Substance quantification in complex mixtures

Abstract

The disclosed invention is in the field of analytical chemistry. In particular, it relates to methods for improved quantification of analytes in complex mixtures, and to compositions comprising precise amounts of said analytes. The improved method uses stable reference standards.

Claims

1. Method for producing a mixture comprising a known quantity of a first analyte, the method comprising the steps of: i) providing a mixture comprising the first analyte; ii) providing a reference standard comprising a known amount of a reference substance, wherein the reference substance is not the first analyte, wherein the reference substance is a small molecule, and wherein the ratio between the molar extinction coefficient of the first analyte and the molar extinction coefficient of the reference substance is known; iii) determining the absorbance of the first analyte and of the reference substance using spectrophotometry to obtain values for first analyte absorbance and for reference substance absorbance; and iv) determining the content of the first analyte in the mixture based on the obtained absorbance values and the known ratio of molar extinction coefficients.

2. The method according to claim 1, wherein the first analyte is a peptide, preferably wherein the first analyte is a peptide having a length from 5 to 100 amino acids.

3. The method according to claim 1, wherein the first analyte is a peptide comprising a sequence represented by any one of SEQ ID NOs: 1-12.

4. The method according to claim 1, wherein the mixture provided in step i) is a UV-transparent solution.

5. The method according to claim 1, wherein the mixture provided in step i) comprises the first analyte and solvents, wherein the solvents are preferably selected from water, isopropyl alcohol, and acetonitrile, and wherein optionally an acid is present such as formic acid or trifluoroacetic acid.

6. The method according to claim 1, wherein the mixture further comprises a second analyte and optionally a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, or fifteenth analyte, preferably wherein the ratios between the molar extinction coefficients of each analyte and the molar extinction coefficient of the reference substance are known.

7. The method according to claim 6, wherein the absorbance of each analyte is determined in step iii), and wherein the content of each analyte is determined in step iv).

8. The method according to claim 1, wherein step iii) is performed using analytical liquid chromatography, such as HPLC or UPLC, and wherein optionally step iii) comprises the following sub-steps: iii-a) adding a known amount of reference substance to the mixture; iii-b) separating the first analyte and the reference substance using liquid chromatography; iii-c) determining the absorbance of the first analyte and of the reference substance using spectrophotometry to obtain values for first analyte absorbance and for reference substance absorbance.

9. The method according to claim 1, wherein the molar extinction coefficient of a reference substance and/or analyte is the molar extinction coefficient determined at a wavelength from 180 to 800 nm.

10. The method according to claim 1, wherein the reference substance has a molar extinction coefficient of from 0.1.Math.10.sup.4 L.Math.mol.sup.−1.Math.cm-.sup.−1 to 3.Math.10.sup.4 L.Math.mol.sup.−1.Math.cm.sup.−1.

11. The method according to claim 1, wherein the reference standard is a United States Pharmacopeia (USP) reference standard.

12. The method according to claim 1, wherein the reference standard is a standard comprising caffeine, acetaminophen, sulfadimethoxine, verapamil, reserpine, amitriptyline, naphthalene, butylparaben, uracil, sulfaguanidine, Val-Tyr-Val, leucine-enkephalin, terfenadine, or a salt thereof, preferably comprising caffeine, acetaminophen, sulfadimethoxine, verapamil, reserpine, amitriptyline, naphthalene, butylparaben, uracil, or a salt thereof, more preferably comprising reserpine or a salt thereof.

13. A composition comprising at least four different peptides as defined in claim 3, wherein the weight percentage of each peptide is from 93% to 103% of the average weight percentage of all different peptides.

14. A plurality of compositions comprising at least a first composition and a second composition, wherein the first and the second composition are each a composition according to claim 13, wherein the composition differ from each other in that the compositions originate from different production batches, wherein the weight percentage of each peptide comprised in the first composition is from 93% to 103% of the weight percentage of that same peptide as comprised in the second composition, wherein preferably the compositions comprise one of: DP-5P) five different peptides, each peptide comprising one of SEQ ID NOs: 1-5; or DP-7P) seven different peptides, each peptide comprising one of SEQ ID NOs: 6-12.

