This invention relates to a process for preparing a hapten-protein-polysaccharide conjugate and a hapten-protein conjugate by reacting a protein with a hapten to produce a hapten-protein conjugate, followed by reacting the hapten-protein conjugate with a polysaccharide to provide a conjugate mixture including the hapten-protein conjugate and a hapten-protein-polysaccharide conjugate. This invention also includes the process described above with the addition of a pharmaceutically acceptable medium or delivery vehicle into the conjugate mixture. The invention further includes the process described above where the hapten is luteinizing hormone releasing hormone peptides derived from E coil, or malaria derived peptides.

SUMMARY OF THE INVENTION

It is an object of this invention to provide a method for producing protein-polysaccharide conjugates that avoids the problems and disadvantages described above. These conjugates can be used as intermediate materials in the production of other conjugates, such as hapten-protein-polysaccharide conjugates. It is a further object of this invention to provide vaccines, immunogens, and other immunological reagents that are produced by this method.

In one embodiment, this invention relates to a process for preparing a protein-polysaccharide conjugate. This process includes reacting a protein with a polysaccharide to produce a mixture including a protein-polysaccharide conjugate and free protein. At least one unreacted reagent or low molecular weight component is removed from this mixture to provide a purified mixture containing the protein-polysaccharide conjugate and free protein.

In another embodiment of the invention, a hapten-protein-polysaccharide conjugate is prepared. In this process, a purified mixture including a protein-polysaccharide conjugate and free protein first is produced in the manner described above. Thereafter, a hapten (e.g., a peptide) is reacted with the purified mixture of the protein-polysaccharide conjugate and the free protein, thereby providing a conjugate mixture including a hapten-protein conjugate, hapten-protein-polysaccharide conjugate, and free hapten. This conjugate mixture can be treated further to remove the free hapten to thereby provide a purified conjugate mixture including the hapten-protein-polysaccharide conjugate and the hapten-protein conjugate.

As another alternative, a hapten-protein conjugate first can be produced. As noted above, the hapten can be, for example, a peptide. The excess free protein and/or free hapten optionally can be removed at this stage. Thereafter, this conjugate, present in excess, is reacted with a polysaccharide to form a hapten-protein-polysaccharide conjugate. It is not necessary to remove the excess hapten-protein conjugate from the resulting conjugate mixture. The conjugate mixture includes the hapten-protein conjugate and the hapten-protein-polysaccharide conjugate.

The invention further relates to the protein-polysaccharide conjugate and free protein mixture, as well as the hapten-protein-polysaccharide conjugate and hapten-protein conjugate mixture, made by the processes of the invention. In addition to vaccines, the conjugates according to this invention can be used as immunogens or immunological reagents.

DETAILED DESCRIPTION OF THE INVENTION

As described above, various techniques and processes for producing protein-polysaccharide conjugates, immunogens, immunological reagents, and vaccines are known. Typically, separating the non-conjugated free protein from the conjugated protein-polysaccharide product represents a major cost in conjugate vaccine production. This separation can be accomplished, for example, using column chromatography (e.g., size exclusion chromatography) or ultrafiltration. These protein separation steps significantly increase the time and expense involved in producing protein-polysaccharide conjugate vaccines, not only because of the time and expense involved in the separation step, but also because of the expense involved in providing a separate chromatography column for each different type of vaccine conjugate. Additionally, costs are increased because the free protein separation step often results in a significant loss of the desired protein-polysaccharide conjugate material.

In some instances, there is no alternative to removing the free protein from the conjugate product. Certain conjugation techniques damage the protein and thereby significantly reduce its antigenicity and immunogenicity. An example of such a technique is carbodiimide coupling of tetanus toxoid to PRP (a capsular polysaccharide from Haemophilus influenza type b). If free protein reduces the immunogenicity of the resulting conjugate, then the unconjugated protein needs to be removed from the conjugate product. Non-immunogenic proteins, such as bovine serum albumin ("BSA"), also may inhibit the anti-protein response to BSA-polysaccharide. In this instance, the unreacted protein should be removed from the conjugate product. As noted above, column chromatography or ultrafiltration typically can be used to remove the unreacted free protein.

