacer molecule bearing at least one amino group, in the presence of a reducing agent, so as to produce an activated polysaccharide which may be further conjugated to a polypeptide according to a variety of conventional conjugation methods.
The polysaccharide terminal reducing sugar is not the only polysaccharide functional groups that can be subjected to reductive amination. Functional groups available for linkage to amino groups also include e.g. carbonyls, such as aldehydes and ketones, present all along the carbohydrate chain. Alternatively, they may even be created on purpose by a chemical treatment.
As described above, reductive amination is a chemical reaction widely used in polysaccharide conjugation methods. However, this reaction is time-consuming, taking on the order of days to perform. It is believed that the imine formation is a very slow reaction that constitutes a limiting step of the global reductive amination reaction.
Also, the ultimate yield of aminated polysaccharide may be compromised by the formation, over time, of undesirable secondary reaction products. Undesirable secondary reaction events include e.g. the reversal to the non-reduced form of the polysaccharide and more importantly, the formation of degradation products. Reducing agents that can be used for reducing the imine bond are also capable of reducing the terminal aldehyde function of the polysaccharide. Indeed, hydrides other than cyanoborohydride can equally reduce the aldehyde and the imine bond. Cyanoborohydride preferentially reduces the imine bond.
That chemical reactions in general can be accelerated by heating is well-known. Indeed, reductive amination of polysaccharides is conventionally carried out at a temperature about 15-50° C., the reaction mixture being placed in a water bath. More elevated temperatures (e.g., above 100° C.) are not appropriate, because polysaccharides could be degraded or denatured.
In a more general manner, the use of microwave ovens, per se, to accelerate certain reactions, such as organic synthesis, has also been described. To our knowledge, Loupy, et al. Tetrahedron Letters (1996) 37:8177 is the only document that discloses the use of rmicrowave radiation in a reductive amination procedure. However, the reaction employed by Loupy et al is a very special reductive amination (namely, the Leuckart reaction), which is only suitable for low molecular weight carbonyl compounds, not for polysaccharides. In particular, the Leuckart reaction is characterized by harsh reaction conditions e.g., high temperatures (maximal reaction temperatures are far above 100° C.). The Leuckart reaction also differs from reductive amination under mild conditions in that reductive amination is accompanied by decarboxylation (see the reaction scheme on page 8180 of Loupy et al). No such decarboxylation occurs when reductive amination is carried out under mild conditions.
Surprisingly, it has now been found that the time necessary for reductive amination of polysaccharides can be dramatically reduced while submitting the reaction mixture to microwave radiation. It is believed that microwave radiation may accelerate both the imine formation and the reductive amination step. In particular, reductive amination using a mild reducing agent such as cyanoborohydride is a slow reaction when performed according to conventional practice and it may dramatically accelerated under microwave radiation.
Also surprising was the finding that this procedure allows for rapid reductive amination of polysaccharides of interest under relatively mild conditions, such that essential antigenic determinants within the polysaccharides are retained. This is indeed extremely important in that conjugate vaccines must be prepared in such a way as to retain, as much as possible, the structure and conformation of the component materials, for example the polysaccharides and proteins. Critical antigenic determinants are often comprised of portions of the structure and/or conformation of such molecules, and if such determinants are not retained then the resulting conjugate vaccine will be incapable of inducing the desired immune response. For example, O-acetyl or phosphate groups on polysaccharide are important antigenic determinants and can be quite labile. Surprisingly, these important determinants are not affected by the microwave-enhanced reductive amination procedure of the present invention.
Even more surprising was the finding that, in contrast to the classical reductive amination procedure, the formation of undesirable secondary reaction products is limited, compared to what is obtained in a conventional reductive amination procedure. The implications of this latter finding are quite significant Indeed, additional reaction cycles can be performed to increase the degree of amination of the polysaccharide. This, in turn, provides the means to prepare conjugates wherein more polysaccharides are added to identical amount of polypeptides and therefore to improve conjugation yield.
In brief, the present invention provides a means for dramatically reducing the overall reaction time for redu
Aventis Pasteur S.A.
Krishnan G.
McDonnell & Boehnen Hulbert & Berghoff
Wilson James O.
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