Description

EXAMPLES

Example 1. Determination of Peptide Content in a Mixture Comprising a Peptide

[0127] A mixture comprising an unknown quantity of a peptide (SEQ ID NO: 1) is provided A reference standard solution comprising 0.1 mg reserpine is further provided. The ratio of molar extinction coefficients is determined by calculation of the relative response factor between the peptide and reserpine, using Formula II. To calculate the relative response factor, the response factor of the peptide is first determined by measuring the absorbance of a peptide solution of known concentration at 220 nm, followed by dividing the measured absorbance by the known concentration of the peptide in said solution. The response factor of reserpine at 220 nm is similarly determined. The absorbance of the peptide in the mixture comprising the unknown quantity of said peptide and of reserpine in the reference standard solution is determined at 220 nm by UPLC-UV. The unknown quantity of the peptide in the mixture is then determined using Formula III.

Example 2. Determination of Peptide Content in a Mixture Comprising Different Peptides Using a Conventional Analytical Method

General

[0128] The purity including individual related substances and the assay of a DP-5P (5 peptides, SEQ ID NOs: 1-5) and DP-7P (7 peptides, SEQ ID NOs: 6-12) mixture were determined by reversed phase rapid HPLC (UPLC) using a mobile phase gradient of acetonitrile and water and UV detection at λ=220 nm.

CHROMATOGRAPHIC CONDITIONS

[0129] Column: RP C18, e.g. Waters Acquity, BEH 130 PST (1.7 μm, 150×2.1 mm) [0130] Sample matrix Eluent A : Eluent B=85:15 (v/v). [0131] Sample concentration 100 μg/mL net peptide (of each drug substance mixture), duplicate [0132] Injection volume: 5 μL [0133] Column temperature: 65° C. [0134] Sample temperature: 5° C. [0135] Detection: UV (λ=220 nm) [0136] Flow rate: 0.3 mL/min [0137] Eluent A: 0.05%v/v TFA and 1%v/v Acetonitrile (ACN) in water [0138] Eluent B: 0.05%v/v TFA in ACN

TABLE-US-00002 TABLE 2 Gradient for conventional analytical method Time (min) Eluent A (% v/v) Eluent B (% v/v) 0 87 13 0.5 87 13 5.5 79.5 20.5 17.0 68 32 22.8 45 55 28.5 45 55 28.6 20 80 30.0 20 80 30.1 87 13 33.0 87 13

Reporting

[0139] The relative retention times (RRTs) assigned to peptides and related substances both in a DP-5P (SEQ ID NO: 1-5) and DP-7P (SEQ ID NO: 6-12) mixture was calculated with reference to the retention time of the peptide eluting in the middle (FIG. 1 and FIG. 2). That retention time was used as reference as the main peak of this drug substance was detected approx. in the center of the chromatogram. The related substances were determined by area normalization. The individual related substances were reported≥0.10%a/a. The total related substances were calculated as the sum of all individual related substances equal to or greater than a disregard limit of 0.0500%a/a. The purity was calculated as the difference between total related substances and 100%.

[0140] The assay of each of the peptides was calculated against a reference standard solution containing either five peptides (DP-5P) or seven peptides (DP-7P) and was expressed in percent and in mg net peptide/vial. For this reference method, the absorption ratio between the reference substance and the analyte is 1, because the reference substance is the analyte itself.

Example 3. Determination of Peptide Content in a Mixture Comprising Different Peptides Using an Analytical Method According to the Invention

General

[0141] The UPLC method similar to Example 1 was used for the quantification of analytes (i.e. the individual peptides) in mixtures DP-5P (SEQ ID NO: 1-5) and DP-7P (SEQ ID NO: 6-12) and for the determination of content uniformity of mixtures DP-5P and DP-7P (lyophilized product). The method is based on the use of an external reference standard. The relative response factor (ratio of molar extinction coefficients, RRF) against this external reference standard has been established for each peptide. The purity, individual related substances, and the assay of DP-5P and DP-7P were determined by reversed phase rapid HPLC (UPLC) using a mobile phase gradient of ACN/IPA and water and UV detection at λ220 nm.

