DESC:FIELD OF THE INVENTION
The present invention relates to compositions with reduced impurity and analytical methods thereof. More particularly, the invention relates to compositions comprising polypeptides with reduced impurity of excipients and methods for determining said impurity.
BACKGROUND OF THE INVENTION
Excipients are formulated alongside an active ingredient to confer stability against unfolding, aggregation, pH changes etc. These include surfactants, sugars, salts, polyols and amino acids, among others. Surfactants confer stability to polypeptides in a high concentration drug formulation against denaturation, unfolding, aggregation and surface adsorption, thereby preserving the activity of the polypeptide for a longer time period. A few examples of approved surfactants in biologic drug formulations are polysorbate 80 (PS80/Tween 80), polysorbate 20 (PS20/Tween 20) and poloxamer.
Interestingly, despite being extensively used and largely accepted, surfactants are not without disadvantages. For example, commercially available polysorbates are a chemically diverse mixture of subspecies, with multiple polymer chain lengths depending upon the synthesis route and raw materials used. For this reason, polysorbate compositions from different manufacturers differ in their relative abundance of subspecies in the solution. In addition, pH changes, thermal stress, auto-oxidation or enzymatic intervention can lead to low-level hydrolysis of polysorbates, giving rise to degradants. These subspecies and degradant impurities can impact the conformation, function, quality, stability and efficacy of the drug product. For example, high levels of PS80 degradants have been reported to destabilize IgG in the solution. (Grabarek, A. D., Bozic, U., Rousel, J., Menzen, T., Kranz, W., Wuchner, K., Jiskoot, W., & Hawe, A. (2020). What makes Polysorbate functional? Impact of Polysorbate 80 grade and quality on IgG stability during mechanical stress. Journal of Pharmaceutical Sciences, 109(1), 871-880. https://doi.org/10.1016/j.xphs.2019.10.015) Thus, degradation of surfactants in a drug composition needs to be monitored.
For this reason, regulators have started to increasingly stress upon impurity profiling of formulations in recent years. However, recommended analytical testing methods so far only give high-level percentages of components, with little clarity on the characterized components under different stressed conditions. Existing analytical methods follow a combination of methods, employing simultaneous runs of reference standards for comparison purposes. However, absence of a universal reference standard for the various components and batch-to-batch and lot-to-lot variability among the said standards are evidently challenging in such impurity determination.
Another barrier in impurity profiling of excipients is interference by the protein itself (usually present in high concentrations) in the solution. Existing methods recommend removal of protein (by for example, solid phase extraction or precipitation) prior to analysis, which can be time-consuming and cumbersome. (Liu, H., Jin, Y., Menon, R., Laskowich, E., Bareford, L., De Vilmorin, P., Kolwyck, D., Yeung, B., & Yi, L. (2021). Characterization of Polysorbate 80 by liquid chromatography-mass spectrometry to understand its susceptibility to degradation and its oxidative degradation pathway. Journal of Pharmaceutical Sciences. https://doi.org/10.1016/j.xphs.2021.08.017). However, complete removal of protein remains a challenge in addition to strong possibility of loss of any adsorbed polysorbate along with protein during the process of such removal.
Hence, there is a need to develop drug compositions comprising polypeptides with a reduced impurity profile of surfactants and analytical methods relating to the same with minimal interference from the polypeptide itself. A further objective of the invention is to develop a method that can qualitatively differentiate subspecies and degradants of the excipient distinctly under different stress conditions. Another objective of present invention is to develop a rapid, less-labour intensive method for the profiling of said impurities.
SUMMARY OF THE INVENTION
Accordingly, present invention discloses a composition comprising polypeptides with reduced impurity of excipients and analytical methods employing chromatography column composed of a porous monolith hybrid material comprising inorganic and organic chromatographic material. Also disclosed is a method for determining impurity of an excipient in a composition comprising polypeptides using enhanced chromatographic separation methods wherein interference from the polypeptide component itself is minimal.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Representative peak profile of standard polysorbate 80 sample
Figure 2: Representative peak profile of sample overlaid over profile of standard polysorbate 80 sample
Figure 3: Representative peak profile of standard Triton X-100 sample
DETAILED DESCRIPTION OF THE INVENTION
Regulatory agencies have emphasized on the need for impurity profiling of drug preparations comprising polypeptides, to establish safety of a drug product. Degradants of the components of the drug product are the major source of said impurities.
In an embodiment, the invention discloses a composition comprising polypeptides with minimal deviation in impurity profile of excipients compared to that of a reference standard.
In another embodiment, the invention discloses a method for determining impurity profile of an excipient in a composition comprising polypeptides wherein the impurity comprises sub-species and degradants of the excipient.
In another embodiment, the invention discloses a method for determining impurity profile of an excipient in a composition comprising polypeptides, wherein the method comprises:
i. applying the composition to a chromatography column composed of a porous monolith hybrid material comprising inorganic and organic chromatographic material in a solution comprising a mobile phase A and a mobile phase B, wherein mobile phase B comprises formic acid and acetonitrile;
ii. eluting bound content from the column with a solution comprising mobile phase A and mobile phase B wherein the ratio of mobile phase A to mobile phase B is 1:19 from retention time of about 10 minutes to about 25 minutes; and
iii. measuring the eluted content of step ii with an aerosol detector;
iv. comparing the output of step iii with that of a reference standard solution
wherein the eluted content comprises impurities;
wherein the impurity comprises sub-species and degradants of the excipient and
wherein the method is devoid of a step to remove polypeptides from the composition.
In a further embodiment, the porous monolith hybrid material has a chromatographically-enhancing pore geometry.
In a further embodiment, the porous inorganic/organic hybrid material having chromatographically-enhancing pore geometry is comprised of homogenous particle homology.
In yet another embodiment, the porous inorganic/organic hybrid material having chromatographically-enhancing pore geometry is surface modified by a surface modifier.
