pH-dependent aggregation behavior of peptides. In a previous blog post, we delved into the complex issue of peptide and protein aggregation and its impact on reverse-phase liquid chromatography. We emphasized the challenge posed by the aggregation and physical instability of peptide-based drugs in the pharmaceutical industry. Peptides tend to aggregate due to various factors such as the amount of peptide/protein, solution pH, impurities, metal particles, salts, ionic strength, temperature, stirring, and shear forces [1].
For instance, let's focus on a critical factor - the pH level for peptide solutions. The common peptide drug
insulin, used to treat type 2 diabetes, has been found to create clumps at acidic pH levels ranging from 1 to 7.0 [1]. Similarly, Glucagon-like peptide-1 (GLP-1) analogues, another essential class of medications for type 2 diabetes and obesity, have been observed to form clumps at both acidic pH levels (pH 3 and 4) [2] and alkaline pH levels (pH 7.4-8.2) [1].
In this blog, we will delve into the impact of pH on the aggregation behavior of GLP-1 and its implications for the reverse-phase chromatography method employed to separate these peptides. To comprehend this implication, it is crucial first to understand the diverse compositions of GLP-1 analogue peptides.
The rationale for the diverse compositions of GLP-1 analogues. Native GLP-1 peptides are broken down quickly in our body's enzymes, so they don't last long. To address this, changes have been made to the GLP-1 sequence to make it stronger against breaking down and to improve its ability to work as a treatment. These changes include using different kinds of amino acids, protecting the N-terminal, making a circular version, cutting parts off the peptide, and stapling additional peptides.
For example, the natural GLP-1 analog GLP-1 (7-36) NH2 has had its C-terminal glycine replaced with —NH2. Another synthetic analog, Val8-GLP-1 (7-37) OH, replaces alanine at position 8 with valine. Similarly, Thr16-Lys18-GLP-1 (7-37) OH replaces valine at position sixteen and serine at position eighteen with threonine and lysine, respectively. These changes have led to the development of twice-daily exenatide and once-daily lixisenatide as similar versions of GLP-1 (7-37) OH.
Even though changing the amino acids has helped to stop the peptide from breaking down, modified GLP-1 analogues still get removed from the body quickly through the kidneys, known as renal clearance. Because of their size, these peptides are removed from the bloodstream through the kidneys (removed from the body via urine), which lowers how well they work.
However, studies have shown that making the peptides heavier (increasing their molecular weight) can help stop them from being removed from the body quickly. Ways to do this include attaching fatty acids to the peptide, combining with human serum albumin, merging with the fragment crystallizable region of a monoclonal antibody, loading them into long-acting drug release capsules, combining to larger proteins, and attaching strands of the polymer polyethylene glycol (PEG, also called PEGylation) to the peptides. These strategies were used to develop the once-daily liraglutide and later once-weekly Semaglutide and dulaglutide.
Aggregation and insolubility of peptides. GLP-1 analogues need to dissolve in water at our body's natural pH of 7.4 to work well. There are two types: active (soluble) analogs that can dissolve at a rate of ≥1 milligram per milliliter of water at pH 7.4, and inactive (insoluble) analogs that can dissolve at a rate of ≤0.5 milligrams per milliliter of water [3].
Research has shown that the inactive, insoluble form of GLP-1 compounds can be transformed into a soluble form by dissolving the inactive form in an aqueous alkaline solution, which increases the solution's pH. This soluble form, having a neutral pH of 7.0, can then be isolated through filtration and lyophilization.
However, increasing the solution's alkalinity or basicity can speed up racemization (changes an amino acid into its mirror-image form, which changes the activity) and other degradation of the compound over time. Therefore, the pH of the alkaline solution is chosen to facilitate compound dissolution without causing amino acid racemization or the formation of other by-products. Studies show that adjusting the pH between about 10.5 and 12.5 is sufficient to prevent significant racemization or degradation, as long as the GLP-1 solution is neutralized within approximately thirty minutes after dissolving the insoluble GLP-1 compound. Investigations also show that the pH value of the neutralized solution can range from about 7 to 9.
Implications on reverse phase liquid chromatography.
The different types of GLP-1 analogues may require specific reverse-phase chromatography methods. The same method cannot be directly transferred for all the different types of peptides.
Scientists, having learned the connection between solubility and aggregation, have developed methods of separating GLP-1 analogues using chromatography under alkaline conditions. This means the pH of the alkaline phase is kept at high levels, for example, a pH of 8.5, to prevent aggregation during the purification phase. Purification is also done at slightly alkaline conditions, for example, pH = 7.5. But, because there may be more insoluble forms at this pH, an additional step of changing to higher alkaline values, followed by immediate neutralization, could be necessary after the purification phase. These additional steps increase the overall production costs.
It’s well known that at high pH levels (>8), the silica C18 stationary phase used in reverse phase chromatography starts to break down. Increasing the temperature speeds up this breakdown process. However, advancements in silica bead technology have shown progress in slowing down this breakdown. The effects of these silica leachings, even in trace amounts, on GLP-1 purification performance, and aggregation needs further investigation.
When GLP-1 solutions are stirred, they can turn into an inactive, aggregated form. Common purification processes, such as mixing or continuous flow through chromatography media, can cause this to happen.
These are some of the challenges in purifying large amounts of active GLP-1 compounds using reverse-phase liquid chromatography.
At Millennial Scientific, we develop next-generation products for separation and purification of GLP-1 peptide purification. Get in touch with us to discuss how we can support your chromatography separation needs. For more information or to request samples, please email us at inquiry@millennialscientific.com, call us at 855 388 2800 or fill in our online form.
References
[1] K.L. Zapadka, F.J. Becher, A.L. Gomes dos Santos, S.E. Jackson, Factors affecting the physical stability (aggregation) of peptide therapeutics, Interface Focus 7(6) (2017) 20170030.
[2] A.D. Keith, E.P. Brichtová, J.G. Barber, D.J. Wales, S.E. Jackson, K. Röder, Energy Landscapes and Structural Ensembles of Glucagon-like Peptide-1 Monomers, The Journal of Physical Chemistry B 128(23) (2024) 5601-5611.
[3] P.J.W. Francis, R.J.J. vincent, Patent US-7598222-B2, Process for solubilizing glucagon-like peptide 1 compounds, Eli Lilly, 2000.
[4] E. P. Brichtová, M. Krupová, P. Bouř, V. Lindo, A. Gomes dos Santos, S.E. Jackson, Glucagon-like peptide 1 aggregates into low-molecular-weight oligomers off-pathway to fibrillation, Biophysical Journal 122(12) (2023) 2475-2488.
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