Materials

[0142] The following lyophilized peptide compositions were used: DP-5P comprising peptides represented herein by SEQ ID NO: 1-5 admixed at equal net weights of 0.40 mg of each peptide per vial (total amount of protein per vial being 2.00 mg) and 0.56 mg TFA per vial; and DP-7P comprising peptides represented herein by SEQ ID NO: 6-12 admixed at equal net weights of 0.40 mg of each peptide per vial (total amount of protein per vial being 2.80 mg) and 0.96 mg TFA per vial. The following chemicals were used: Acetonitrile, (Biosolve, 01204102); UPLC water, (Type 1, milli-Q); trifluoroacetic acid, (Chem Lab, CLoo.2094.0001), 2-propanol, (UPLC/MS grade, Biosolve, 16264102); Reserpine, (USP, Sigma Aldrich, 1601000). The following equipment was used: QuanRecovery vials, (Waters, 186009186); UPLC pump equipped with a gradient system, autosampler, and UV detection (Waters Acquity or equivalent) and mixer volume 425 μL.

Preparation of solvents and solutions:

[0143] Mobile phase A: 0.1% (v/v) TFA in water [0144] Mobile phase B: 0.1% (v/v) TFA in 70/30 ACN/IPA [0145] Diluent A: 0.05% (v/v) TFA in water [0146] Diluent B: 0.05% (v/v) TFA in ACN [0147] Blank solution: 80/20 (v/v) diluent A/diluent B [0148] Purge solvent: 80/20 (v/v) water/ACN [0149] Needle wash: 0.05% (v/v) TFA in 80/20 (v/v) water/ACN

Stock reference solution (100 μg/mL Reserpine)

[0150] 5.00 mg Reserpine reference material was accurately weighed and added into a 50 mL volumetric flask. 40.0 mL of blank solution was added, by means of a volumetric pipette. The solution was stirred for 10 min, by means of a magnetic stirrer, until fully dissolved. The solution was diluted to volume with blank solution and mixed well.

Working reference solution (10 μg/mL Reserpine)

[0151] Transfer of 2.0 mL, by means of a volumetric pipette, of stock reference solution 1 into a 20 mL volumetric flask. Diluted to volume with blank solution and homogenized.

LOQ solution (20 ng/mL Reserpine)

[0152] Transfer of 2.0 mL, by means of a volumetric pipette, of working reference solution 1 into a 100 mL volumetric flask. Diluted to volume with blank solution and homogenized. Transfer of 5 mL, by means of a volumetric pipette, of the above solution into a 50 mL volumetric flask. Diluted to volume with blank solution and homogenized.

Sample Solution

[0153] This sample preparation was setup for lyophilized products DP-5P and DP-7P in an initially sealed container. Before opening and dissolving the drug product, the vials were equilibrated at room temperature for at least 1 hour. The content of the vial was dissolved with 0.8 mL of diluent B, and stirred for 20 min, using a magnetic stirrer. 3.2 mL of diluent A were added. The solution was stirred well for 20 min, using a magnetic stirrer. After dissolving the content of the vial, the sample vial was kept on the lab bench for 30 minutes. The solution was stirred briefly and transferred into an HPLC vial.