Applicants have observed, however, that many other conjugation techniques do not damage the free protein. Examples of these techniques include conjugating via CDAP activation of the polysaccharide or coupling via thio-ether linked spacers. When the free protein is not damaged during conjugation, typically there is no reduction in its antigenicity or its immunogenicity.

In new generation vaccines, the antibody response to the protein also is important. One example is the lipoprotein D-PRP conjugate vaccine ("PRP" means "polyribosylribitol phosphate). In this vaccine, the anti-PRP response is for Haemophilus type b, and the lipoprotein D response is expected to provide protection against non-typable Haemophilus (ref.: Akkoyunlu, et al., Infection & Immunity, Vol. 64, 1996, beginning at pg. 4586, which article is entirely incorporated herein by reference). In still other instances, the immune response to the protein carrier coupled to the polysaccharide is not considered critical, per se, but if an immune response is generated to this carrier, it may be helpful. An example of this is the combination vaccine including a tetanus toxoid ("TT")-PRP conjugate vaccine mixed with tetanus toxoid, pertussis and diphtheria toxoid (Hib DPT).

Considering these observations, applicants have developed an improved method for manufacturing conjugate vaccines. In this method, instead of removing the unconjugated or free protein that remains after producing the protein-polysaccharide conjugates, only certain reagents and low molecular weight polysaccharides are removed from the reaction mixture. The free unconjugated protein remains in solution with the protein-polysaccharide conjugate. By this improved process, the resulting conjugate vaccine can have improved immune response due to the free protein while reducing production costs, equipment costs, and time expenditure in conjugate production.

In addition to the protein and polysaccharide components, during conjugate production, various reagents and low molecular weight components typically are present in the reaction mixture (e.g., cross-linking reagents, buffering components, low molecular weight oligosaccharides, etc.). These excess reagents and low molecular weight components can be removed from the reaction mixture by any suitable process known in the art, such as through dialysis, ultrafiltration, or desalting columns. Typically, at least any materials having a molecular weight below 10,000 are removed, and preferably, materials having a molecular weight below 30,000 are removed. This removal provides a purified mixture including the protein-polysaccharide conjugate and the free protein. Preferably, little or no free protein is removed from the mixture during this initial purification step. The purified mixture preferably contains protein-polysaccharide conjugate and free protein in a weight ratio of 0.95 mg conjugated protein per 0.05 mg free protein to 0.1 mg conjugated protein per 0.9 mg free protein, and advantageously this ratio is in the range of 0.7 mg conjugated protein per 0.3 mg free protein to 0.95 mg conjugated protein per 0.05 mg free protein. These ratios correspond to 5 90% free protein, and preferably 5 30% free protein, by weight, based on the entire protein content. While these free protein ratios are preferred, with high conjugate yield during the conjugation reaction, the free protein content can be as low as 1% in the invention, such that the purified mixture contains 1 90% free protein, with 1 30% free protein preferred (based on the entire protein content). In one embodiment of the invention, the ratio of conjugated to free protein is about 1:1, by weight.

The purified mixture, including the free protein and the protein-polysaccharide conjugate, can be combined with a pharmaceutically acceptable medium or delivery vehicle. As will be discussed in more detail below, the pharmaceutically acceptable medium or delivery vehicle can include at least one member selected from the group consisting of water, petroleum oil, animal based oil, vegetable oil, peanut oil, soybean oil, mineral oil, sesame oil, saline, aqueous dextrose, and glycerol solutions.

In accordance with the process of the invention, the polysaccharide can be activated, for example, using an organic cyanylating reagent during the step of producing the conjugate. Suitable cyanylating reagents include 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate ("CDAP"), N-cyanotriethyl-ammonium tetrafluoroborate ("CTEA"), and p-nitrophenylcyanate. As noted above, the use of such organic cyanylating reagents is described in U.S. Pat. application Ser. No. 08/124,491 (filed Sep. 22, 1993, now abandoned), U.S. Pat. No. 5,651,971; and U.S. Pat. application Ser. No. 08/482,666, now U.S. Pat. No. 5,849,301 (filed Jun. 7, 1995). CDAP is particularly preferred as an organic cyanylating reagent.

