Tirzepatide Impurities

Tirzepatide Impurities | Tilpeptide Impurity Control | Impurity Custom Synthesis and Structure Confirmation | Science-Peptide

Tirzepatide Impurities control - for GLP-1/GIP dual-target drug quality research, generic drug reporting.Science-Peptide provides custom synthesis of tilpeptide-related impurities, including deletion peptides, oxidized impurities, fatty acid exfoliation products, dimers, etc., with structural confirmation data to support your We can support your impurity spectroscopy study with structural confirmation data. Welcome to contact us.

Tirzepatide (Tirzepatide) is the GLP-1/GIP dual agonist that Eli Lilly came up with, and it has been in full swing these two years. It has a good effect of lowering sugar and a strong effect of reducing weight, and is called the upgraded version of "the miracle drug for weight loss". Domestic manufacturers of peptide generic drugs, many are staring at it - after the expiration of the patent, who can first run through the quality of research, who will have the opportunity to grab the first generic.

 

However, the quality of tilpeptide is not that easy to study. It is more complex than simethicone, with 39 amino acids, a fatty acid side chain (eicosanedioic acid) and a linker. The impurities that may appear during the synthesis process are much higher than those of ordinary peptides. Missing peptides, oxidative impurities, fatty acid shedding products, dimers, linker-related impurities each have to have a control, each have to be clear.

 

We have accumulated a lot of experience in the synthesis of complex peptide impurities. We have specialized in the study of tilpeptide, and we know what are the difficulties in its impurity profile and what are the essential control items. We can customize the synthesis of various tilpeptide impurity controls according to your needs, with structure confirmation data, and help you establish the method so that you will be less likely to be issued a supplement when filing.

The molecular structure of tilpeptide determines that its impurity profile is much more complex than that of general peptides. When we do impurity studies, we mainly focus on these categories:

Missing peptide
The 39 amino acids, any one of which is missed during the synthesis process, is an impurity. The retention times of the missing peptides on HPLC were widely distributed, with different polarity of the missing peptides in different positions. Some are missing at the N-terminus, some at the middle, and some near the fatty acid attachment site. We have received an order from a customer for 15 missing peptides in different positions to localize all possible missing impurities in their process.

Oxidizing impurities
Met in the sequence of tilpeptide is susceptible to oxidation to sulfoxide and then to sulfone. Oxidized impurities often appear in stability studies and grow after a period of time. We have done a comparison, under accelerated conditions for a month, the oxidation peak can grow to more than 0.3%, without control.

Fatty acid shedding products
This is an impurity unique to tilpeptide. Its fatty acid side chain (eicosanedioic acid) is attached to lysine through a linker, and this modification may be partially shed during synthesis or storage, resulting in fatty acid-removing impurities. More problematically, the shedding may occur at different locations - between the fatty acid and the linker, or between the linker and the peptide - resulting in different impurities. We have synthesized several fatty acid-related impurities for our customers, ranging from complete shedding to just a small section.

Dimers/polymers
Two or more tilpeptide molecules cluster together to form a dimer or higher. The molecular weight of the dimer is twice that of the main peak, and it is often a broad peak on HPLC, so it has to be specially separated and purified before it can be used as a control. We have done the preparation of dimers, and it took several rounds of purification to achieve a purity of more than 95%.

Linker-related impurities
The fatty acid modification of tilpeptide uses a linker (e.g., γ-Glu, PEG, etc.), and this linker itself may produce side reactions during synthesis, generating impurities with missing or incomplete linkers. This kind of impurity is not found in common peptides, but is unique to tilpeptide, which needs to be studied specifically.

Deamidated impurities
Asparagine (Asn) and glutamine (Gln) are easily deamidated under acidic or heating conditions, resulting in isomers. There are several Asn and Gln in the sequence of tilpeptide, and the deamidation impurity often appears in the forced degradation test, so the control product has to be used to locate the impurity.

Diastereoisomers
During the long chain synthesis, individual amino acids may undergo racemization, resulting in diastereoisomers. The content of this kind of impurity is usually not high, but experts will ask when declaring, and it is better to have a control product for confirmation.

Many of these impurities cannot be purchased and need to be custom synthesized. We have done a lot of projects on the customization of tilpeptide impurities, covering all the above categories.

The deletion of the peptide series
We synthesize whatever is missing. For example, the lysine at position 20 is missing, or the amino acid near the fatty acid attachment site is missing. We will synthesize according to your process impurity profile, and the purity is usually >95%, with MS and NMR confirmation. We have synthesized a set of peptides with 5 amino acid spacing from N-terminal to C-terminal.