Chromatographic Conditions

[0154] Column: Advance Bio Peptide Map (2.7 μm, 250 x 2.1 mm) [0155] Sample matrix 0.05%v/v TFA in ACN : water (1:4 v/v) [0156] Sample concentration 100 μg/mL net peptide (of each drug substance), duplicate [0157] Injection volume: 10 μL [0158] Column temperature: 40° C. [0159] Sample temperature: 5° C. [0160] Detection: UV (λ=220 nm) [0161] Flow rate: 0.2 mL/min [0162] Run time: 65 min [0163] Purge solvent: 80/20 (v/v) water/ACN [0164] Needle wash: 0.05% (v/v) TFA in 80/20 water/ACN (v/v) (20 s) [0165] Seal wash: 90/10 (v/v) water/MeOH [0166] Elution mode: Gradient elution [0167] Eluent A: 0.1%v/v TFA in water [0168] Eluent B: 0.1%v/v TFA in ACN/IPA (70:30 v/v)

TABLE-US-00003 TABLE 3 Gradient for analytical method according to the invention Time (min) Eluent A (% v/v) Eluent B (% v/v) 0 80 20 1 80 20 50 35 65 50.1 1 99 55 1 99 55.1 80 20 65 80 20

TABLE-US-00004 TABLE 4 Injection sequence Injec- tions Solution (#) Acceptance criteria Blank min. 2 Blank peaks preferably absent or if present, solution preferably separated from the peptides and all related impurities LOQ solution 1 S/N ≥ 10 Identification 1 The chromatogram is visually comparable with solution the example chromatogram in the method Working 1 Recovery towards mean injections (n = 5) of reference working reference solution 1: 98.0%-102.0% solution 2 Working 5 RSD on area: % RSD(n = 5) ≤ 1.0 reference solution 1 Sample 1 Maximum 12 sample injections between solutions reference solutions Control 1 Recovery towards mean injections (n = 5) of working working reference solution 1: 98.0%-102.0% reference solution 1

Relative Response Factor (RRF)

[0169] The RRF was determined for all 12 peptides, enabling the quantification of each peptide against a reserpine as shown above. For the determination, the absorbance of each peptide in a solution comprising said peptide at a known concentration, and of a reference standard solution comprising reserpine at a known concentration, were measured at 220 nm. Response factors for each peptide and for reserpine were determined as described above, by dividing the measured absorbance by the known concentration value (Table 5). The relative response factor was then determined using Formula II.

[00004]$\begin{array}{cc}RRF=\frac{R{F}_{\mathrm{peptide}}}{R{F}_{\mathrm{reserpine}}},& \mathrm{Formula}\mathrm{II}\end{array}$

TABLE-US-00005 TABLE 5 Absorbance at 220 nm, concentration of measured solutions and relative response factors (RRFs) between each peptide and reserpine     Compound   Response (μV*sec)   Concentration (mg/mL) RF [00005]$\left(\frac{\mathrm{Response}}{\mathrm{Concentration}}\right)$ RRF.sub.reserpine [00006]$\left(\frac{{\mathrm{RF}}_{\mathrm{peptide}}}{{\mathrm{RF}}_{\mathrm{reserpine}}}\right)$ Reserpine 3001390 0.0099 303275005 N/A Peptide 6 (SEQ ID NO 6) 4396082 0.0974 45122916 0.148 Peptide 7 (SEQ ID NO 7) 5164489 0.1019 50692679 0.165 Peptide 8 (SEQ ID NO 8) 3476444 0.0966 36004134 0.116 Peptide 9 (SEQ ID NO 9) 3353840 0.0984 34092402 0.110 Peptide 10 (SEQ ID NO 10) 3068353 0.0994 30878932 0.101 Peptide 11 (SEQ ID NO 11) 4455737 0.1011 44066034 0.146 Peptide 12 (SEQ ID NO 12) 3376228 0.1009 33453703 0.108     DP-5P   Response (μV*sec)   Concentration (mg/mL) RF [00007]$\left(\frac{\mathrm{Response}}{\mathrm{Concentration}}\right)$ RRF.sub.reserpine [00008]$\left(\frac{{\mathrm{RF}}_{\mathrm{peptide}}}{{\mathrm{RF}}_{\mathrm{reserpine}}}\right)$ Reserpine 3166278 0.0104 304008664 N/A Peptide 1 (SEQ ID NO 1) 5901793 0.1019 57907269 0.190 Peptide 2 (SEQ ID NO 2) 3781376 0.0986 38358917 0.125 Peptide 3 (SEQ ID NO 3) 3852495 0.0974 39571863 0.132 Peptide 4 (SEQ ID NO 4) 5030590 0.0995 50537361  0.168* Peptide 5 (SEQ ID NO 5) 2258101 0.1019 22169413 0.073 Note: *A different reserpine solution was used for the determination of the RRF, this solution has an RF of 301932379