The protein and polysaccharide also can be conjugated together via a spacer in the process according to the invention. As one example, a thio-ether spacer can be used in this process. Processes for using a spacer during production of a protein-polysaccharide conjugate, as noted above, are described in U.S. Provisional Patent Appln. No. 60/017,103 filed on May 9, 1996 and U.S. Pat. application Ser. No. 08/852,733, now U.S. Pat. No. 6,309,646, filed May 7, 1997. These applications describe, for example, the use of homobifunctional or heterobifunctional vinylsulfones to provide a spacer in the protein-polysaccharide conjugate. The protein and/or the polysaccharide can be derivatized or functionalized prior to the conjugation reaction procedure (e.g., with thiols, amines, or hydrazides). Other suitable protein/polysaccharide conjugation techniques for use with this invention are described, for example, in U.S. Provisional Patent Appln. Nos. 60/041,781 and 60/042,379, as mentioned above.

In another aspect of the invention, a hapten-protein-polysaccharide conjugate can be prepared using a mixture including a protein-polysaccharide conjugate and free protein produced in the manner described above. After the purified mixture containing the conjugate and the free protein is prepared, a hapten is reacted with the purified mixture to thereby provide a conjugate mixture including a hapten-protein conjugate and a hapten-protein-polysaccharide conjugate. This conjugate mixture can be treated further to remove the free hapten to thereby provide a purified conjugate mixture. This purified conjugate mixture can be mixed with a pharmaceutically acceptable medium or delivery vehicle.

The excess hapten can be removed from the conjugate mixture by any suitable process known in the art. As one specific example, the excess peptide is removed by dialysis to provide the purified conjugate mixture. Peptides are the particularly preferred haptens for use in this embodiment of the invention.

An alternative procedure for producing a conjugate mixture including a hapten-protein-polysaccharide conjugate and a hapten-protein conjugate is described below. A hapten-protein conjugate first is produced by reacting a hapten (such as a peptide) with a protein. The excess free protein and/or free hapten optionally (and preferably) is removed at this stage. Thereafter, this conjugate is reacted with a polysaccharide to form a hapten-protein-polysaccharide conjugate. In this reaction, the hapten-protein conjugate is used in excess to produce a conjugate mixture including the excess hapten-protein conjugate and a hapten-protein-polysaccharide conjugate. The purified conjugate mixture includes the hapten-protein conjugate and the hapten-protein-polysaccharide conjugate. This conjugate mixture can be combined with a pharmaceutically acceptable medium or delivery vehicle.

While any amount of protein can be included in the conjugates according to the invention, generally about 0.1 to 1.0 mg protein is present per mg polysaccharide in the conjugate mixture. Also, in conjugates that include peptides, generally there will be about 5 30 moles peptides per mole of protein.

The process in accordance with the invention can be used on any suitable protein. Examples of suitable proteins include microbial proteins or bacterial proteins. Specific examples of suitable proteins include diphtheria, pertussis toxoid, lipoprotein D, lipoprotein OspA, tetanus toxoid, and gD protein (derived from herpes). Likewise, the processes of the invention can be used on any suitable polysaccharide, such as microbial polysaccharides, fungal polysaccharides, or bacterial polysaccharides. Specific examples of suitable polysaccharides include PRP, dextran, Neisseria meningiditis polysaccharide type C ("Neisseria PsC"), Vi antigen, and pneumococcal polysaccharide. Where a peptide or other hapten is included in the conjugate, any suitable peptide or other hapten can be used. Examples of suitable peptides include luteinizing hormone releasing hormone ("LHRH"); peptides derived from E Coil bacteria (such as ETEC as described in "Antibody to N-Terminal Consensus Peptide is Cross-Reactive with all Six Members of the Entero-Enterotoxigenic E coil CFA/I Family," F. J. Cassels, et al., Abstract, 31st Joint Conference, U.S./Japan Cooperative Medical Science Program, Kiwa Island, S.C. Dec. 1, 1995, which document is entirely incorporated herein by reference); and malaria derived peptides, such as SPf66. Other suitable proteins, polysaccharides and haptens for use in this invention are described in the above-noted U.S. patents and patent applications (e.g., U.S. Pat. application Ser. Nos. 08/124,491; 08/402,565, now U.S. Pat. No. 5,585,100; 08/444,727; 08/468,060; and 08/482,666, now U.S. Pat. No. 5,849,301, and U.S. Pat. No. 5,651,971).