In a further embodiment, the aerosol detector is preferably an evaporative liquid scattering detector, or preferably a condensation nucleation light scattering detector, or more preferably charged aerosol detector.
In yet another embodiment, the excipient is a polysorbate, preferably polysorbate-20, or preferably polysorbate-80; or the excipient is Triton X-100.
In an embodiment, the invention discloses a method for determining impurity profile of polysorbates in a composition comprising polypeptides, wherein the method comprises:
i. applying the composition to a chromatography column composed of a porous monolith hybrid material comprising inorganic and organic chromatographic material in a solution comprising a mobile phase A and a mobile phase B, wherein mobile phase A comprises ammonium acetate and mobile phase B comprises formic acid and acetonitrile;
ii. eluting bound content from the column with a solution comprising mobile phase A and mobile phase B wherein the ratio of mobile phase A to mobile phase B is 1:19 from retention time of about 10 minutes to about 25 minutes;
iii. measuring the eluted content of step ii with an aerosol detector;
iv. comparing the output of step iii with that of a reference polysorbate solution
wherein the eluted content comprises impurities;
wherein the impurity comprises sub-species and degradants of the excipient and
wherein the method is devoid of a step to remove polypeptides from the composition.
In an embodiment, the invention discloses a method for determining impurity profile of Triton X-100 in a composition comprising polypeptides, wherein the method comprises:
i. applying the composition to a chromatography column composed of a porous monolith hybrid material comprising inorganic and organic chromatographic material in a solution comprising a mobile phase A and a mobile phase B, wherein mobile phase A comprises formic acid and water and mobile phase B comprises formic acid and acetonitrile;
ii. eluting bound content from the column with a solution comprising mobile phase A and mobile phase B wherein the ratio of mobile phase A to mobile phase B is 1:19 from retention time of about 10 minutes to about 25 minutes;
iii. measuring the eluted content of step ii with an aerosol detector; and
iv. comparing the output of step iii with that of a reference Triton X-100 solution
wherein the eluted content comprises impurities;
wherein the impurity comprises sub-species and degradants of the excipient and
wherein the method is devoid of a step to remove polypeptides from the composition.
In yet another embodiment, the invention discloses a method for testing stability of excipients in a dispensed drug solution intended for in-process testing or commercial release wherein, the method comprises:
i. applying the solution to a chromatography column composed of a porous monolith hybrid material comprising inorganic and organic chromatographic material in a solution comprising a mobile phase A and a mobile phase B, wherein mobile phase B comprises formic acid and acetonitrile;
ii. eluting bound content from the column with a solution comprising mobile phase A and mobile phase B wherein the ratio of mobile phase A to mobile phase B is 1:19 from retention time of about 10 minutes to about 25 minutes;
iii. measuring the eluted content of step ii with an aerosol detector; and
iv. comparing the output of step iii with that of a reference standard solution
wherein the eluted content comprises impurities;
wherein the impurity comprises sub-species and degradants of the excipient and
wherein the method is devoid of a step to remove polypeptides from the composition.
Stress conditions such as temperature and pH changes can give insight into the degradation behaviour of excipients (that includes surfactants) and possible impact on the stability and activity of the protein.
Definitions:
The term “chromatographically-enhancing pore geometry” as used herein refers to a pore geometry that enhances chromatographic separation ability of the material in terms of properties such as separation efficiency, column life etc.
The term “composition comprising polypeptides” refers to a diagnostic, preventive, or therapeutic preparation comprising polypeptides derived from a biological source. For example, this includes a population of antibody molecules or antibody fragments thereof or fusion protein molecules that is produced by mammalian cell culture.
The term “hybrid” as used herein includes inorganic-based chromatographic material wherein an organic functionality is integral to the internal and surface aspects of the structure. For example, in a case where the inorganic component is silica, ‘hybrid silica’ refers to chemically modified one or more silica molecules bearing aliphatic and/or aromatic moieties, which may additionally be substituted with desired functional groups. The said desired functional groups may include, but not limited to, alkyl, aryl, cyano, amino, hydroxyl, diol, nitro, ester, ion exchange moieties of polar functional groups.
The term “impurity” as used herein pertains to impurity relating to excipients, for example, surfactant. This includes the molecules structurally related to a given excipient present in the solution. The impurity may, in addition to other reasons, arise owing to stressed conditions such as heat or oxidation stress and may include undesired sub-species and degradants of the excipient.
The term “minimal deviation in impurity profile” refers to a condition wherein the impurity profile of the chromatogram of a given preparation has no or minimal number of additional peaks when compared to the reference standard in a given experiment.
The term “monolith” as used herein refers to monolith materials that are characterized by a continuous interconnected phase of porous material. The pores, referred to as macropores allow liquid flow with minimal resistance, low back pressures, resulting in highly efficient chromatographic separation.
The term “non-ionic surfactant” as used herein invention refers to a surface-active agent consisting of a hydrophilic head group and a hydrophobic tail, carry no charge and are relatively non-toxic. Examples of non-ionic surfactants are Polysorbate 20, Polysorbate 80, Triton X-100 and Poloxamer.
The term "reference standard" refers to a known standard solution comprising the excipient, serving as a comparator in the studies.
The term “surface modifier” as used herein refers to substances attached to the base material via derivatization or crosslinking, thereby imparting the chemical nature of the modifier to the said base material. The surface modifier can be selected from a group consisting of an organic group surface modifier, a silanol group surface modifier, a polymeric coating surface modifier, and combinations thereof.
EXAMPLES
Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of this invention. The invention will now be described in greater detail by reference to the following non-limiting examples. The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
Example 1: Impurity profiling of polysorbate 80 in composition