Assay calculation

[0170] The content of each peptide in each analysis was determined using a single level external standard method with working reference solution 1. The formula to calculate the content of an individual peptide per vial was adapted from Formula III as follows.

[00009]$\begin{array}{c}\mathrm{Assay}\left(\mathrm{mg}\mathrm{peptide}/\mathrm{vial}\right)=\frac{A_{\mathrm{SLP}}\times Q_{\mathrm{RS}}\times P_{\mathrm{RS}}\times \mathrm{DF}}{A_{\mathrm{RS}}\times Q_S\times \mathrm{RRF}}\end{array}$ [0171] A.sub.SLP: Response of individual peptide (area) (SLP stands for peptide solution) [0172] A.sub.RS: Average response Reserpine (RS) in the 5 injections of the working reference solution 1 (area) [0173] Q.sub.RS: Quantity of Reserpine in working reference solution 1 (mg) [0174] Q.sub.S: Quantity of the DP: 1 (vial) [0175] P.sub.RS: Purity of Reserpine (RS) (expressed in ratio of 1, e.g. 0.99) [0176] DF: Dilution factor of the SLP-solution

[00010]$DF=\frac{V_{\mathrm{SLP}}}{V_{\mathrm{stock}\mathrm{solution}\mathrm{RS}}\times D{F}_1}=0.008$$V_{\mathrm{SLP}}=\mathrm{volume}\mathrm{used}\mathrm{to}\mathrm{dissolve}\mathrm{the}\mathrm{content}\mathrm{of}\mathrm{the}\mathrm{vial}\left(=4\mathrm{mL}\right)$$V_{\mathrm{stock}\mathrm{solution}\mathrm{RS}}=\mathrm{volume}\mathrm{of}\mathrm{stock}\mathrm{reference}\mathrm{solution}\left(=50\mathrm{mL}\right)$$D{F}_1=\mathrm{Dilution}\mathrm{factor}\mathrm{of}\mathrm{the}\mathrm{stock}\mathrm{reference}\mathrm{solution}\mathrm{to}\mathrm{obtain}\mathrm{working}\mathrm{reference}\mathrm{solution}\left(=10\right)$ [0177] RRF: Relative response factor between Reserpine and SLP (ratio of response factors, shown in Table 5)

Reporting

[0178] The relative retention times (RRTs) assigned to peptides and related substances in both DP-5P and DP-7P were calculated with reference to the retention time of the peptide eluting in the middle (FIG. 3 and FIG. 4). That retention time was used as reference as the main peak of this drug substance is detected approx. in the center of the chromatogram. The related substances were determined by normalization of the total response.

[0179] The individual related substances are reported ≥0.10%w/w. The total related substances were calculated as the sum of all individual related substances equal to or greater than a disregard limit of 0.05%w/w. The purity was calculated as the difference between total related substances and 100%w/w. The assay of each of the drug substances is calculated against the USP reference standard reserpine and is expressed in mg net peptide/vial.