This invention is particularly suitable for conjugation methods where the unconjugated protein is unmodified or minimally modified by the conjugation reaction procedure. CDAP coupling to produce the protein-polysaccharide conjugate is one such conjugation technique where the method according to the invention may be used. The method according to the invention, however, also may be used with other conjugation techniques where there are minimal modifications in the uncoupled protein. The unconjugated protein fraction in the protein-polysaccharide conjugate vaccine can be just as immunogenic as the native protein.

The process of the invention also can be used in producing a combination vaccine. Typically, to produce a combination vaccine, such as a vaccine including a tetanus toxoid ("TT")-PRP conjugate vaccine mixed with tetanus toxoid, pertussis and diphtheria toxoid (Hib DPT), one adds the conjugate back to free protein. In preparing this combination vaccine, first a purified TT-PRP conjugate is prepared (without free protein), and this material is then added to a tetanus toxoid, pertussis, diphtheria toxoid mixture to formulate the combination vaccine. In the process of the invention, however, this combination vaccine is produced by a different process. First, the tetanus toxoid and PRP are conjugated together. One vaccine manufacturer has indicated that it can obtain about 90% coupling efficiency of tetanus toxoid to PRP when CDAP is used to activate the polysaccharide and prepare the TT-PRP conjugates. Accordingly, after this conjugate production process, there may be about 10% free protein remaining in the mixture with the conjugate. In accordance with the process of the invention, there is no need to separate this free protein from the conjugate. Rather, only the excess CDAP and any other reagents are removed from the conjugate-free protein mixture (TT-PRP+TT). This conjugate-free protein mixture (TT-PRP+TT) is then added to a mixture including diphtheria toxoid and pertussis. If necessary or desired, the total amount of tetanus toxoid in the original conjugation reaction procedure can be adjusted so that a predetermined desired amount of tetanus toxoid is present in the final combination vaccine product. Alternatively, additional tetanus toxoid can be included in the diphtheria toxoid and pertussis mixture.

As another alternative, the process of the invention also can be used in the preparation of peptide-protein-polysaccharide conjugates or other hapten-protein-polysaccharide conjugates. Typically, when making such conjugates, a protein-polysaccharide conjugate first is prepared, and thereafter, a peptide is coupled to this conjugate. Applicants have observed, however, that the peptide-protein conjugate and the peptide-protein-polysaccharide conjugate mixture induces anti-polysaccharide, anti-protein, and anti-peptide responses. Accordingly, in an example of this process according to the invention, the protein-polysaccharide conjugate is produced, and the free protein is allowed to remain with the protein-polysaccharide conjugate. Thereafter, the peptide is reacted with this conjugate mixture, including the protein-polysaccharide conjugate and the free protein, to thereby produce a conjugate mixture including a peptide-protein-polysaccharide conjugate and a peptide-protein conjugate. The free peptide can be removed by dialysis to provide a purified conjugate mixture including the peptide-protein conjugate and the peptide-protein-polysaccharide conjugate. By eliminating the free protein removing step, the peptide-protein-polysaccharide conjugate can be produced in a more cost effective manner, and the resulting purified conjugate mixture produces enhanced anti-protein and anti-peptide responses.

Alternatively, as described above, a conjugate mixture including a peptide-protein conjugate and a peptide-protein-polysaccharide conjugate can be prepared by first conjugating the peptide and protein, and thereafter reacting this conjugate with a polysaccharide reagent.
 

Claim 1 of 9 Claims

1. A process for preparing a hapten-protein-polysaccharide conjugate comprising: a) reacting a protein with a hapten to produce at least a hapten-protein conjugate, and any unreacted protein and any unreacted hapten; b) reacting the hapten-protein conjugate with a polysaccharide to produce at least a hapten-protein-polysaccharide conjugate, and unreacted hapten-protein conjugate; c) removing at least one unreacted hapten or low-molecular weight component from the mixture without removing unreacted protein or unreacted hapten-protein conjugate, if present, to provide a purified mixture that contains the hapten-protein-polysaccharide conjugate and at least one of unreacted protein and unreacted hapten-protein conjugate, to thereby provide a conjugate mixture including the hapten-protein-polysaccharide conjugate, and at least one of hapten-protein conjugate and unreacted protein.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.