10mM Ammonium acetate and (Acetonitrile + 0.2% (v/v) Formic acid) was used as mobile phase A and mobile phase B respectively. Standards were prepared by first making a stock solution of polysorbate 80 (2 mg/mL). From this, working standards were prepared by diluting with water. A standard curve was generated by injecting 15 µL of injection volume from different standard stock concentrations.
For sample run, 15 µL sample was loaded onto XBridge BEH C18 column (Waters, 5µ, 4.6 * 250mm) and run at a flow rate of 1.0 mL/min at a column temperature of 40°C as per gradient shown in table 1:
Time Flow rate (mL/min) %A %B
1 0.01 1 50 50
2 10 1 5 95
3 25 1 50 50
4 30 1 50 50
Table 1
Signal was collected using a charged aerosol detector by applying an evaporation temperature of 35°C. Blanks were run by injecting 15 ?L milli Q water. For system suitability testing, 15 µL of the 0.7 mg/mL polysorbate 80 was injected in duplicates in the beginning of the sample set, singlet after 10 sample injections and again as flank at the end of the sample set. The flank system suitability is run to ensure the successful completion of the sample set.
A typical chromatographic profile for qualitative analysis of polysorbate 80 shall have three distinct peaks at the retention time (RT) of ~11.7 min, ~13.0 min and ~14.7 min as shown in below Figure 1. Chromatographic profile of sample is overlaid with the representative peak profile of polysorbate 80 for a qualitative assessment of impurities if any as depicted in Figure 2 and matched against probable moiety as in Table 2.
As seen in Figure 2, the method can help assess thermal stability of polysorbate 80 solution as such or in formulation buffer. Figure 2(a) shows a dominant additional peak RT ~15.9 mins & Figure 2(b) displays additional dominant peaks marked as peaks 7 and 9, when compared with peak profile of standard solutions. Thus, the extent of degradation of polysorbate 80 due to temperature and storage conditions at different conditions can be easily assessed by given method.