Example 4. Exemplary Peptide Content Determination and Calculation

[0180] For the analysis the following solutions/ samples are prepared and injected according to the method: [0181] I. Reference solution [0182] 1) A stock solution is prepared from 5.08 mg Reserpine in 50 mL. [0183] 2) 2 mL of the stock is diluted to a total of 20 mL (dilution factor (DF,) =10) [0184] The purity of reserpine is taken from its CoA: 98.2% (purity factor 0.982) [0185] The mean response over 5 injections is 3088845 μV*sec [0186] II. The sample solution: [0187] 1) One vial that contains approximately 0.4 mg of each peptide present in DP-5P is dissolved in 4 mL [0188] 2) The response for peptide 4 (SEQ ID NO. 4) for injection is 4984380 μV*sec [0189] III. The dilution factor (DF) for the solution of peptide 4 (SEQ ID NO. 4) is calculated as follows:

[00011]$DF=\frac{V_{\mathrm{SLP}}}{V_{\mathrm{stock}\mathrm{solution}\mathrm{RS}}\times D{F}_1}=\frac{4}{50\times 10}=0.008$ [0190] IV. Now the assay for peptide 4 (SEQ ID NO. 4) can be calculated by filling out formula III:

[00012]$\mathrm{Assay}\mathrm{peptide}\mathrm{\#4}=\frac{A_{\mathrm{SLP}}\times Q_{\mathrm{RS}}\times P_{\mathrm{RS}}\times \mathrm{DF}}{A_{\mathrm{RS}}\times Q_S\times {\mathrm{RRF}}_{\mathrm{peptide}4/\mathrm{reserpine}}}=\frac{4984380\times 5.08\times 0.982\times 0.008}{3088845\times 1\times 0.168}=0.38\mathrm{mg}/\mathrm{vial}$

Example 5. Improved Results are Obtained with a Method of the Invention

DP-5P

[0191] The DP-5P batch shown in Table 6 was manufactured by combining 5 separate peptides in solution in a 1:1 ratio each. Subsequently the solution of filled in vials was lyophilized, ending up with a vial that contained 0.400 mg of each of the peptides (i.e. 2.00 mg in total). The content of each of the peptides can be expressed in several ways, amongst which the actual amount present in a vial in mg or the percentage of the amount in relation to the manufacturing target of 0.400 mg per vial.

At T=0 months (TOM) the peptide content was measured using the conventional method of Example 2. From T=48 months (T48M) onwards the peptide content was measured with the conventional method of Example 2 and the method according to the invention of Example 3. The results are shown in Table 6:

TABLE-US-00006 TABLE 6 Comparison of data from the same DP-5P batch Method according to the Conventional method (peptide as RS) invention (USP RS) Identity T0M T48M T54M T60M T48M T54M T60M Label claim (% target 0.40 mg/vial) Peptide 4 (SEQ ID NO 4) 98%  98%  98%  98% 96% 99% 96% Peptide 2 (SEQ ID NO 2) 102%  104% 104% 104% 99% 98% 97% Peptide 1 (SEQ ID NO 1) 101%  103% 102% 102% 98% 98% 93% Peptide 3 (SEQ ID NO 3) 99% 108% 105% 104% 95% 95% 97% Peptide 5 (SEQ ID NO 5) 99% 103%  99% 100% 101%  101%  99% Assay (mg/vial) Peptide 4 (SEQ ID NO 4) 0.3936 0.3937 0.3911 0.3913 0.3820 0.3966 0.3842 Peptide 2 (SEQ ID NO 2) 0.4085 0.4141 0.4149 0.4174 0.3950 0.3932 0.3880 Peptide 1 (SEQ ID NO 1) 0.4047 0.4107 0.4088 0.4082 0.3910 0.3932 0.3701 Peptide 3 (SEQ ID NO 3) 0.3942 0.4311 0.4194 0.4170 0.3798 0.3789 0.3883 Peptide 5 (SEQ ID NO 5) 0.3976 0.4107 0.3948 0.4011 0.4028 0.4021 0.3968 Total net peptide 1.9986 2.0603 2.0290 2.0350 1.9506 1.9641 1.9274

DP-7P

[0192] The DP-7P batch shown in Table 7 manufactured by combining 7 separate peptides in solution in a 1:1 ratio each. Subsequently the solution of filled in vials was lyophilized, ending up with a vial that contained 0.400 mg of each of the peptides (i.e. 2.80 mg in total). The content of each of the peptides can be expressed in several ways, amongst which the actually amount present in a vial in mg or the percentage of the amount in relation to the manufacturing target of 0.400 mg per vial.