Oxidizing impurities
Met in Tilpeptide is oxidized to sulfoxide or sulfone, and we can control the oxidation conditions and synthesize the oxidized control product. We can control the oxidation conditions and synthesize the oxidized control. The purity is >95% and we can provide the LC-MS spectrum to confirm the degree of oxidation. Sulfoxide and sulfone are separated by HPLC, and we can do both.

Fatty acid shedding products
This is one of the most difficult impurities to work with in tilpeptide. The fatty acid chain may be completely off, or it may be partially off (e.g. the last few carbons are missing). We can synthesize a series of variants for different positions of the chain. In one of the most complicated projects we have done, the customer asked us to synthesize an impurity with 8 carbons missing from the fatty acid chain, and it took us two months to get it right.

Dimer
Dimers formed by cross-linking or non-covalent interactions can be prepared by different methods and isolated and purified to provide high purity controls. Dimers are laborious to purify, but we have the experience to know how to separate the monomers.

Linker-related impurities
The linker (γ-Glu, PEG, etc.) may be missing or incomplete, resulting in a series of related impurities. We can synthesize various variants with missing linkers, shortened linkers, or mismatched linkers, and help you to build up the spectra of such impurities.

Deamidated impurities
For the Asn and Gln sites in the sequence, we can prepare deamidated impurity controls by forced degradation or targeted synthesis. The difference in molecular weight after deamidation is only 1, which is not easy to be distinguished by MS, so we usually use NMR or special chromatographic conditions to confirm.

Other customized impurities
If you find a new unknown impurity in your research, we can help you analyze the structure and synthesize it. Send us the samples, we will do LC-MS analysis, deduce the structure, then directional synthesis, and then compare the retention time and mass spectrum to confirm the agreement. We have done this process many times and the success rate is quite high.

Analytical method development and validation
When setting up an HPLC or LC-MS method, an impurity control is required for localization, determination of separation, verification of linear range and recovery. Without the control, the method validation is missing a piece. Many customers ask us to do a few key impurities first to set up the method, and then slowly make up the rest.

Heterogeneous mass spectrometry
When a generic drug is declared, a comprehensive qualitative and quantitative analysis of process impurities and degradation products is required. The location, content and structure of each impurity need to be confirmed by a control product. We have seen the declaration of the information was sent to fill in, nine times out of ten is the impurities of this piece did not understand.

Stability studies
In long-term stability and accelerated tests, it is necessary to determine which degradation products are dominant and what the trend in content is. With the impurity control, we can accurately quantify and monitor. A customer was doing the stability of tilpeptide preparation, and found that a degradation peak grew very fast under accelerated conditions, so we analyzed and synthesized it by MS to confirm the structure, and then the customer was relieved to report it.

Batch release testing
After commercialization, each batch of API or formulation needs to be tested for the substance in question. Impurity controls are essential tools for release testing.

Information on generic drug declarations
Impurity studies are a key section of the filing information. Providing complete impurity profiling and control data significantly reduces the risk of a review release.

Case 1: a pharmaceutical company, tilpeptide generic drug program
They are developing a generic version of tilpeptide and need a complete set of impurity controls for method validation. We customized 12 impurities for them: 5 missing peptides, 2 oxidized impurities, 1 dimer, 2 fatty acid shedding products, and 2 linker-related impurities. Each of them has a purity of more than 95%, and is accompanied by MS and NMR data. The project leader said, "The impurities of tilpeptide are twice as many as other peptides, but you can make all of them, and the data are complete, which saves us a lot of work." Later on, the validation of their method was successfully completed, and now they are preparing for the declaration.

Case 2: A CRO company, impurity profiling
They undertook a quality research project on tilpeptide, which required a comprehensive analysis of process impurities. They sent us the crude peptide samples, and we performed LC-MS analysis, deduced the possible structures of several unknown impurities, then synthesized them in a targeted manner, and compared retention times and mass spectra, and confirmed four new impurities - two of them were linker-related, and two of them were variants of fatty acid shedding. The client used these data to refine the impurity profiles and successfully passed the client's acceptance.

Case 3: Stability studies, a biotechnology company
They were examining the stability of a tilpeptide formulation and found that the amount of a degradation product increased rapidly under accelerated conditions, but they did not know what it was. We analyzed the sample by LC-MS and assumed that it was a deamidation impurity at a certain Asn site, and then synthesized it in a targeted manner and confirmed it by comparison. They established an assay using this control and accurately monitored the trend of the degradation product. Later, when they submitted the report, this part of the data was emphasized by the experts, and because of the solid data, no compensation was issued.

Tilpeptide is a popular product, but impurity study is a delicate job, especially fatty acid exfoliation products and linker-related impurities, which many manufacturers can't handle. If you are doing quality research or generic development of tilpeptide and need impurity controls, please feel free to contact us. We have done a lot of work, we know what are the difficulties, and we know how to make it out.