Peak Approximate RT (min) Probable Moiety
1 1.5 POE
2 2.2 POE isosorbide
3 2.6 POE sorbitan
4 10.5 POE sorbitan epoxy-ester (stearate)
5 11.6 POE sorbitan ester (palimitate or palmitoleate)
6 13 POE sorbitan ester (oleate)
7 13.8 POE sorbitan ester (stearate or linoleate)
8 14.7 POE isosorbide ester (oleate)
9 15.9 POE ester (oleate)
10 16.4 POE sorbitan di-ester (di-oleate)
11 18.5 POE isosorbitan di-ester (di-oleate) or
POE sorbitan tii-ester (tri-oleate)

Table 2: Fingerprint of chromatographic peak profile for PS80 by HPLC-CAD

Example 2: Impurity profiling of Triton X-100 in composition

0.2% Formic acid in MilliQ water and (Acetonitrile+ 0.2% Formic acid) was used as mobile phase A and mobile phase B respectively. As standard stock, 1 mg/mL and 2 mg/mL Triton-X 100 was prepared. The standards remain stable at 2-8°C for about 19 days. Working standard of 0.2 mg/mL Triton-X 100 was prepared. Standard curve was generated by injecting 15 µL of injection volume from different standard stock concentrations.
For sample run, 15 µl sample was loaded onto XBridge BEH C18 column (Waters, 5µ, 4.6 * 250mm) and run at a flow rate of 1.0 mL/min at a column temperature of 40°C as per gradient shown in Table 3.

Time Flow %A %B
1 0.01 1 50 50
2 7 1 5 95
3 12 1 50 50
4 15 1 50 50
Table 3
Signal was collected using a charged aerosol detector by applying an evaporation temperature of 35°C. Blanks were run by injecting 15 ?L milli Q water. For system suitability testing, 15 µL of the 0.05 mg/mL Triton-X 100 was injected in duplicates in the beginning of the sample set, singlet after 10 sample injections and again as flank at the end of the sample set. The flank system suitability is run to ensure the successful completion of the sample set.
A typical chromatographic profile of Triton-X 100 will have one major peak at RT ~ 8.5 ± 0.5 minutes as shown in Figure 3. Chromatographic profile of sample can be overlaid with the representative peak profile of polysorbate 80 to check for qualitative assessment of impurities if any.

,CLAIMS:CLAIMS
We claim:
1. A composition comprising polypeptides with minimal deviation in impurity profile of excipients compared to that of a reference standard.
2. A method for determining impurity profile of an excipient in a composition comprising polypeptides, wherein the method comprises:
v. applying the composition to a chromatography column composed of a porous monolith hybrid material comprising inorganic and organic chromatographic material in a solution comprising a mobile phase A and a mobile phase B, wherein mobile phase B comprises formic acid and acetonitrile;
vi. eluting bound content from the column with a solution comprising mobile phase A and mobile phase B wherein the ratio of mobile phase A to mobile phase B is 1:19 from retention time of about 10 minutes to about 25 minutes; and
vii. measuring the eluted content of step ii with an aerosol detector;
viii. comparing the output of step iii with that of a reference standard solution
wherein the eluted content comprises impurities;
wherein the impurity comprises sub-species and degradants of the excipient and
wherein the method is devoid of a step to remove polypeptides from the composition.
3. The method as claimed in claim 2 wherein, the porous monolith hybrid material has a chromatographically-enhancing pore geometry comprising of homogenous particle homology and surface modified by a surface modifier.
4. The method as claimed in claimed 2 wherein, the aerosol detector is an evaporative liquid scattering detector, or a condensation nucleation light scattering detector, or a charged aerosol detector.
5. The method as claimed in claimed 2 wherein, the excipient is polysorbate-20 or polysorbate-80 or Triton X-100.
6. A method for testing stability of excipients in a dispensed drug composition comprising polypeptides intended for in-process testing or commercial release wherein, the method comprises:
v. applying the solution to a chromatography column composed of a porous monolith hybrid material comprising inorganic and organic chromatographic material in a solution comprising a mobile phase A and a mobile phase B, wherein mobile phase B comprises formic acid and acetonitrile;
vi. eluting bound content from the column with a solution comprising mobile phase A and mobile phase B wherein the ratio of mobile phase A to mobile phase B is 1:19 from retention time of about 10 minutes to about 25 minutes;
vii. measuring the eluted content of step ii with an aerosol detector; and
viii. comparing the output of step iii with that of a reference standard solution
wherein the eluted content comprises impurities;
wherein the impurity comprises sub-species and degradants of the excipient and
wherein the method is devoid of a step to remove polypeptides from the composition.