At T=0 months (TOM) the peptide content was measured using the conventional method of Example 2. From T=48 months (T48M) onwards the peptide content was measured with the conventional method of Example 2 and the method according to the invention of Example 3. The results are shown in Table 7:

TABLE-US-00007 TABLE 7 Comparison of data from the same DP-7P batch Method according to the Conventional method (peptide as RS) invention (USP RS Identity T0M T48M T54M T60M T48M T54M T60M Label claim (% target 0.40 mg/vial) Peptide 6 (SEQ ID NO 6) 108% 110% 107% 107% 103% 101%  100%  Peptide 10 (SEQ ID NO 10) 105% 108% 107% 109% 101% 99% 99% Peptide 11 (SEQ ID NO 11) 106% 107% 105% 105%  97% 95% 95% Peptide 8 (SEQ ID NO 8) 109% 108% 107% 107%  99% 98% 98% Peptide 7 (SEQ ID NO 7) 107% 107% 105% 106% 101% 99% 99% Peptide 12 (SEQ ID NO 12) 107% 108% 105% 105% 103% 101%  100%  Peptide 9(SEQ ID NO 9) 108% 108% 107% 109%  98% 93% 93% Assay (mg/vial) Peptide 6 (SEQ ID NO 6) 0.4315 0.4390 0.4280 0.4286 0.4100 0.4033 0.4010 Peptide 10 (SEQ ID NO 10) 0.4212 0.4329 0.4271 0.4362 0.4060 0.3966 0.3945 Peptide 11 (SEQ ID NO 11) 0.4235 0.4293 0.4192 0.4209 0.3866 0.3815 0.3788 Peptide 8 (SEQ ID NO 8) 0.4357 0.4323 0.4289 0.4294 0.3980 0.3928 0.3900 Peptide 7 (SEQ ID NO 7) 0.4393 0.4279 0.4219 0.4222 0.4036 0.3980 0.3967 Peptide 12 (SEQ ID NO 12) 0.4261 0.4323 0.4215 0.4219 0.4132 0.4034 0.4010 Peptide 9 (SEQ ID NO 9) 0.4328 0.4325 0.4262 0.4361 0.3901 0.3718 0.3700 Total net peptide 3.0101 3.0262 2.9728 2.9953 2.8074 2.7473 2.7320

Assessment

[0193] Several facts can be considered: [0194] From a manufacturing perspective values that are higher than the target of 0.4 mg per vial are not expected; and similarly a value higher than 2.0 mg (DP-5P) and 2.8 mg (DP-7P) is not expected. The manufacturing process warrants the amount of peptide-input per peptide is according to the target; if any, results expected might be slightly lower due to e.g. potential of adsorption to filter or other surface. Therefore values over 100% have a higher probability of not being accurate, while values under 100% do not have this issue. [0195] From a stability perspective assay values are not expected to increase over time, but rather decrease due to potential degradation taking place over time. Values that increase over time have a higher probability of not being accurate. [0196] Per peptide a trend can exist in level over time, presenting a downwards level. In general it is not to be expected that levels increase (other than slightly due to method variability) or that values do not present a trend (e.g. values alternately increasing and decreasing over time).

[0197] As illustrated by the results (and summary in Table 8) the method of the invention shows significantly improved quantification over the conventional method.

TABLE-US-00008 TABLE 8 Comparison of results and their variation by both methods for DP-5P and DP-7P DP-5P DP-7P Method Method Target Conventional according to Conventional according to Variation value method invention method invention 48M Individual 100  98-108  95-101 107-110  98-103 peptides assay (% label claim) Individual peptides 0.4000 0.3911-0.4311 0.3701-0.4028 0.4192-0.4390 0.3700-0.4132 assay (mg/vial) Total peptides 2.000 1.9986-2.0603 1.9274-1.9506 assay (mg/vial) 2.800 2.9728-3.0101 2.7473-2.8074